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MED Phoenix

Volume (1), Issue (1), July 2016ISSN: 2392 – 425X (Print)

A Peer Reviewed International Medical Journal

An Official Journal of National Medical College

Birgunj, Nepal

NatioNal Medical collegeBirgunj, Nepal

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any means, electronic,mechanical, photocopying, recording or otherwise without the prior permission of MEd PhOENIx.

An Official Journal of National Medical College

Volume 1, Issue 1, July 2016

MED PhoenixA Peer Reviewed International Medical Journal

MED PHoEnix : An Official Journal of NMC, Birgunj, Nepal, Volume (1), Issue (1) July 2016, ISSN: 2392 – 425X (Print)

Editor-in- Chief Prof.Dr.JainuddinAnsari Executive Editor Dr.MukhtarAnsari

EditorsDr.AnandKumarJhaDr.DipakBhargavaMrs.T.MalarvaniDr.AmarnathMishraDr.Md.ParwezAhmadDr.M.RahullahDr.M.IzharMr.AnirbanMajumderMr.ManishKumarSinghDr.SarojDeoMr.RakeshKumarGuptaDr.AkhtarA.AnsariDr.RaviShankarGupta

Assistant EditorsMrs.ShrutiSahDr.TarannumKhatunDr.SujitaBhandariMr.RakeshSinghMr.SatyendraPrakashSingh

Statistical Editor Prof.Dr.S.P.Patel

Editorial Advisory BoardMd.BasaruddinAnsari Prof.Dr.A.K.Koshy Mr.AbidHussainAnsari Prof.Dr.K.N.SinghProf.Dr.M.AslamProf.Dr.P.N.SinghProf.Dr.J.N.VyasProf.Dr.R.S.P.SinhaProf.Dr.AwadheshKumarJhaProf.SavitriBhattaraiProf.Dr.J.N.ShivapuriProf.Dr.B.K.VermaProf.Dr.A.HassanProf.Dr.KedarPd.BaralDr.SabihaYashmineDr.P.K.Sarraf

International Editors Dr.MohamedIzham,QatarDr.AzharHussain,PakistanDr.AzmiHassali,MalasiyaDr.H.WaseemSheriff,USADr.S.Jeevanandham,EthiopiaDr.M.Adak,MauritiusDr.SaffarMansoor,USADr.P.RaviShankar,Aruba Technical Support Er.AshwiniKumarMr.SajadHawari

EDITorIAL BoArD MEMBErS


This inaugural issue of the journal, is dedicated to our former Medical Education director - Late dr. S. N. hissaria & former Principal - Late Prof. dr. L. Sharma.

Late dr. S. N. hissaria Late Prof. dr. L. Sharma

A tribute


TABLE of CoNTENTS

Message from Editor-in-Chief iMessage from Principal ii

Editorial• Gifted authorship in scientific writings 1-2

Ansari Mukhtar

research Articles• Anti Escherichia coli activity of some medicinal plants of high altitude of Nepal 3-9

Bhargava Dipak, Abhilasha Saha, Mondal Keshab Chandra, Pandit Bijay Raj, Shidiki Amrullah,

Chaudhary Navin Kumar, Gupta Ravi Shankar, Jha Awadhesh Kumar

• Lipid peroxidation and antioxidant status in male and female patients with Type 1 and Type 2 Diabetes Mellitus 10-14Madhikarmi Nirjala Laxmi, Singh Prem Prakash, Khatun Tarannum

• Bacteriological and physicochemical analysis of drinking water in Tokha, Kathmandu, Nepal 15-18Shidiki Amrullah, Bhargava Dipak, Gupta Ravi Shankar, Ansari Akhtar Alam, Pandit Bijay Raj

Review Articles• A review on congenital Tuberculosis 19-22

Jha Anand, Bhandari Sujeeta, Kadel Binod, Kadel Anjeela, Shah Dhiraj,

Sah Arun Kumar

• Oligohydramnios and fetal outcome: A review 23-30Khatun Tarannum, Ansari Akhtar Alam, Hamid Irfan, Gupta Ravi Shankar,

Ahmad Md. Parwez

A review on Pharmacological and chemical documentation of Euphorbia hirta Linn (Asthama herb) 31-38 Ansari Akhtar Alam, Khatun Tarannum, Ahmad Md. Parwez1, Gupta Ravi Shankar,

Ansari Mukhtar, Madhikarmi Nirjala Laxmi


Case reports• Acute intestinal obstruction due to Ascaris 39-40

Bhatt Pashupati N

• Problems and solution to diagnose Extrapulmonary Tuberculosis in central region of Nepal 41-43Gupta Ravi Shankar, Khatun Tarannum, Ansari Akhtar Alam, Shidiki Amrullah, Bhargava Dipak, Gupta Bidhya, Majumder Anirban

• Role of DNA fingerprinting in disputed paternity 44-46Mishra Amarnath, Sathyan Sukumaran

Letter to the Editor• Can you grow grain in Jumla? 47-48

Shankar Pathiyil Ravi


DearReaders,

Greetings!

NationalMedicalCollegeisaprivateMedicalCollegewhichcameinexistencein2001ADdespiteseveralobstacles.Weworkeddaytonighttomakethisasanestablishedinstitutionandoneofthelargest private Medical College and Teaching Hospital of Nepal. NMC is committed to producecompetentmedicaldoctorsandprovidequalityhealthcareservicesandwesucceededinthisdirection.However,welaggedintermsofresearchandpublicationsintheformof‘Journal’. Today, I feel proud to announce that after untiring efforts of the editorial team especially ChiefExecutiveEditor,wefinallysucceededtolaunchtheinauguralissueof‘MEDPHOENIX’2016.MEDPHOENIXisapeerreviewedinternationalmedicaljournalpublishedbyNationalMedicalCollege,Birgunj,Nepal.

AsanEditor-in-Chiefofthejournal,IwouldliketoassureyouthatIwon’tcompromiseinthequalityofjournal.Ihopeyouwillenjoyreadingthepapersandhavegreatlearningandupdatesinmedicaleducationandpractice.

With thanks

Prof. (Dr) Jainuddin AnsariEditor-in-Chief

MESSAgE

i


DearReaders,

Greetingsandwelcometotheinauguralissueof“MEDPHOENIX”,theOfficialMedicalJournalofNationalMedicalCollege,Birgunj,Nepal.IcommendthecollectiveeffortsandvisionofthemembersoftheEditorialBoardinrealizingthepublicationofthisjournal.

NationalMedicalCollege has come a longway since its inception and publication of the officialjournal isa landmark testimonyofendorsementof scientific researchamong facultymembersandstudentsofthisinstitution.

IexpressfullconvictionthatthisjournalwillserveasaneminentcommunicationchannelforresearchersconcernedwithallaspectsofMedicine.

MayIhavethesefewwordstoourreadership:thesuccessofthisjournalintheyearstocomedependsonyou.Yourcontributionintermsofsubmissionofresearcharticlesandsharingyourideastohelpimprovethisjournalintheforthcomingissuesmatteralottous.

Finally,IwishtosharewithallmembersoftheEditorialBoardandourreadershipmycherishedjoytohavelearntaboutthelaunchoftheOfficialMedicalJournalofthispremierinstitutioninthefieldofMedicalEducation.

Prof(Dr.)AnilKumarKoshyPrincipal

MESSAgE

ii

MED PHoEnix : An Official Journal of NMC, Birgunj, Nepal, Volume (1), Issue (1) July 2016, ISSN: 2392 – 425X (Print) 1

Ansari Mukhtar

ChiefExecutiveEditor,MEDPHOENIX

ABSTrACTThetrendofhonoraryauthorshipisproliferatingmainlyinresearchpapersfromdevelopingnationswhere resources are scarce, and the concept of research and publication is in growing stage.Thepracticeofgiftedauthorshipisunethicalandunacceptable,andthusitshouldbediscouraged.

Keywords: Authorship,Gift,Medicalwriting

Corresponding Author: Dr. MukhtarAnsari, Chief Executive Editor, MED PHOENIX; E-mail:[email protected];[email protected]

It is imperative to disseminate new ideas, thoughts, research findings and innovations among thescientificcommunitiesof theworld tomake themusefulandfruitful.And,scientificwriting is themostfavoredmeanstodoso.Forthis,researchoutcomes,ideasorconceptsneedtobepublishedinpeerreviewedscholarlyjournals.Thereisaverypopularquotation“PublishorPerish”toemphasizethecultureofpublishingtheresearchfindings.

Inrecenttime,asthenumberofjournalsisincreasingdaybyday,thepracticeofincludinggiftedauthorsinpapersisalsoproliferating.Inmanyresearchpaperspublishedinandfromdevelopingcountries,alonglistofauthorsareincludedwhoareeitherfromthesamedepartmentorotherdepartmentsofthesameordifferentinstitutions.Infact,theresearchisdesignedandcarriedoutbyoneortwoauthorsandtherestarehonoraryauthors.Sometimes,anauthorhasnotcontributedatallbutbecomesthefirstorsecondauthorofthepaperandthisconceptisbasedonmutualunderstandingbetweentheactualcontributorandthegiftedone.

Therecanbeseveralreasonsforgiftedauthorship.Publicationsareimportantforpromotioninacademicpostorranking,juniorresearchersmaybepressuredbysenioranddepartmentalheadtoincludetheirname,youngresearchersmaybelievethatincludingexperiencedcolleaguesorrenouncedfacultiesasauthorswillincreasetheirchancesofpublication,andalsotoincreasecollaborationamongauthors.1

Authorshipmattersbecauseauthorsareresponsibleandaccountableforthepublishedwork.However,therearecertaincriteriathatneedtobefulfilledbyallofthecontributorstobecreditedasauthorsofthemanuscript,andtheirsequenceshouldappearaspertheircontributions.Toavoidconfusionaboutauthorship,theInternationalCommitteeofMedicalJournalEditors(ICMJE)recommendsfourcriteriatobemetbyeachofthecontributorstobecomeauthorofapublishedwork.2 Substantialcontributions to theconceptionordesignof thework;oracquisitionofdata;or the

analysis,orinterpretationofdataforthework.

gIfTED AuTHorSHIP IN SCIENTIfIC wrITINgSEDITorIAL

MED PHoEnix : An Official Journal of NMC, Birgunj, Nepal, Volume (1), Issue (1) July 2016, ISSN: 2392 – 425X (Print)2

Draftingtheworkorrevisingitcriticallyforimportantintellectualcontent. Finalapprovaloftheversiontobepublished. Agreementtobeaccountableforallaspectsoftheworkinensuringthatquestionsrelatedtothe

accuracyorintegrityofanypartoftheworkareappropriatelyinvestigatedandresolved.

Itisnotnecessarythatonecanonlybeconsideredaspartofapublishedpaperorscientificwritingifheisoneoftheauthors.Inreality,thosewhodonotmeettheabovefourcriteriacanalsobepartofthepaperandtheycanbeacknowledgedintheacknowledgementsectionofthemanuscript.Examplesof activities that alone (without other contributions) do not qualify a contributor for authorship isacquisitionoffunding;generalsupervisionofaresearchgrouporgeneraladministrativesupport;andwritingassistance,technicalediting,languageediting,andproofreading.

Regardless of the cause, gifted authorship is an unethical and unacceptable practice.Therefore, itshouldbediscouraged.

rEfErENCES1. StreetJM,RogersWA,IsraelM,Braunack-MayerAJ.Creditwherecreditisdue?Regulation,research

integrityandtheattributionofauthorshipinthehealthsciences.SocSciMed.2010;70(9):1458-65.

2. Definingtheroleofauthorsandcontributors[Internet].InternationalCommitteeofMedicalJournalEditors. [http://www.icmje.org/recommendations/browse/roles-and-responsibilities/defining-the-role-of-authors-and-contributors.html]

Cite this article as: Ansari M. Gifted authorship in scientific writings. MED PHOENIX 2016;1(1):1-2


Bhargava Dipak1*, Abhilasha Saha2, Mondal Keshab Chandra3, Pandit Bijay Raj1, Shidiki Amrullah1 Chaudhary Navin Kumar1, Gupta Ravi Shankar1

, Jha Awadhesh Kumar4

1DepartmentofMicrobiology,NationalMedicalCollege,Birgunj,Nepal2DepartmentofPediatricNursing,NationalMedicalCollege,NursingCampus,Birgunj,Nepal3DepartmentofMicrobiology,VidyasagarUniversity,PaschimMedinipur-721102,WB,India4DepartmentofPharmacology,NationalMedicalCollege,Birgunj,Nepal

Anti Escherichia Coli Activity of Some Medicinal Plants of High Altitude of NepalrESEArCH

ABSTrACTBackground: Plants and plant-basedmedicamentsarethebasisofmanyofthemodernpharmaceuticals we use today for our variousailments. The aim of the present study was tofind out the bioactive chemical constituentssuchasflavonoids,alkaloids, tannins,saponins,carbohydrate and to find out the anti E. coli activityoftheethanolicextractsoftraditionallyusedtenmedicinalplantsofNepalatanaltitudeof1500ftfromabovethesealevel.

Methods:Ethanolicextractsoftencommonlyusedmedicinalplantswereanalyzedphytochemicallyandevaluated for their significantantimicrobialactivityagainsttheclinicalisolatesofEscherichia coli. Mean zones of inhibition were calculatedforeachoftheextracts.

results: Theresultsrevealedthatthoughalltheplantsof thehighaltitudeshowedsomedegreeof antimicrobial activity, the leaf extract ofSyzygium cumini (5.7±0.3 cm), Chromolaenaodorata(5.2±0.4cm),Ocimumsanctum(4.7±0.6cm) and Justicia adhatoda (3.2±0.3 cm) weremosteffectiveagainst theclinical isolatesofE.coli, whereas the other six plant extracts wereleast effective against the clinical isolates ofE. coli. Qualitative phytochemical analysis of

the extracts revealed the presence of bioactivecomponents.Sevenoftheplantextractscontainalkaloids,sixofthemcontainglycosides,fourofthem contain flavonoids, three of them containcarbohydrate, oil and fats, twoof themcontaintannins, whereas only one of them containssaponins.

Conclusion: The result of this study justifiedthe folkloric usage of the studied plants andconcluded that these plants extract have greatpotential in finding new clinically effectiveantimicrobialcompounds.

Keywords: Analysis, Antimicrobial, Effect,Phytochemical,Plantextracts

Corresponding Author: Dr. Dipak Bhargava,Department ofMicrobiology,NationalMedicalCollege, Birgunj, Nepal; E-mail: [email protected]

INTroDuCTIoNHerbal medicine contributes immensely to thequalityoflifeforthousandsofyears.Plantsareanessentialpartoftraditionalmedicineinalmostanyculture.Currentlytheworldpopulationisabout5-6 billion and the increase in this populationisexpected to riseup to7.5billionby theyear


2020.1,2AccordingtoWorldHealthOrganization(WHO),morethan80%oftheworld’spopulationreliesontraditionalmedicinesfor theirprimaryhealth careneeds. Themedicinal propertiesofplantsareduetotheactivechemicalconstituentspresentindifferentpartsoftheplant.3

These chemical constituents produce a definitephysiologic action on the human body. Themost important bioactive compounds of plantsare alkaloids, flavonoids, tannins and phenoliccompounds. The phytochemical researchbased on ethno-pharmacological information isgenerally considered an effective approach inthediscoveryofnewanti-infectiveagents fromhigherplants.4

Medicinalplantsareasourceofgreateconomicvalue all over the world. The World HealthOrganizationalsosupportstheuseofmedicinalplants provided it is proven to be efficaciousand safe.5 Nature has bestowed on us a veryrich botanical wealth and a large number ofdiverse types of plants grow in different partsof the country. In high altitude country likeNepal thousands of species are known to havemedicinalvalueandtheuseofdifferentpartsofseveralmedicinalplantstocurespecificailmentshasbeeninvaguesinceancienttimes.Amongthe7000speciesofmedicinalplants recognizedallovertheworld,morethan900typesofpreciousmedicinalplantsaresaidtobefoundinNepal.6Unfortunately, the medicinal values of all theplantsarenotyetexplored.

Escherichia coliareGram-negative,facultativelyanaerobicbacilli,commonlyfoundinthelowerintestine of warm-blooded organisms.7 Theyconstitute0.1%ofgutflora,andthepathogenicstrains of the bacterium can cause disease bytransmitting through the faeco-oral route.8 VirulentstrainsofE. coli cancauseUrinarytractinfection(uropathogenicE. coli),gastroenteritis,and neonatal meningitis. In rare instances theyare also responsible for septicemia, mastitis,haemolytic-uremicsyndromeandGram-negativepneumonia.9

According to WHO and Centre for DiseaseControl(CDC)E. coli isoneofthemostcommonly

BhargavaDipaketal.

isolated organisms from different samples indifferent diseases.10 The problem is furthercompounded by the emergence of resistance toantimicrobial agents that are commonly usedagainstE. coli,making the treatmentexpensiveandprolonged.

Therefore,inthepresentstudy,anefforthasbeenmadetoanalyse thephytochemicalconstituentsandantiE. coliactivityoftheethanolicextractsof the ten medicinal plants found in the highaltitudesofNepal.

MATErIALS AND METHoDSThe fresh plants were collected from differentsites of Parsa (Birgunj), Bara (Simra) andMakwanpur (Hetauda) districts (altitude about1500ft from above the sea level), during May2012toAugust2012,andprocessedatClinicalMicrobiology laboratory of National MedicalCollegeandTeachingHospital,Birgunj,Nepal.These plants were identified on the basis ofavailable literatures11, 12 and in the DepartmentofBotany,VidyasagarUniversity,India.Leaveswere washed with distilled water, shade dried,powderedandstoredinanairtightcontaineruntilfurtheruse.

Preparation of extractsSolventextractswerepreparedbytransferring1g of the powder to sterile wide-mouthedscrew-capped bottles containing the solvent(50%,v/v).Itwasallowedtosoakfor24hoursat room temperature then heated for an hourat 100°C. The mixture was then centrifugedat 2000 rpm for 10 minutes at 4°C.13 Thesupernatants were filtered through a sterilefunnelcontainingsterilewhatmannfilterpaperno.1 and then filtrate was sterilized usingsyringefiltercontaining0.2μcelluloseacetatemembrane(Sartorius,India).13Thepercentageyieldof the extractwasdeterminedusing theexpression:

Percentage yield = Weight of extract X 100/ Weight of ground plant Material

Phytochemical Evaluation of the ExtractThephytochemicalevaluationoftheplantextractsforthemajorconstituentswasundertakenasperthe


standardmethods.14, 15 Themethods for detectingthebioactiveconstituentsweredescribedbelow.

Detection of Alkaloids 5ml plant extracts were dissolved individuallyin dilute (2%) Hydrochloric acid and filtered.Thefiltrateswereusedtotestforthepresenceofalkaloids.

a) Mayer’s Test: Filtrates were treated withMayer’s reagent (Potassium Mercuric iodide).FormationofayellowcreamprecipitateindicatesthepresenceofAlkaloids.

b)Dragendroff’s test:FiltratesweretreatedwithDragendroff’s reagent (solution of potassiumbismuth iodide). Formation of red precipitateindicatesthepresenceofalkaloids.

Detection of Carbohydrates The plant extracts (5ml) were dissolvedindividually in5mldistilledwaterandfiltered.Thefiltrateswereusedtotestforthepresenceofcarbohydrates.

a)Molisch’s Test:2mlofthefiltratesweretreatedwith2dropsofalcoholicα-naphtholsolutioninatesttubeand2mlofConc.Sulphuricacidwasaddedcarefullyalongthesidesof the test tube.FormationofvioletringatthejunctionindicatesthepresenceofCarbohydrates.

b)Benedict’s test:ThefiltratesweretreatedwithBenedict’s reagent and heated on water bath.Formationoforangeredprecipitateindicatesthepresenceofreducingsugars.

Detection of glycosidesTheplantextractswerehydrolysedwithdilutedHCl(2%)andthensubjectedtotestforglycosides.

Modified Borntrager’s Test: Extracts weretreated with Ferric Chloride solution andimmersed inboilingwater forabout5minutes.The mixture was cooled and shaken with anequalvolumeofbenzene.Thebenzenelayerwasseparated and treated with ammonia solution.Formationofrose-pinkcolourintheammoniacallayer indicates the presence of anthranolglycosides.

AntiEscherichiaColiActivity

Detection of Phenolsferric Chloride Test:5mloftheextractsweretreatedwithfewdropsofferricchloridesolution.Formation of bluish black color indicates thepresenceofphenols.

Detection of oils and fatsStain Test:2mloftheplantextractswerepressedbetweentwofilterpapers.Anoilystainonfilterpaperindicatesthepresenceoffixedoil.

Detection of Saponinsfroth Test:5mloftheextractsweredilutedwith20ml. distilled water and this was shaken in agraduated cylinder for 15 minutes. Formationof1cmlayeroffoamindicatesthepresenceofsaponins.

foam test:5mloftheextractswereshakenwith10mlofwater.Iffoamproducedpersistsfortenminutesitindicatesthepresenceofsaponins.

Detection of Tanningelatin Test: 1% gelatin solution containing2% sodium chloride was added to the extract.Formation of white precipitate indicates thepresenceoftannins.

Detection of flavonoidsa)Alkaline reagent Test: 5ml of the extractsweretreatedwithfewdropsofsodiumhydroxidesolution. Formation of intense yellow color,which becomes colorless on addition of diluteacid,indicatesthepresenceofflavonoids.

b)Lead acetate Test:Extractsweretreatedwithfewdropsofleadacetatesolution.Observation of yellow color indicated thepresenceofflavonoids.

Isolation of E. coliTheclinicalisolatesofE. coli wereisolatedfromtheurine,pusandfecalsamplesinthedepartmentofmicrobiologyofNationalMedicalCollege&TeachingHospitalofBirgunj,Nepal.Forisolationof E. coli;clinicalsamplessuchasurine,pusandfeces were inoculated onto MacConkey’s agarmedia(HiMedia,India).Theinoculatedcultureplates were incubated at 37°C in an incubator


for24hours.Theseconsecutiveclinical isolatesof E. coliwere identifiedon thebasisofcolonymorphology, gram staining, catalase, indoleproductionandmethylredpositivetests.16

Preparation of the InoculumFive colonies grown onMacConkey’s agar (HiMedia,India)(E. coli)mediawereobtained. Thesecolonieswereinoculatedin2.5mlMuller-Hintonbrothinatesttubebyrotatingtheinoculatingloopatleasttentimeswiththetiptouchingthebottomofthetesttube.Thetesttubewasincubatedat37°Cfor3hours.Theturbidityisalsomatchedwith0.5McFarlandStandard.

Statistical analysesToensureconsistencyofallfindings,antimicrobialactivityofplantextractswasperformedintriplicateunder aseptic condition. Data were statisticallyanalyzed and expressed as mean ± Standarddeviation(SD)bySPSS10,Chicago,Inc.

rESuLTSTen medicinal plants (as shown in Table 1)used in this study are widely used in folkloricmedicineofNepal in treatingdifferentdiseases.The percentage yields of extracts and the

phytochemicalconstituentsoftheplantsareshowninTable2and3respectively.ThehighestyieldofethanolicextractwasfoundincaseofC. odorata (L.) (32.4%± 0.65) and the lowest inCurcuma longa (L.)(9.3%±0.65).Phytochemicalanalysisconducted for the ten plant extracts reveals thepresence of flavonoids, alkaloids and saponinsas the major constituents (Table 3). The othersecondary metabolites like tannins, glycosidesandcarbohydrateswerepresent in trace amountin some of the plant extracts (Table 3). In thisstudy,ethanolicextractsofthesetenplantswerestudied. The clinically isolated gram negativebacteriaE. coli was found maximally inhibitedbytheethanolicextractofSyzugium cuminii(L)Chromolaena odorata (L.)Ocimum sanctum(L.), and Justicia adhatoda (L.).

The zones of inhibition of ethanolic extract ofdifferent medicinal plants, which are able toproducezoneofinhibitionareshowninTable4.Thoughalltheplantsofthehighaltitudeshowedsomedegreeof antimicrobial activityagainstE. coli,itwasseenfromtheTable4thatC. odorata (L.), S. cumini (L.), O. sanctum (L.) and J. adhatoda (L.) possess significant antimicrobialactivityagainst E. coli.

Table 1: List of medicinal plants used in antimicrobial assay

Botanical Name Vernacular Name family Month of collection

Curcuma longa(L.) Haledo Zingiberaceae May,2012Emblica officinalis (L.) Amlaki Euphorbiaceae June,2012Ocimum sanctum (L.) Tulsi lamiaceae June,2012Sapindus mukorossi (Gaertn.) Reetha Sapindaceae July,2012Chromolaena odorata(L.) Banmara asteraceae July,2012Justicia adhatoda(L.) asuro Apocynaceae July,2012Origanum majorana(L.) Ramtulsi lamiaceae August,2012Syzygium cumini(L.) Jamun Myrtaceae August,2012Rauvolfia serpentina(L.) Sarpagandha Apocynaceae August,2012Allium sativum(L.) Lasun Liliaceae August,2012

BhargavaDipaketal.


Table 2: Percentage yield of extracts

Plant name Weight of raw material (gm)

weight of the extract (gm)

% yield *Extractive value

Curcuma longa(L.) 90 8.4 9.3±0.65Emblica officinalis(L.) 78 18.3 23.4± 0.54Ocimum sanctum(L.) 77.4 17.2 22.2±0.79Sapindus mukorossi (Gaertn.) 105 22.4 21.3±0.65Origanum majorana(L.) 64 19.4 30.3 ±0.61Syzygium cumini(L.) 115 27.8 24.2±0.78Chromolaena odorata(L.) 120 38.9 32.4±0.65Justicia adhatoda(L.) 100 21.1 21.1±0.48Rauvolfia serpentina(L.) 96 15.6 16.2±0.61Allium sativum(L.) 80 16.4 20.5± 0.57

*ValuesareintermsofMean±SEMofresultsdoneintriplicate

DISCuSSIoNThe present study was conducted to obtainpreliminary information on the antimicrobialactivity of ten traditional medicinal plants ofNepal. Plant extracts are potential sources ofnovelantimicrobialcompoundsespeciallyagainstbacterialpathogens.Through this in vitro study,wecouldseethattenplantextractsinhibitedthegrowth of clinical isolates of E. coli but theireffectivenessvaried.

Based on the mean zone of inhibition, the testorganismE. coliwasfoundtobemostsusceptibleto Syzygium cumini(L.)(5.7±0.3cm)extractandleastsusceptibletoEmblica officinalis (L.)(1.1± 0.4cm)(Table4).SimilarlyastudyconductedbyChhetriet al.,in2008foundthat E. coli was most susceptible to Rhododendron setosum (L.) andleasttowardsCurcuma longa(L.).17 Thefindingsindicatetheexistenceofantimicrobialcompoundsinthecrudeethanolicextractsoftenplantsunderstudyandshowedagoodcorrelationbetweenthereported medicinal uses of these plants againstdifferentdiseasesinlocalcommunitiesofNepal.The inhibitory effect of these ten plants against

theclinicalisolatesofE. coli mightbeattributedtothepresenceofsomeactiveconstituentsintheplantasinotherherbalplants.

Thephytochemicalanalysisof thestudyrevealsthat the extract of the ten plants (ethanolic)contained bioactive compounds (flavonoids,alkaloids, saponins, tannins, carbohydrates,glycosides & oils) (Table 3). These bioactivecompoundsarebelievedtoberesponsiblefortheobserved antimicrobial effects. Some workershave observed the antimicrobial effects ofplant extracts to the presenceof these bioactivecompounds.18,19 The presence of these bioactivecompounds is an indicator that the ten plantscan be a potential source of precursors in thedevelopmentofsyntheticdrugs.

The result of the present study provides ascientific evidence for the local use of the tenmedicinal plants studied and ushers for theselection of plants with antimicrobial activitiesfor further phytochemical work in the isolationandidentificationoftheactivecompounds.

AntiEscherichiaColiActivity


Table 3: Phytochemical constituents of the ten plant extracts

Components Plant extracts

Alkaloids flavonoids Saponins Tanins Carbohydrates glycosides oils& fats

Chromolaena odorata(L.) ++ +++ - - - - -Ocimum sanctum(L.) ++ - - +++ - + -Sapindus mukorossi (Gaertn.) + - +++ - + - -

Syzygium cuminii(L.) ++ - - +++ + - -Allium sativum(L.) - - - - - + +++Curcuma longa(L.) - +++ - - - ++ -Rauvolfia serpentina (L.) +++ ++ - - - - +Justicia adhatoda(L.) ++ + - - + + -Emblica officinalis(L.) - - - - - + +++Origanum majorana(L.) + - - - - + +++

‘+’ = Positive; ‘++’ = Moderate Positive; ‘+++’ = Strongly Positive; ‘-’= Not Detected

CoNCLuSIoNTheplantextractsunderstudydemonstratedgreatpotentialagainsttheclinicalisolatesofE. coliandsupportedtheirfolkloricuseagainstthediseasescausedbythisorganism.Furthermore,ithasopenedthe avenues for the discovery of new clinically effective antimicrobial compounds.Therefore, thefutureinvestigationsshouldbedirectedtowardsthedeterminationofchemicalstructureoftheactiveconstituentsandtoxicologicalevaluationwiththeaimofformulatingnovelchemotherapeuticagentstocopeupwithincreasingprevalenceofdrugresistantdiseasescausedbyvirulentstrainsofE. coli.

Table 4: Zone of inhibition of the plant extracts against Escherichia coli

Plants Extract solution (ZoI) *Curcuma longa(L.) 2.1± 0.3 cmEmblica officinalis (L.) 1.1 ± 0.4 cmOcimum sanctum (L.) 4.7 ±0.6cmSapindus mukorossi (Gaertn.) 1.6± 0.3 cmOriganum majorana (L.) 2.3± 0.5 cmSyzygium cumini (L.) 5.7 ± 0.3 cmChromolaena odorata (L.) 5.2± 0.4 cmJusticia adhatoda (L.) 3.2± 0.3 cmRauvolfia serpentina (L.) 2.1± 0.3 cmAllium sativum (L.) 1.1 ± 0.4 cm

*Values are in terms of Mean ± SEM of results done in triplicate.

BhargavaDipaketal.


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2. Ramawat SKC, Sharma MC. Biotechnology ofmedicinalplants,In:Ramawat,KG,editors.VitalizerandTherapeutics.USA:SciencePublisherInc;2004.p.1-18.

3. MitscherLA,ParkYH,ClarkD.Antimicrobialagentsfrom higher plants, antimicrobial isoflavonoids andrelatedsubstancesfromGlycyrhizaglabra.JNatProd.1980;43:259-69.

4. Duraipandiyan V, Ayyanar M, Ignacimuthu S.Antimicrobial activity of some ethnomedicalplants used by Paliyar Tribe from Tamil Nadu,India. BMC Complement Altern Med. 2006;6:35.doi: 10.1186/1472-6882-6-35

5. WHO:WorldHealthOrganization.TheWorldHealthReport.Bridgingthegap.Geneva:1995.1:p.118,

6. Manandhar NP. Plants and People of Nepal. USA:TimberPress;2000.p.50.

7. Singleton P. Bacteria in Biology. In: BiotechnologyandMedicine.(5thed.).USA:Wiley;1999.p.444-54.

8. Eckburg PB, Bik EM, Bernstein CN, Purdom E,Dethlefsenl,SargentM,GillSR,NelsonKE,RelmanDA.Diversityofthehumanintestinalmicrobialflora.Science.2005;308(5728):1635-8.

9. Todar K. Pathogenic E. Coli. Online textbook ofBacteriology. University of Wisconsin-Madison.DepartmentofBacteriology.Retrieved;2007.11.30.

10. CDC National Center for Emerging and Zoonotic InfectiousDiseases.Escherichiacoli.Retrieved;2012.10.02.

11. HMG/N.Medicinal Plants ofNepal. In:Ministry ofForestandSoilConservation,editors.DepartmentofPlantResources.Kathmandu:1993.

12. Rajbhandari KR. Ethnobotany of Nepal. In:Ethnobotanical Society of Nepal, editors.Kathmandu:2000.p.98.

13. BansoA,Adeyemo S. Phytochemical screening andantimicrobial assessment of Abutilon mauritianum,Bacopa monnifera and Datura stramonium.BIOKEMISTRI.2006;18(1):39-44.

14. Brain KR, Turner TD. The practical evaluation ofphytopharmaceuticals. In: Wright Sciencetechnica,editors.Bristol:1975.p.81–2.

15. Evans WC. Pharmacognosy. In: Trease, Evans,editors. London: W.B. Sounders companylimited;1996.p.545-6.

16. Collee JG, Miles RS, Watt B. Practical MedicalMicrobiology. In: Mackie, McCartney, editors.London:ChurchillLivingstone;1996.p.140-1.

17. Chhetri HP, Yogol NS, Sherchan J, AnupaKC, Mansoor S, Thapa P. Phytochemical AndAntimicrobialEvaluationsOfSomeMedicinalPlantsOfNepal.KathmanduUniversityJournalofScience,EngineeringAndTechnology.2008;1(5):49-54.

18. SofoworaA.MedicinalplantsandTraditionalMedicineinAfrica.Nigeria:SpectrumBooksLimited;1993.

19. SuksamrarnA,ChotipongA, SuavansriT,BoongirdS, Timsuksai P, Vimuttipong S, Chuaynugul A.Antimycobacterial activity and cytotoxicity ofFlavonoidsfromtheflowersofChromolaenaodorata.ArchPharmRes.2004;27(5):507-11.

Cite this article as: Bhargava D, Abhilasha S, Mondal KC, Pandit BR, Shidiki A, Chaudhary NK, Gupta RS, Jha AK. Anti Escherichia coli activity of some medicinal plants of high altitude of Nepal. MED PHOENIX 2016;1(1):3-9


Madhikarmi Nirjala Laxmi1*, Singh Prem Prakash2, Khatun Tarannum 3

1 DepartmentofBiochemistry,M.B.KediaDentalCollege,Chapakaiya,Birgunj,Nepal2DepartmentofBiochemistry,ManipalCollegeofMedicalSciences,Pokhara,Nepal3Departmentofobstetrics&Gynaecology,NationalMedicalcollegeandTeachinghospital,birgunj,Nepal

Lipid Peroxidation and Antioxidant Status in Male and Female Patients with Type 1 and Type 2 Diabetes Mellitus

rESEArCH

ABSTrACTBackground: Free radicals are reactiveoxygen species which cause lipid peroxidationprecipitatingmanymetabolicdiseasesincludingDiabetesMellitus.However, these free radicalsarequenchedbysubstancesknownasantioxidantslike vitamin C, vitamin E and several othercompounds.Lipid peroxidation and antioxidantstatuswereinvestigatedinpatientswithType1andType2Diabetesmellitus-Pokhara,Nepal.

Methods:The extent of lipid peroxidationwasassessedbythiobarbituricacidreactivesubstancesand the antioxidant parameter estimationsweretotalantioxidantactivity,VitaminCandVitaminE assessed in Type 1 and 2 diabetes mellituspatientsalongwithmatchedhealthycounterparts.

results: The lipid peroxidation was increasedinmaleType1and2diabeticpatientswhereasfemale group showed decreased level ascompared to itshealthycounterparts.Similarly,the total antioxidant activity was found tobe decreased in the diabetic group. The lipidperoxidation parameter and antioxidant statuswerestatisticallysignificantatp<0.05.

Conclusion: Oxidative stress and antioxidantstatusvariedinmaleandfemalepatientssufferingfrom diabetes either Type 1 or Type 2. Apartfromgenderbasisofevaluatingoxidativestress,variables based on diet, habitat, socioeconomicstatus,education,etc.canalsobeconsidered.

Keywords: Antioxidant, Diabetes Mellitus,Lipidperoxidation,Oxidativestress

Corresponding Author: Dr.NirjalaLaxmiMadhikarmi,DepartmentofBiochemistry,M.B.KediaDentalCollege,Chapakaiya,Birgunj,Nepal;E-mail: [email protected]

INTroDuCTIoNThegrowingglobalprevalenceandhealthburdenassociatedwiththediseasehasrecentlyledtotheWorldHealthOrganization toclassifyhighbloodglucoselevelasthethirdleadingcauseofprematuremortality globally. The annual global healthcareburden associated with diabetes is currentlyestimatedtobeatleast376billionUSD,accountingfor 7, 10, 11 and 14 percents of total healthcareexpenditureinSub-SaharanAfrica,Europe,SouthEast Asia and North America, respectively.1, 2 Furthermore, the projected growth in the globalprevalenceandeconomicburdenofType2diabeteswillfurtherdisproportionatelyaffectlowandmiddleincomecountriesinfuture.InIndia,theprevalenceofdiabetesisprojectedtoincreaseby60percentto80millioncasesby2030anditseconomicburdenisprojectedtoincreasebyalmost50%toatleast4.8billionUSDoverthesameperiod.

Lipid peroxidation by reactive oxygen species(ROS)appearstoprecipitatemanydiseaseswhichinclude Diabetes Mellitus (DM). Substancesknownasantioxidantsreducethepoweroffreeradicals.Thereareseveralcompoundsfunctioningasantioxidants includingvitaminC,vitaminE,beta-carotene and vitamin A.3, 4 The quantumof lipid peroxidation in plasma is generallydetermined by measurement of thiobarbituricacid reactive substances (TBARS). Vitamin CservesasantioxidantsintheaqueousphasewhilevitaminEfunctionsinthelipidphase.


Oxidativestressisdefinedasdisturbanceintheprooxidant-antioxidant balance in favor of theprooxidant,leadingtopotentialdamageproducingoxidativestress.Freeradicalisanyatomwithatleastoneunpairedelectronintheoutermostshell,andiscapableofindependentexistence.Raisedoxidative stress has been implicated not onlyforchronicdiseaseslikecardiovasculardisease,cancer, cirrhosis, atherosclerosis, Alzheimer’sdiseaseandParkinson’sdisease;ithasalsobeenfoundalteredindiabetesmellitus.5-8

Oxidative stress is defined as an imbalancebetween the production of reactive oxygenspeciesorfreeradicalsandantioxidantdefense,which may induce tissue injury. In diabetes,oxidative stress is caused by both an increasedformationofplasmafreeradicalsandareductionin antioxidant defense. An unbalanced excessoffreeradicalsdueto lackofantioxidantsmayincreasetheriskofcomplicationsofdiabetes.4,5

Therefore,theaimofpresentstudywastostudytheroleofoxidativestressandantioxidantsinthepathogenesisoftype1andtype2diabetesmellitus.

MATErIALS AND METHoDSInthepresentCase-Controlinvestigationpatientssufferingfromtype1andtype2DMandattendingManipalTeachingHospital,Pokhara,Nepalwasselected.Thestudywasconducted fromMarch2004toFebruary2016.Thenumberofpatientsintype1diabetesmellitus(DM)waslimitedto20(10malesand10females)and40type2DM(20maleand20 female)alongwith60healthyindividualsascontrol.

Both patients and controls were selected alongwithmatchedcontrols (individualswhohadnohistory of illness or disease). Type 1 Diabeticpatients were individuals treated with insulininjections, followed by elevated ketone bodieson diagnosis, less body mass index whereas,type 2 Diabetic patients were associated withhigher body mass, high cholesterol and bloodpressure at diagnosis. All the subjects afterobtaining their informed written consent wereexamined clinically and information pertainingto age, habits and health statuswere collected.Using standardprotocolsglucoseestimationbyglucose oxidasemethod-Randox diagnostic kit,

total antioxidant activity (TAA), thiobarbituricacid reactive substances (TBARS), Vitamin C,VitaminElevelswereestimatedinplasmausingUV-visibleSpectrophotometer.

Collection of blood sampleAllglasswareandplasticwaresweresoaked in20%nitricacidfor24hrstomakethemmineralfree.About 6ml of venous bloodwas collectedby vein puncture by standard blood collectiontechnique in acid washed vials containingethylene diamine tetraacetic acid (EDTA).Plasma was separated by centrifugation for 10minutesat3000 revolutionperminuteandwasanalyzedwithminimumpossibledelay.Plasma TBARS (thiobarbituric acid reactivesubstances)levelwascarriedoutbyBuegeandAustmethod9,TAA(totalantioxidantactivity)levelwasmeasuredbyBenzieandStrainmethod10,vitaminC levelwas estimatedbyNatelsonMethod11 andvitaminEwasestimatedby themethodofBakerandFrank.12

Statistical analyses The resultswere expresses inmean ± standarddeviation, the level of significance wasdetermined by Student’s Independent t-test atp<0.05.StatisticalanalysiswascarriedoutusingSPSSversion13.0.

rESuLTSAsperinclusioncriterion,weobservedsignificantdifferences in lipid peroxidation and antioxidantstatusinmaleandfemalepatientsasgiveninFigure1,whichrevealstheplasmaglucoseconcentrationoffasting(FBS)andpost-prandial(PPBS)ofType1 and Type 2 diabetic patients based on gender.MalepatientsrevealedhigherFBSandPPBSlevelas compared to healthy individuals.On the otherhand,femalediabeticpatientsexhibiteddecreasedFBS and increased PPBS level as compared tohealthycontrols.

The various biochemical parameters in plasmawere estimated in healthy controls,Type 1 andType2diabeticpatients.The lipidperoxidationwas assessed by thiobarbituric acid reactivesubstances(TBARS)andantioxidantassaysweretotal antioxidant activity (TAA),vitaminCandE.Thebasalmetabolicrate(BMI)andsurfacearea (SA) was decreased in Type 1 DM, but

MadhikarmiNirjalaLaxmietal.


LipidPeroxidationandAntioxidant

Type2DMpatientsshowedincreasedBMIandSAascomparedtohealthymales.LipidperoxidationparameterTBARSwasincreasedbothinType1andType2DMwhencomparedwithhealthymalecontrols.Thetotalantioxidantactivity(TAA)waslowerinbothtype1andtype2DM.TheantioxidantvitaminsCandEwerealsofoundtobeinnormalrangeinfemalediabeticpatients.

Table 1: Plasma levels of TBARS, TAA, Vitamin C and Vitamin E in male control, Type 1 and Type 2 DM patients

group (male)

BMI (kg/m2) SA (m2)

TBArS (nmol/ml)

TAA (µmol/L) Vit C (mg/dl)

Vit E (mg/dl)

Control 22.35±2.32 1.71±0.24 1.47±1.38 787.79±16.62 0.76±0.29 0.77±0.49Type 1 21.16±1.59 1.69±0.27 2.23±1.38a 548.80±10.37c 0.91±0.33e 0.82±0.72g

Type 2 25.67±8.02 1.92±0.02 2.01±1.81b 647.41±18.92d 0.82±0.32f 0.74±0.41h

Note: SA= surface area, p<0.05 (level of significance) i.e. 95% confidence interval; a=0.04, b=0.002, c=0.015, d=0.039, f=0.023, g=0.034, h=0.02

PlasmaTBARSwas increased inboth type1and2DMpatientscompared tocontrols.Between type1andType2DM,thechangeinthevalueofTBARSwasnotstatisticallysignificant.TheplasmaTAAwassignificantlydecreasedinbothtype1andtype2DM.TherewasanincreaseofTAAintype2overtype1DMwhichwasstatisticallysignificant.VitaminCantioxidantisincreasedinbothtype1andtype2DM.VitaminEisnotsignificantlyalteredinbothtype1andtype2DM.TheincreaseofvitaminCintype2waslessthanthatintype1DM.TheplasmalevelsofTBARSinfemalegroupissignificantlydecreasedintype1withoutanysignificantchangeintype2DM.PlasmaTAAisdecreasedintype1butnotsignificantlyalteredintype2DM.VitaminCinplasmaisdecreasedintype1withoutanychangeintype2DM.SimilarlyvitaminEdidnotalterintype1butdecreasedintype2DM.OnIndependentt-testanalysis,maleandfemalepatients(Table:1and2)showedstatisticalsignificancewithrespecttotheirhealthyindividualsatp<0.05(levelofsignificance).

Table 2: Plasma levels of TBARS, TAA, Vitamin C and Vitamin E in female control, Type 1 and Type 2 DM patients

group (female)

BMI (kg/m2)

SA (m2)

TBArS (nmol/ml)

TAA (µmol/L)

Vit C (mg/dl)

Vit E (mg/dl)

Control 24.45±2.29 1.74±0.11 2.12±0.21 615.39±18.07 0.99±0.31 0.81±0.34Type 1 22.38±3.36 1.72±0.36b 0.95±0.54d 585.50±16.24f 0.83±0.38h 0.82±0.06Type 2 28.36±3.64a 1.82±0.09c 1.62±0.15e 647.10±28.56g 0.98±0.40 0.70±0.14i

Note: SA=Surface area, p<0.05 (level of significance) i.e. 95% confidence interval, a<0.001, b=0.005, c=0.012, d=0.019, f=0.001, g=0.034, h=0.02, i=0.033

GlucoseConcentration(mg/dl)

250

200

150

100

50

FBS FBSPPBS PPBS0 0

20

40

60

80

100

120

140

160

180

MALE fEMALE

fig 1: glucose concentration of Type 1 and Type 2 of Diabetic patients based on genderNote: FBS- Fasting blood sugar, PPBS- Post-prandial blood sugar level



DISCuSSIoNThe present study covers patients with diabetesmellitusboth in type1and type2whoattendedManipal Teaching Hospital, Pokhara, Nepalduring the studyperiod.Thenumberof patientsin type 1 is relatively less compared to type 2DM,duetothefactthatincidenceoftype2DMisgloballymorecommonthantype2andNepalisnoexception.Theresultsofthisstudyhaveindicatedthat lipid peroxidation is increased in both thetypesofthediseasesinmalepatients,butnotinthefemalepatients.Theantioxidantstatusisgenerallyrelated to theprooxidant status.Thismeans thatanincreaseofplasmaTBARSshouldnecessarilybe associated with an increase in antioxidantlevels. But, the present study has indicated thattheantioxidantsaremarginallyincreasedinmalepatients but not significantly altered in femalegroup.Surprisingly,thefemalepatientsoftype2DMshoweddecreasedvitaminElevelofplasma.Thereasonforthisisnotknown.

The prooxidant and antioxidant status during anystressneedtobebalancedinpatientswithDMbutsuch a balance is not encountered in the currentinvestigation.Aquestionariseswhetherthedietarystatusofbothvitaminshavetobetakencareoftobring about a counter effect of lipid peroxidationwhich does exist in these patients. Experimentalevidences suggest the involvementof free radicalsin the onset of diabetes and more importantly inthe development of diabetic complications.7-9, 13-16 Scavengersoffreeradicalsareeffectiveinpreventingexperimentaldiabetesinanimalmodelsandintype1 (IDDM) and type 2 (NIDDM) patients as wellas reducing severity of diabetic complications.Persistent hyperglycemia in the diabetic patientsleadstogenerationofoxidativestressduetoauto-oxidation of glucose, non-enzymatic glycosylationand polyol pathway. 1-3Auto-oxidation of glucoseinvolvesspontaneousreductionofmolecularoxygentosuperoxideandhydroxylradicals,whicharehighlyreactiveandinteractwithallthebiomolecules.15,16

Lipidperoxidationwasincreasedintheplasmaofdiabeticpatientsby50%(TBARS).Theactivityof superoxide dismutasewas enhanced by 9.4%intheerythrocytesofpatients.Ourresultsshowedhigher oxidative stress and generation of freeradicals in the RBCs of diabetic patients. 17-20 Parthiban (1995), Oberley (1988) has reported

oxidative stress is associated with developmentof diabetic complications. In diabetes mellitus,GSH formation is hampered due to diminishedefficiencyoftheHMPshuntpathwayofglucoseoxidation,thesecondenzymeintheredbloodcellrisestothepassionanddisposesoffthehydrogenperoxide formed in the cell so that internalenvironmentintheerythrocyteistakencareoftothemaximumpossibleextent.Butinspiteofthismechanism, lipid peroxidation and free radicalgenerationprevailsintheRBCsofdiabetics.21-23

Thedevelopment andexistenceof anorganism inthepresenceofO2isassociatedwiththegenerationof reactive oxygen species (ROS), even underphysiological conditions. Twenty four ROS areresponsible for theoxidativedamageof biologicalmacromolecules such as DNA, carbohydratesandproteins.Someof themost relevantROSare:peroxylradicals(ROO.),nitricoxideradical(NO.),superoxideanionradical(O2

.-),singletoxygen(1o2),peroxynitrite (ONOO--) and hydrogen peroxide(H2o2). ROS are either radicals (molecules thatcontainatleastoneunpairedelectron)orreactivenon-radicalcompoundcapableofoxidizingbiomolecules.ROSarealsoproducedintheorganismasapartoftheprimaryimmunedefense.22-26Phagocyticcellssuch as neutrophils, monocytes, or macrophagesdefend against foreign organism by synthesizinglargeamountofsuperoxideanionradicalandnitricoxide radical as apart of theirkillingmechanism.To counteract the prooxidant load a diversityof antioxidant defense systems are operative inbiological systems including enzymatic and non-enzymatic antioxidants. Major enzymes directlyinvolvedindetoxificationaresuperoxidedismutaseaswellascatalaseandglutathioneperoxidase.Thehumandietcontainsanarrayofdifferentcompoundsthat possess antioxidant activities or have beensuggestedtoscavengeROSbasedontheirstructuralproperties. The most prominent representativesof dietary antioxidants are ascorbate, tocopherols,carotenoidsandflavonoids.26,27

ROS are known to be involved in pathogenicprocesses of numerous diseases, such asCVD, cancer, cataract, rheumatoid arthritis,neurodegenerativedisease.Oxidativedamagetoimportantbiomoleculesisadeleteriouspathway,butalsoinfluencesofROSongeneregulationandimmunesystemmightimpairbodilyfunctions.4-8

MadhikarmiNirjalaLaxmietal.


CoNCLuSIoNAccounting all the information and data gatheredin this study indicate that oxidative stress is notthe etiologic factor in diabetes mellitus of localpopulation. By implication, antioxidant defensesystemdidnotappeartobeinvolved.Interestingly,the local population seems to be well endowedwithantioxidantdefensesystem.Whetherthegoodantioxidant defense system or well antioxidant-balanced diet, habitat, socioeconomic status,educationareresponsibletokeeptheoxidativestress,incheckformsagoodplatformforfuturestudy.

ACKNowLEDgEMENTSWeareindebtedtoManipalCollegeofMedicalSciences, Pokhara, Nepal, Department ofBiochemistry,Samplecollectioncenter,patientsandstaffsofManipalTeachingHospital,Pokharaforallowingustoconductthisresearchwork.

rEfErENCES1. World Health Organization. Global health risks:

mortalityandburdenofdiseaseattributabletoselectedmajorriskfactors.Geneva,Switzerland:WorldHealthOrganization:2009.

2. ZhangP,ZhangX,BrownJ,VisisenD,SicreeR,ShawJ,etal.Globalhealthcareexpenditureondiabetesfor2010and2030.DiabetesResClinPract2010;87:293-301.

3. GuptaMM,ChariS.Lipidperoxidationandantioxidantstatus inpatientswithdiabeticnephropathy. IndianJPhysiolPharmacol2005;49:187.

4. Madhikarmi NL,Murthy KRS, Rajagopal G, SinghPP. Lipid peroxidation and antioxidant status inpatients with type 2 diabetes in relation to obesityin Pokhara – Nepal. J Diabetol [Internet]. 2013[cited 2016 Feb12];1:3.Available from: http://www.journalofdiabetology.org/

5. HalliwellB,CrossCE,GutteridgeJMC.Freeradicals,antioxidantsandhumandisease:wherearewenow?JLabMed1992;119:598.

6. KumarN,ChandhoikN,DhillonBS,KumarP.Roleofoxidativestresswhilecontrollingirondeficiencyanemiaduring pregnancy-Indian scenario. Indian J ClinicalBiochemistry.2009;24(1):5-14.

7. JohansenJS,HarrisAK,RychlyDJ,ErgulA.Oxidativestressandtheuseofantioxidantsindiabetes:linkingbasicsciencetoclinicalpractice.CardiovascDiabetol2005;4:5.

8. MemsoullariR,TaysS,BakanE,CapogluI.Antioxidantstatusandlipidperoxidationintype2diabeticmellitus.CellBiochemistryandFunction2003;21:291.

9. Buege JA, Aust SD. Thiobarbituric acid assay.MethodsinEnzymology.1978;52:306.

10. Benzie IFF, Strain JJ. The ferric reducing ability ofplasma(FRAP)asameasureofantioxidantpower-theFRAPassay.AnnalBiochem1996;239:70.

11. NatelsonS.DeterminationofAscorbicacidusing2,4-Dinitrophenylhydrazine.In:TechniquesofClinical

Chemistry. Charles C Thomas, Spring Field, NewYork,USA.3rded,USA:Illionois;1971:165-6.

12. BakerH,FrankO.DeterminationofVitaminE.In:Clinicalvitaminology.NewYork:Wiley,USA.1968;172-3.

13. PrasadS,SinhaAK.Freeradicalactivityinhypertensivetype2diabetesmellitus.InternationalJDiabetesMellitus2010;2:141-3.

14. Davi G, FalcoA, Patrono C. Lipid peroxidation indiabetesmellitus.AntioxidRedoxSignal2005;7:256.

15. Devasagayam TPA, Tilak JC, Bloor KK, Sane KS,GhaskadbiSS,LeleRD.Freeradicalsandantioxidantsinhumanhealth:currentstatusandfutureprospectus.JAPI2004;52:794.

16. EvansJL,GoldfineID,MadduxBA.Oxidativestressandstressactivatedsignalingpathways:aunifyinghypothesisoftype2diabetesmellitus.EndocrRev2002;2:599.

17. Ghosh R, MehtaA, Ramavataram DVSS, BarjatiyaMK,SinghPP.Prooxidantandantioxidantbalanceinaging.InFreeradicalandantioxidants:sortoutfactsfromfiction, editedbySinghPP,PendseAK,BombBS,BarjatiyaMK.1999;127

18. ChertowB,PloughMS.Advancesindiabetesforthemillennium:vitaminsandoxidativestressindiabetesanditscomplications.MedGenMed2004;6:4.

19. AlbertiKGMM,ZimmetPZ.Definition,diagnosisandclassificationofdiabetesmellitusanditscomplications.DiabetMed1998;15:539.

20. Baynes JW, Thorpe SR. Role of oxidative stress indiabetic complications- a newperspective on an oldparadigm.Diabetes1999;48:1.

21. Parthiban A, Vijayalingam S, ShanmugasundaramKR, Mohan S. Oxidative stress and developmentof diabetic complications- antioxidants and lipidperoxidationinerythrocytesandcellmembrane.CellBiolInt.1995;19(2):987-90.

22. OberelyLW.Freeradicalsanddiabetes.FreeradicalsBiol.ed.,1988;5:113.

23. Kumar PA, Rajagopal G. Lipid peroxidation inerythrocytesofpatientswithType2diabetesmellitus.IndianJClinBiochem.2003;18(1):71-4.

24. WildS,RoglicG,GreenA,SicreeR,KingH.Globalprevalenceofdiabetes:estimatesfortheyear2000andprojectionsfor2030.DiabetesCare2004;27:1047-53.

25. PasaogluH,SancakB,BuskanN.Lipidperoxidationand resistance to oxidation in patients with type 2diabetesmellitus.TohokuJExpMed2004;203:211.

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Cite this article as:MadhikarmiNL,SinghPP,KhatunT.LipidperoxidationandantioxidantstatusinmaleandfemalepatientswithType1andType2DiabetesMellitusinPokhara,Nepal.MEDPHOENIX2016;1(1):10-14


Shidiki Amrullah*1, Bhargava Dipak1, Gupta Ravi Shankar1, Ansari Akhtar Alam2 Pandit Bijay Raj1

1DepartmentofMicrobiology,NationalMedicalCollege,Birgunj,Parsa,Nepal2DepartmentofPharmacology,NationalMedicalCollege,Birgunj,Nepal

Bacteriological and Physicochemical Analysis of Drinking Water in Tokha, Kathmandu, Nepal

rESEArCH

ABSTrACTBackground:Thisstudyreportsthecomparativestudiesandmicrobialriskassessmentofdifferentwatersamplesusedfordrinkingwater.Theresultsobtained were compared with WHO and EPAstandardsfordrinkingandrecreationalwater.

Methods: Physicochemical and bacteriologicalanalysis of water samples were carried out fromsource,taps,wellandstonespouts usedfordrinkingpurpose in Tokha (Saraswati and ChandeswariVillage Development Committee). Total viablecountwascarriedoutbypourplatetechnique.Totalcoliform and fecal coliform were performed bymembranefiltrationtechnique. Theresultsobtainedwere compared with World Health Organization(WHO), National Agency for Food and DrugAdministrationandControl(NAFDAC)andNepalStandard of Drinking Water Quality (NSDWQ)standardsfordrinkingwater.

results: ThepH,totalhardness,chloride,nitrateandarseniccontentofsampleswerefoundwithinpermissibleguidelinevaluehoweverwellsamplewas found to exceedNepal standardvalues forcalcium hardness and ammonia content. Thetotalviablecountsforallthewatersampleswerehighexceedingthelimitforwater(1.0×102cfu/ml).AllthewatersampleswerefoundtocontaincoliformsandfecalorganismsinnumbersgreaterthantherequiredWHO/FAOstandardsforwater. ThefecalcoliformcoloniesonM-endoagarplaterangedbetween143and152andtotalcoliformfrom110to248per100mlwateralsoexceedthestandardlimitforwater. TheIsolatedorganisms

wereidentifiedtobeE.coli,Klebsiella spp. andCitrobacter spp.

Keywords:Analysis, Bacteriological, Drinkingwater,Nepal,Physicochemical

Corresponding Author: Amrullah Shidiki,Department of Microbiology, National MedicalCollege and Teaching Hospital, Birgunj (Parsa),Nepal;E-mail: [email protected]

INTroDuCTIoNWater of good drinking quality is of basicimportance to human physiology.1 The provisionofportablewatertotheruralandurbanpopulationis necessary to prevent health hazards.2,3 Beforewatercanbedescribedaspotable,ithastocomplywithcertainphysical,chemicalandmicrobiologicalstandards, which are designed to ensure that thewaterispalatableandsafefordrinking.4

Unfortunatelyclean,pureandsafewateronlyexists briefly in nature and is immediatelypolluted by prevailing environmental factorsandhumanactivities.Waterfrommostsourcesis thereforeunfitfor immediateconsumptionwithout some sort of treatment.5 Theconsequencesofwaterbornebacteriaandvirusinfections; polio, hepatitis, cholera, typhoid,diarrhoea, stomach cramps have been wellestablished but nitrate contamination is justas deadly. Contamination of drinking waterfrom any source is of primary importancebecauseofthedangerandriskofwaterbornediseases.6,7


AccordingtoWHOestimate,about80%ofthethirdworld diseases are transmitted by pollutedwater.Duetothelackofsafeandprotectivewatersupplyandsanitation,morethan15millionchildrenbelowfiveyearsdieeachyear.8Accordingtopublichealthdepartment of the Government of Nepal (1990),about 443,000 children die every year due togastroenteritisbydrinkingcontaminatedwater.Therecent exampleof the epidemic is in thewesternandmidwesternregionofNepalwheremorethan500deathshaveoccurred.

InNepal,TokhaVillageDevelopmentCommittee(VDC)doesn’thaveproperwatersupplysystem.Drinkingwaterisobtainedfromsurfacesource-river. Such naturalwater supply is likely to bepolluted with domestic and industrial wastes.Thisworkistherefore,inanattempttoexaminethe different sources of drinking water in thisVDC and to compare with standard tablewater for conformity to microbiological andphysicochemical standards for supply watersamples as well as to examine the differentdomesticandindustrialeffluents/wastewaterforconformitytostandardsforeffluentdischarges.

MATErIALS AND METHoDSThe water samples were collected randomlyfromtaps,sourcesandreservoirfromtheareaofVDC Tokha, Kathmandu. Water was collectedin the pre-sterilized biological oxygen demandbottleandcleanplasticbottleformicrobialandphysic-chemical analysis. For tap and sourcewater, bottlewas cleanedwith alcohol and leftwaterfor2minutesandfinallyfilledfromslowflowofwater.Forreservoirwater,thebottlewasplacedinthetankatadepthof15to30cm.Aftercollection,sampleswereprocessedatlaboratoryofNationalCollegeKathmandu,NepalfollowingstandardmethodswithAPHAAmerican publichealthassociation,1995.

For microbial analysis, water samples wereprocessedforserialdilution,standardplatecountand standard total coliform count bymembranefilterprocedure.ColoniesfromM-Endoagar,XLDagar and TCBS agar after 48 hours incubationweresub-culturedontoNAagarforpureculture.Isolated bacteria were identified on the basisof their colonial characteristic, morphological

characteristics and biochemical propertiesaccording to Bergey’sManual of DeterminativeBacteriology,1994.Physicochemicalanalysisliketemperature,pH,totalhardness,calcium,chloride,nitrate, arsenic and ammonia were identifiedaccordingtoWHOguideline,1998. rESuLTSOut of twenty three samples from public taps,wells,stone-spoutsandsource,pHvaluesofallwater sampleswere found similar in levelwithNepal Standard values. From well, the valuesofcalciumhardnessandammoniacontentweremorethanNepalstandard.Totalhardnesscontentwas found samewithin the standard guide-linevalues. The nitrate and arsenic content of allsamples were same within permissible level.The bacteriological analysis for total coliformand faecal coliformrevealed most frequentlyE.coli and Citrobacter.WaterfromtapsandInar(T1,T5,T6,T11,T16,T18,T19,and I1& I2)containedcoliforms(>300cfu/100ml).

DISCuSSIoNThetemperatureofthewaterwasfoundintherangeof20°Cto22°C.Thistemperaturerangeof 280c–300c of waterhasbeen influencedby the intensity of the sunlight as temperaturerise from 280c -300c on relatively hot days.9 The temperature range of 260c- 300c is due totheinsulatingeffectofincreasednutrientloadresultingfromindustrialdischarge.10Thesamples have neutral pH (7-7∙89). The pH ofmost natural waters ranges from 6.5-8.5 whiledeviationfromtheneutralasaresultoftheCO2/bicarbonate/carbonateequilibrium.11

0

5

10

15

20

Num

bers

Tap Water

Well Water

Source Water

Stone Spout

figure 1: Sample Size

ShidikiAmrullahetal.


Table 1: Physicochemical parameters of waterSample Tem

°CpH

7.0-8.5Total

Hardness500mg/l

Calcium hardness200mg/l

MagnesiumHardness

mg/l

Nitrate 50mg/l

Arsenic 0.05mg/l

Chloride250mg/l

Ammonia1.5mg/l

t1 20 7.5 23 7 16 2 0 12.78 0T2 20 7.25 14 5 9 2 0 9.23 0.5t3 21 7.47 9 7 2 2 0 8.52 0t4 21 7.6 11 26 15 2 0 9.94 0t5 20 7.19 24 32 8 2 0 7 0T6 21 7.2 24 10 14 2 0 7.1 0t7 21 7.67 124 64 60 2 0 46.86 0T8 21 7.64 140 0 0 0 0 0 0W1 21 7.89 324.6 242 82.6 8 0 68.16 1W2 20 7.56 64 378 226 8 0 231.46 >3T9 20 7.45 11 8 3 2 0 9.23 0t10 20 7.05 10 15 5 2 0 7.81 0t11 20 7.05 11 8 3 2 0 10.65 0T12 21 7.52 27 96 69 2 0 7.1 0t13 21 7.27 23 31 8 2 0 7.1 0t14 21 7.56 21 34 13 2 0 6.39 0t15 20 7.57 21 35 14 2 0 7.81 0T16 21 7.55 24 0 0 0 0 0 0t17 20 7.57 24 13 11 2 0 8.52 0T18 20 7.17 26 18 8 ≤2 0 9.94 0T19 20 7.51 24 16 8 ≤2 0 7.1 0S 20 7.56 18 26 8 - 0 - 0SS 22 7.64 - 55 - 2 0 6.39 0.2

Table 2: Microbiological parametersSN Sample Total bacteria

count(Cfu/ml)Total Coliform at 370c ( per

100ml)fecal coliforms at 440c

(per100ml)1 t1 141×104 tMtc 1402 T2 67×104 113 1043 t3 241×104 228 1994 t4 100×104 128 1125 t5 8×104 tMtc 1126 T6 10×104 tMtc 1567 t7 101×104 144 1128 T8 150×104 144 1049 T9 134×104 240 15210 t10 tMtc 168 14511 t11 120×104 tMtc 14512 T12 2×104 165 13613 t13 2×104 168 15414 t14 8×104 184 104

BacteriologicalandPhysicochemicalAnalysisofDrinkingWater


SN Sample Total bacteria count(Cfu/ml)

Total Coliform at 370c ( per 100ml)

fecal coliforms at 440c (per100ml)

15 t15 15×104 136 6016 T16 25×104 tMtc 22217 t17 65×104 264 17218 T18 20×104 tMtc 14519 T19 1×104 tMtc 25020 W1 tMtc tMtc 14821 W2 tMtc tMtc 17822 S 204×104 248 152

The fluctuations in pH lead to an increase ordecrease in the toxicity of poisons in waterbodies.12 The samples showed hardness withinthenormallevelwhileoneofthesample(I1)showedgreatervaluethannormal.Thecalciumcontent of the water bodies fluctuates directlywith bicarbonates and both of these movesinverselywithcarbonatesandPH.NH3 wasfoundwithinnormal rangeexceptonesample I2 (≥3)whichexceededWHOguidelinevalue(1.5mg/l).Similar resultswere found in the groundwatersamples of Kathmandu valley. Water sampleswerepositivefortotalcoliforms.Thequalityofwaterhasdeteriotedduetopoormanagementandnomonitoringofwaterquality.IsolatedbacteriawerethefamilyofEnterobacteriacea.

The identified organisms include E.coli,Klebsiella species,Citrobacter species.Isolationof E.coliwas highest among isolates since thisorganism is abundant in human and animalfaeces.Thewaterwasfaecallycontaminatedandtreatmentwasineffective.

CoNCLuSIoN Thepathogenicorganicandtheindicatororganismspresent in all thewater samples rendered themunfit for human consumption though they canbe used for other purposes.Water shouldmeetdifferent quality specifications depending onthe particular uses.Thus, potable and domesticwatershouldbeharmlessfor thehealthofmanand should have proper organoleptic propertiesand should be suitable for domestic use.Waterqualityshouldbecontrolledinordertominimizeacute problem ofwater related diseases,whichareendemictothehealthofman.

rEfErENCES1. LamikaranA. EssentialMicrobiologyforstudentsand

PractitionersofPharmacy,MedicineandMicrobiology.2ndEdn.Amkrabooks;1999.p.406.

2. Nikoladze GDM, Akastal S. Water treatment forPublicandIndustrialSupplyMIRPublisherMoscoul;1989.p.163.

3. LemoO. BacteriologyDetermination ofWater withlongtermstorage[dissertation];TribhuvanUniversity,2002.

4. TebuttTHY.PrinciplesofQualityControl.Pergamon,England;1983.p.235.

5. Raymond F. Le Problame dis ean dans le monde(problems of water), EB and Sons Ltd., UK;1992.p.123-6.

6. EdemaMO,OmemuAM,FapetuOM.Microbiologyand Physicochemical Analysis of different sourcesof drinking water in Abeokuta. Nigeria. Niger. JMicrobiol2001;15(1):57-61.

7. FapetuOM.ComparativeAnalysisofDifferentSourcesofDrinkingWaterinAbeokutaSouthL.GA.,OgunState[dissertation]UNAABAbeokuta;2000.p.44.

8. WHO. Guidelines for drinkingwater quality, 1999Volume 2, 2nd edition, A.I.T.B.S. Publishers anddistributors,Delhi.

9. Mulusky DS. Ecology of Estuaries. HeinemannEducationalBooks,London;1974.pp.5-103.

10. BanwoK.NutrientLoadandPollutionStudyofSomeSelected Stations along Ogunpa River in Ibadan,Nigeria. [Dissertation].University of Ibadan; 2006.p.107.

11. Medera V, Allen HE, Minear RC. Non metallicconstituents;ExaminationofWaterPollutionControl.A reference handbook Physical, Chem. Radiol.Exam1982;2:169-357.

12. AliJ.AnAssessmentoftheWaterQualityofOgunpaRiver Ibadan, Nigeria. [Dissertation]. University ofIbadan;1991.

ShidikiAmrullahetal.

Cite this article as:ShidikiA,BhargavaD,GuptaRS,AnsariAA,PanditBR.BacteriologicalandphysicochemicalanalysisofdrinkingwaterinTokha,Kathmandu,Nepal.MEDPHOENIX2016;1(1):15-18


A Review on Congenital TuberculosisrEVIEw

Jha Anand1*, Bhandari Sujeeta2, Kadel Binod3, Kadel Anjeela4, Shah Dhiraj5, Sah Arun Kumar6

1,2DepartmentofPediatricsandChildHealth,NationalMedicalCollege,Birgunj,Nepal3DepartmentofMedicine,NationalMedicalCollege,Birgunj,Nepal4,5,6DepartmentofPediatrics,NepalMedicalCollege,Jorpati,Kathmandu,Nepal

ABSTrACTBackground: Congenital tuberculosis isdefinedasinfectionacquiredtoanewbornfrominfectedmotherbyMycobacteriumtuberculosisbacilli during the intrauterine period or duringnormal birth. Though tuberculosis infection isvery common all over the world, congenitaltuberculosis is rare andmortality is50%.Non-specific symptoms in congenital tuberculosisand difficulties encountered in the diagnosisof tuberculosis in general, make it difficult toreachafinaldiagnosissocongenitaltuberculosisis generally known clinically during the firstpostnatal month. Maternal tuberculosis iscommonbutcongenital tuberculosis is rareandfatal.Alsotheclinicalfeaturesarenotspecificbutdiagnosisisdifficult.Soscreeningofallpregnantladiescanhelpinearlydiagnosisandpreventionofcongenitaltuberculosis.

Methods: This article has been produced byanalyzing various publications since 1998 tilldate,andbyusingsearchgear,pubmed,hinariandgoogle.

result: Around 350 cases have been reportedsofarfromdifferentpartoftheworld.Thereispaucityofdatafromourpartofworld.

Conclusion: The difficulties in diagnostic andtherapeuticconductofthisdisease,whichareofgreatinteresttopublichealth,pointstotheneedtodevelopspecificprotocols.

Key words:Congenital,Review,TuberculosisCorresponding Author: Dr. Anand Jha,

Department of Pediatrics and Child Health,National Medical College, Birgunj, Nepal;E-mail: [email protected]

INTroDuCTIoNCongenital tuberculosis is rare despitetuberculosis being a common infection worldwide.1 Three hundred cases were reported inthe literature till 1989.2 Fifty eight caseswerereviewedbyAbughalietal in19943,andfrom2001 to 2005, 18more caseswere reported.4-10 Tuberculosis (TB) among pregnant women iscommon, but congenital TB are rare becausefirstly tuberculosis primarily causes infertility11 and also secondly placenta forms a protectivebarrier against the invasion of the fetus by thetuberculous organisms but it has been assumedthat the infection acquires in uterus, becauseof: (i) theageof the infant, (ii) absenceofanyknown contact with an open case of TB, and(iii) generalized dissemination of the disease.The risk of TB in pregnancy has increasedowingtorecentchangesintheepidemiologyofthe disease,which has led to an increased riskof congenital TB 4, 12.Although a rare disease,congenital TB should be distinguished fromthe more frequent acquired neonatal TB, inwhich the infant is infected after birth by anadultsufferingfromthedisease.CongenitalTBmay occur as a result of maternal TB when itinvolves thegenital tractorplacenta.Thesignsand symptoms are non-specific; the atypicalclinicalmanifestationsofcongenitalTBandthedevastating consequences in absence of earlytherapysignifytheimportanceofearlydiagnosisand treatmentduring theneonatalperiod.4,12,13,14


Infections acquired early in neonatal life alsohassimilarclinicalmanifestationsandtreatment.Thus, clinical differentiation of neonatal andcongenitaltuberculosisisepidemiological.15

PATHoPHySIoLogy As described earlier the tubercular bacilli aretransmittedtransplacentallythroughtheumbilicalveingivingtoprimarylocalizationinliverorlung.ItcanalsobetransmittedbyaspirationofinfectedamnioticmaterialeitherinuteroorduringpassagethroughbirthcanalleadingtoprimaryinfectioninlungsorinGIT.Transplacentalisusuallylateinpregnancywithprimarylesioninliverleadingto characteristic periportal lymphadenopathy.Furthermore,hematogenousspreadmay lead tolesions in the lungandbrain.Caseous lesionintheplacentacauseamnioticfluidtobeinfected.This infected amniotic material aspirated/

ingested will lead to multiple foci in lung andgut resulting in enlarged mesenteric, bronchialor both lymphnodes.16, 17 Eustachion tube inthe newborn is patent and does not collapse.Bacteria can readily access the middle ear viathe eustachion tube and spread the disease tomiddleearviapharyngealsecretion.Thisformofdiseasepresentsasmultipleperforationor totaldestruction of tympanic membrane.18 So, it isworthwhile todohistopathologicalexaminationofplacentainmotherwithtuberculosis.

DIAgNoSTIC CrITErIADiagnosticcriteriaforthediagnosisofcongenitaltuberculosiswerelaiddownbyBeitzki in1935andsubsequentlyrevisedbyCantwellin1994.19ThesearesummarizedinTable1.

Table 1: Diagnostic criteria for congenital

tuberculosisA. Beitzki criteria (12, 14)IsolationofM.tuberculosisfromtheinfantDemonstrationoftheprimarycomplexintheliverIntheabsenceofprimarycomplexintheliver:EvidenceoftuberculosiswithindaysafterbirthAbsenceofcontactwithacaseoftuberculosisafterbirth

B. Revised criteria by Cantwell (19)Proventuberculosislesionsintheinfantplusoneofthefollowing:LesionsoccurringinthefirstweekoflifeAprimaryhepaticcomplexMaternalgenitaltractorplacentaltuberculosis

CLINICAL MANIfESTATIoN Theaffectedinfantisfrequentlybornpremature,but signs of disease usually do not appear forseveral days or weeks. The most commonpresentationiswithrespiratorydistress,lethargy,poor feeding, fever, irritability, abdominaldistension and failure to thrive, seizure.Hepatospleenomegaly and lymphadenopathyarecommon.Meningitisisuncommon,asisthejaundice. In a small percentage of cases otitismedia with or without mastoiditis is the firstsignofcongenitalTB.Obstructivejaundicedueto glands in the porta hepatitis may occur andpapular or pustular skin lesions may be foundin few cases2,4,6,12,14somemay have progressiveliver dysfunction in absence of respiratory

symptoms7finally,thecourseisoftenfulminant,characterizedinmanycasesbydisseminationoftheinfection.4

INVESTIgATIoNSCongenitalTBisparticularlydifficulttodiagnose.Themothersareoftenapparentlyhealthy.Inonereview, 24 of 32mothers were asymptomatic3,becausethesignsandsymptomsoftuberculosisin neonates are nonspecific; they are initiallyattributed to other causes like prematurity,congenital viral infections or sepsis21, 22. So thediagnostictestingfortuberculosisisnecessary.

1.Mantoux test is frequently negative 2,4,12,14 . IntheclassicalstudyofHagemanetal23only2of

JhaAnandetal.


JhaAnandetal.

the14 infantswithcongenitalTBhadpositivetuberculintests.Similarlyinanotherstudyof9infantswithcongenitalTB,only2showed thepositivereactions(>10mm).6

2.Chestradiographyandcomputedtomographyshowed the presence of scattered infiltrates,bronchopneumonia,consolidationorperiportalhypodensity.4,10,12

3.Positive smear and/ or culture results canoftenbeobtainedfromgastricwashings,openliverbiopsy,lymphnodebiopsy,spinalfluid,ear discharge, endotracheal aspirate or bonemarrow.2,4,14

4.Newermodalitieslikepolymerasechainreaction(PCR) 4,13 and T-SPOT.TB assay are highlybeneficial in the diagnosis of congenital TB.TheT-SPOT.TBassayisatypeofcommerciallyavailable interferon-gamma (IFN-gamma)released assays (IGRAs) which are based onIFN-gammareleasedbyantigen-specificT-cells.OthertypesofIGRAareQuantiferonTBGoldand Quantiferon In-Tube tests. T-SPOT.TBassay proved to have greater sensitivity thanTuberculin Skin Tests and also T-SPOT.TBassay have greater specificity than TuberculinSkinTestsinBCG-vaccinatedindividuals.24

5.Recentlyphagetypingisalsousedtoestablishthe identity of mycobacteria isolated frommotherandtheinfant.2,14

6.Mother’shistoryregardingtuberculosisduringpregnancyespeciallypleuraleffusion,militaryor meningeal disease suggesting primarylymphohematogenous spread or history ofendometritis.Work up of themother shouldincludeahistologicalexaminationofplacentaat birth, Montoux test, chest X-ray andendometrialaspirationandcurettage.25-28

Thediagnosisisbasedonpositivesmearand/orculture results obtained from gastric washings,liverbiopsy,lymphnodebiopsy,spinalfluid,eardischarge,endotrachealaspirateorbonemarrowbiopsy and mother’s history of tuberculosis inantenatalperiod.

TrEATMENT Congenital TB is a rare entity, even in humanimmunodeficiency virus (HIV) endemicpopulations,andisuniformlyfatalifuntreated.4,5 Treatment of the infant should begin as soonas the diagnosis is suspected without waiting

for laboratory confirmation, while appropriatespecimens should be obtained fast forbacteriologicalandhistologicalexamination.2,12,14

SincecongenitalTBisrare,notherapeutictrialshavedeterminedtheoptimaltreatment,however,several regimens have been evaluated andestablished.29,30Treatmentregimensshouldcontainat least 2 and preferably 3 drugs to which theorganismsarelikelytobesusceptible.9,14Completerecovery has been obtained by combination ofisoniazid, rifampin and pyrazinamide for 18months with intravenous amikacin for initial 2months.4 A 6 month course of isoniazid (H),rifampin(R),pyrazinamide(Z)andstreptomycin(S) for 2months andbiweekly (R andH) for 4monthshasshowngoodresultswitharelapseofonly1%,andnodeathsfromthedisease.TherearealsocasereportsofsuccessfultreatmentwithHZS,HSandHRZ.25,28,31Streptomycin(20-30mg/kg/day)canbeusedininfantsbutiscontraindicatedinpregnantwomen.19Currentlytheacceptedmodeof treatment is isoniazid (10-15mg / kg / day),rifampin(10-20mg/kg/day)andpyrazinamide(15-30 mg / kg / day) and either streptomycinor ethambutol (15-25mg / kg / day) for first 2monthsfollowedbyisoniazidandrifampinfor4to 10 months.21,26 Corticosteroids may be givenempirically if thebabyisvery ill.19,30Supportivetherapy such as oxygen,mechanical ventilatorand Extracorporeal membrane oxygenationmayberequiredforrespiratoryfailure.8

ProgNoSISTheprognosis ispoor. In thepre-chemotherapyerathereportedsurvivalratewasverylow(around50%),butsincetheadventofchemotherapy,thechances of successful treatment have improvedthe overall survival. Delay in the diagnosiscontributestotheincreasedmortality.2,14,30

PrEVENTIoN Prevention should be possible through earlydetection of disease during pregnancy andinstitutionofappropriatetherapy.4,14

rECoMMENDATIoNSInviewoftheincreasingburdenoftuberculosis,chances of congenital TB are also likely toincrease.Asmostofthewomenareasymptomatic

CongenitalTuberculosis


forthediseaseduringpregnancy,werecommendthatscreeningofallpossiblepregnantwomenfortuberculosisshouldbemadeanecessaryprotocol.

rEfErENCES1. Smith MHD, Tele DW. Tuberculosis acquired in

utero.In:RemingtonJS,KleinJO(Eds).InInfectiousDiseases of the fetus and Newborn, (4th edn).Philadelphia, WB Saunders Publication 1995;1077-80.

2. SeatonA,SeatonD,LeitchAG.ClinicalFeaturesofTuberculosis.In:CroftonandDoughlas’sRespiratoryDiseases. 4th Edition, Blackwell ScientificPublications,London.1989pp.395-422.

3. Abughali N, Vander Kuyp F, Annable W, KumarML. Congenital tuberculosis. Pediatr Infect Dis J1994;13:738-41.

4. Massik TS, Carrel T, Duppenthaler A, ZeilingerG,Gnehm HE. Congenital tuberculosis in a prematureinfant.SwissMedWkly2002;132:598-602.

5. GroverSB,PatiNK,MehtaR,MahajanH.Congenitaltuberculosis:earlydiagnosisbyimagingstudies.AmJPerinatol2003;20(3):147-52.

6. Chotpitayasunondh T, Sangtawesin V. Congenitaltuberculosis.JMedAssocThai2003;86:689-95.

7. Berk DR, Sylvester KG . Congenital tuberculosispresenting as progressive liver dysfunction. PediatrInfectDisJ2004;23(1):78-80.

8. Weisoly DL, Khan AM, Elidemir O, Smith KC.Congenital tuberculosis requiring extracorporealmembraneoxygenation.PediatrPulmonol2004;37(5):470-3.

9. Chanta C, Jariyapongpaibul Y ,Triratanapa K.Congenitaltuberculosispresentingassepsissyndrome.JMedAssocThai2004;87(5):573-7.

10. KondoS, ItoM,NishimuraG.CT imagingfindingsin congenital tuberculosis, Part I: Usefulness ofperiportalhypodensity in thediagnosisofcongenitaltuberculosis.Kekkaku2004;79(6):391-5.

11. KGhosh,KGhosh,JRChowdhury.Tuberculosisandfemale reproductive health. Journal of postgraduatemedicine2011;55(4):307-13.

12. Sri SS. Congenital Tuberculosis. In: Text Book ofPulmonary and Extra pulmonary Tuberculosis, 2ndEdition,Interprint,NewDelhi.1995pp.205.

13. Hatzistamatiou Z, Kaleyias J, Ikonomidou U,Papathoma E, Prifti E, Kostalos C. Congenitaltuberculous lymphadeninitis in a preterm infant inGreece.ActaPaediatr2003;92(3):392-4.

14. BaheraD.Tuberculosis.In:TextBookofPulmonaryMedicine. Ist Edition, Jaypee Brothers Publishers,NewDelhi.1995pp.233-86.

15. SparkRP, PockNA,PedronSL, FoxC, OpulskiA. Perinataltuberculosisanditspublichealthimpact:Acasereport.TexMed1996;92:50-3.

16. VermaM,ChhatwalJ,SarinYK,SinghVP,SinghT,AnejaR.CongenitalTuberculosis:AnunderstandingEntity.IndianPediatrics1996;33:51-4.

17. Scnbil N, Sahin F, CaglarMK, OksalA, Yilmaz F,

TunaF.Congenitaltuberculosisoftheearandparotidgland.PediatrInfectDis1997;16:1090-91.

18. NaranbaiRC.Congenitaltuberculosisoftheear.ArchDisChild1989;64:738-40.

19. Cantwell MF, Sehab ZM, Costello AM, Sandsl, GreenWF, EwingEPJr etal.Briefreport:congenitaltuberculosis.NewEnglJMed1994;330:1051-4.

20. Hageman J, Shulman S, Schreiber M, Luck Susan,YogevR.Congenitaltuberculosis:Criticalreappraisalof clinical findings and diagnostic procedures.Pediatrics1980;66:980-4.

21. StarkeRSandSmithMH.Tuberculosis.In:RemingtonJS,Klein JO, editors. Infectious disease of the fetusand newborn.Philadelphia:WB Saunders Company;2001pp.1184-7.

22. Mazade MA, Evans ME, Starke JR, Correa AG.Congenital tuberculosispresentingas sepsis syndrome:casereportandreviewoftheliterature.PediatrInfectDisJ2001;20:439-42.

23. HagemanJ,ShulmanS,SchreiberM,LuckS,YogevR. Congenital tuberculosis: critical reappraisal ofclinicalfindingsanddiagnosticprocedures.Pediatrics1980;66:980-4.

24. ZhengY,BaiGandZhangH.CongenitaltuberculosisdetectedbyT-SPOT.TBassayinamaleinfantafterinvitro fertilization and followed upwith radiography.ItalianjournalofPediatrics2014;40:96.

25. BeitzkeH.Uberdieangeborenetuberculoseinfection.ErgebnTuberkForsch1935;7:1-30.

26. RamosA,HibbardL,CraigL.Congenitaltuberculosis.ObstetGynecol1974;43:61-4.

27. Kabra SK, JainY,Madhulika, BaggaA. Congenitaltuberculosis.NEngJMed1994;331:548.

28. VuciccuicZ,SuskoviT,FerencicZ.Afemalepatientwith tuberculosis, polyserositis, and congenitaltuberculosis in her newborn child. Tuber Lung Dis1995;76:460-2.

29. NemirR,O’HareD.Congenital tuberculosis.AmJDisChild1985;139:284-7.

30. KhilnaniGC.Tuberculosisandpregnancy.IndJChestDisAlliedSci2004;46(2):105-11.

31. Gordon-Nesbitt D, Rajan G. Congenital tuberculosissuccessfullytreated.BritishMedJ1973;1:233-4.

JhaAnandetal.

Cite this article as:JhaA,BhandariS,KadelB,KadelA,ShahD,SahAK.AreviewoncongenitalTuberculosis.MEDPHOENIX2016;1(1):19-22


oligohydramnios and fetal outcome: A Review

Khatun Tarannum1, Ansari Akhtar Alam2, Hamid Irfan3, Gupta Ravi Shankar4, Ahmad Md. Parwez2

1DepartmentofObstetrics&Gynaecology,NationalMedicalCollegeandTeachingHospital,Birgunj,Nepal2DepartmentofPharmacology,NationalMedicalCollegeandTeachingHospital,Birgunj,Nepal3DepartmentofPharmacy,TheUniversityofLahore,Pakistan4DepartmentofMicrobiology,NationalMedicalCollegeandTeachingHospital,Birgunj,Nepal

rEVIEw

ABSTrACTOligohydramnios is a threatening conditionto fetal health and is associatedwith increasedfetal morbidity. These conditions are oftenmissed and patient may not seek appropriatetreatmentatappropriatetimethatoftenincreasesriskofnumerousconditions.Earlydetectionofoligohydramniosand itsmanagementmayhelpinreductionofperinatalmorbidityandmortalityone side and decreased caesarean deliveries ontheotherside.

A search inGoogle scholar, PubMed,Medline,EMBASE was performed using key words.Inclusion criteria for articles selection weresingletonpregnancy,definitionofolgohydramniosasAFI<5cm,AFassessmentat37-42gestationalweeks.Thesearchedrevealednumerousresearcharticleswhichisfurtherrefined.It isfoundthatoligohydramnios is associatedwith IntrauterineGrowthRestriction(IUGR),smallforgestationalage (SGA), prolonged labour, caesarian section(C/S) for fetaldistress (FD),meconiumstainedliquor,LowApgarscoreandNeonatalIntensiveCareUnit(NICU)admission.

Women with oligohydramnios are associatedwith higher fetal risk but can expect a safedeliveryandgoodoutcomeforwhichproperfetalsurveillance and regularAntenatal care (ANC)visitsarerequired.

Keywords:AmnioticFluidIndex;Apgarscore;Fetaloutcome,Oligohydramnios

Corresponding Author: Dr. Tarannum Khatun,Department of Obstetrics & Gynaecology, NationalMedical College, Birgunj; E-mail: [email protected]

INTroDuCTIoNOligohydramnios is a relatively commoncomplication of pregnancy and such case isoften encounter in clinical practice.1 it refers to amniotic fluid volume that is less than expectedfor gestational age. It is typically diagnosed byultrasound examination and may be describedqualitatively(e.g,normal,reduced)orquantitatively(e.g,amnioticfluid index [AFI]<5).2Diminishedfluidvolumemaybefoundoftenwithpregnanciesthatcontinuebeyondterm.3

Oligohydramnios is often to describepregnancies with AFI <5 cm and borderline/low normal amniotic fluid volume to describepregnancieswithAFI5to8cm.4Alternatively,someclinicianspreferthesingleverticalpocket(SVP)with severeoligohydramniosdefinedasSVPless than1cmandmildoligohydramniosdefinedasSVP1to2cm.1Anadequatevolumeofamnioticfluidiscriticaltoallownormalfetalmovementandgrowth,andtocushionthefetusand umbilical cord.5 Oligohydramnios mayinhibit these processes and can lead to fetal


deformation, umbilical cord compression anddeath.6

INCIDENCEReportedratesofoligohydramniosareinfluencedbyvariationsindiagnosticcriteria,thepopulationstudied(loworhighrisk,screeningorindicatedultrasoundexamination),thethresholdusedandthegestationalageatthetimeoftheultrasoundexamination(preterm,termorpostterm).7

Astudyof3050uncomplicatedpregnancieswithsingletonnon-anomalousfetusesbetween40and41.6weeksofgestationnotedoligohydramnios(definedasAFIless than5) in11percent.8Theincidence is high in laboring women, largelyduetoruptureoffetalmembranesduringorjustbeforelabor.9

Aprospective studyconducted atUniversityofTexas Southwestern Medical Center showedthe incidenceof oligohydramnios to be2.3%.10 Similar resultwasobtained inUSAamong953women over a period of 12 months in thirdtrimester.11 In contrast, a study conducted atUniversity of Milano-Bicocca, Monza, Italyamong3050womenwhounderwentsonographicassessments of AFI after 40.0 weeks showedoligohydramnios of 11.18%.12 Higher rates ofoligohydramnios were found in the summermonthsascomparedintherestoftheyear.7

Higher rate of isolated oligohydramnios (24%) wasdetectedinastudyinterm8andthisisdoubleincidence as compared to other studies.5-7 TheAFVof135womenwasevaluatedbetween70women(52%)intheuppergreatergroupthatwasestablishedbyAFIand65women(48%)inthelowergreaterAFIgroup.9

AMNIoTIC fLuID VoLuMETheamnioticfluidvolume(AFV)isregulatedbyseveralsystems,includingthein-tramembranouspathway, fetal production (fetal urine and lungfluid),uptake(fetalswallowing)andthebalanceoffluidmovementviaosmoticgradients.13

The evaluation of Amniotic fluid volumehas become an integral component of thefetoplacental assessment and surveillance of

pregnanciesthatareconsideredtobeatriskforanadversepregnancyoutcome.14DecreasedAFVin those pregnancieswithout premature ruptureof themembranescan reflecta fetus inchronicstress, shunting blood to its brain, adrenal glandsand heart are away from other organs, whichincludesthekidneyandresultsindecreasedfetalperfusionandurinaryoutput.15TheadequacyofAFVisestimatedwithultrasonicmeasurements.4 Its evaluation has been used at the time ofadmission to labor and delivery to recognizea pregnancy that is at risk for a poor perinataloutcome; vulnerable to variable decelerations,late decelerations, caesarian deliveries for fetaldistress,meconiumstainedAF,lowApgarscoresandlowumbilicalcordarterypH.9

A study examined the usefulness of umbilicalarteryDopplervelocimetry,amnioticfluidvolumeassessment and fetal heart rate data in the earlyintra-partum period found as predictors ofsubsequent fetal distress.16 Both an abnormalinitialfetalheartrateandanamnioticfluidindexlessthanorequalto5.0cmwereassociatedwitha significant increase in the incidence of intra-partumfetaldistress.6,17Thefetalheartratetracingand the assessment of amniotic fluid volumein the early intra-partum period are reasonablepredictorsofsubsequentfetalcondition.18

METHoDS of AfI ESTIMATIoNWhenAFIisusedtodefinefluidstatus;amnioticfluidincreasesfrom14to31weeksanddeclinesthereafter.11Useof singledepestpocket (SDP)ortwo depest pocket suggest that fluid increasesfrom 14 to 20 weeks, plateaus between 20 to37 weeks and thereafter declines gradually. In1990, Moore and Cayle noted that the meanAFI changed weekly.12 The identification andevaluation of abnormal amount of amnioticfluid is an important component of antenatalassessment. Current techniques for estimatingAFV range from subjective clinical assessmenttomoreelaborateultrasonicallyderived indicesofAFvolume.

The overall accuracy of subjective estimatesof amniotic fluid volume range from 65-70%.12 The ultrasonographic measurements usingthree ultrasound indices correctly identifiesthatdyedeterminedvolumewithanaccuracy

KhatunTarannumetal.


of 87%.13 Oligohydramnios was recognizedsignificantly more than using two diameterpocket technique (72%) than using the othersonographicmeasurements(17%)ofamnioticfluidvolume.19

The ultrasound estimation of amniotic fluidvolume is used with both the Non Stress Test(NST) as the AFI and the biophysical profileas the 2x1 pocket technique.14 Both of theseultrasonic techniques have primarily usedfixedcutoff values for theAFI and SDP. The cutoffvalue for theAFI commonly used are anAFIof 0-5cm labeled as low fluid, 5.1-18 cm asnormal fluid and greater than 18 as high fluidvolume.14 TheAFI is observed to overestimateand SDP underestimate dye determined ordirectly measured low fluid volume.15 Becausethe same cutoff values are used throughout thesecond half of pregnancy to identify abnormalamnioticvolumes, investigatorshave suggestedthatgestational age-specificpercentilesbeusedinstead to define the upper and lower limits ofnormality.20

Invasive methods such as indicator dilutiontechniques are the most accurate measures ofAFV; but are impractical for clinical use.15 Ultrasound is non-invasive and hence widelyused.Severalmethodsareusedtoassessamnioticfluid.Thefirstmethodisasubjectiveassessmentwherethevolumeisdescribedasaverage,aboveaverage, below average or scanty. Ultrasonicassessment of amniotic fluid can be viewed asa semi-quantitativemethod.AFI and the singledeepest vertical pocket are themost commonlyemployed techniques for assessingadequacyofamnioticfluid.15

Amnioticfluidindex,whichsummatesthedeepestverticalpoolineachofthefourquadrants,mightbereferredtoasamoresensitiveestimateofamnioticfluid volume throughout gestation.17 It has beensuggestedthatamnioticfluidindexisreasonablyreliable in determining normal or increasedamnioticfluidbutislessaccurateindeterminingoligohydramnios.19Assessment of amniotic fluidvolumebyAFItechniqueispreferredbymanytothesingledeepestverticalpooltechniquebecausethe deepest vertical pool does not allow for anasymmetricalfetalpositionwithintheuterusand

because the regression curve between amnioticfluid index and gestational age is similar inshapetothatbetweenamnioticfluidvolumeandgestational age.21 It is found that amniotic fluidindexissuperiortoameasureofthesingledeepestpoolasanassessmentof the fetusatorafter40weeks.22AFI is certainly the most widely usedparameterofalland it alsocomesclosest to theactualamountofamnioticfluid.16Whenamnioticfluidisusedtomonitortermpregnancies,theAFIshouldbemeasuredweeklyinpregnanciesunder41weeksifitexceeds8cmandtwiceaweekinpregnanciesover41weeksorifitisbelow8cm.16MooreetalclaimedthataAFI<5cmwaspresentonlyin1%ofthenormalpopulation.17

Theabdominalpressureexertedbytheultrasoundtransducermay also induce changes in theAFIandintheSDP.Indeedlowpressureresultsina13%increaseinAFI,whilehighpressurecouldleadto21%AFIdecrease.18AstudybyMagnannetaltoassesswhethertheAFIorSDPisthebesttechniquetoestimatetheamnioticfluidvolumerevealed post-term pregnancy and advancedmaternalagewereassociatedwiththeoccurrenceofoligohydramnios.23

Congenitalanomaliesofthekidneyandurinarytract (CAKUT) are detected frequently in upto 1% of newborns and nowadays diagnosis isoftenestablishedbeforebyfetalultrasound.18 if CAKUToccursunilaterallyeg.Hydronephrosisdue to ureterovesical junction obstructionor multicystic renal dysplasia, prognosis isgenerally good. Bilateral renal disease withOligohydramnios indicates significant globalfetalrenaldysfunctionandisariskfactorforthedevelopment of pulmonary hypoplasia. Outcomeof fetus with renal oligohydramnios thereforeis regarded as poor.24 In a series from Mayoclinic;18of52(32%)childrenwithCAKUThadoligohydramniosandallchildrendied,includingsix intrauterine death.25 Recent advances intreatment of infants and children with chronickidney disease and end stage renal disease hasimproved prognosis also for infants with renalinsufficiencyconsiderably.24, 26

The recent use of color Doppler sonographyhas not improved the diagnostic accuracy of

OligohydramniosandFetalOutcome


sonographic estimates of the AFV but insteadhasledtooverdiagnosisofoligohydramnios.27,28 ThustheuseofAFItoidentifyoligohydramniosinat-riskpregnanciesseemstobeabetterchoicebecausetheuseoftheAFIleadstoanincreaseinthediagnosisofoligohydramnios.29

MoDE of DELIVEryOligohydramnios at term may be managedactively via induction of labour or expectantlyvia hydration, fetal surveillance and or regularultrasounds assessing amniotic fluid volume.30 AnisolatedborderlineAFI,i.e.5-8cmisnotanindication for labor induction.22A retrospectivecasecontrolstudywasperformedattheLiverpoolMaternity Hospital, 103 pregnancies withreducedAFI in third trimester were taken intoaccountwhichshowedahigherriskofinductionforfetalreasons.31

Intrapartum oligohydramnios was associatedwithanincreasedriskofC/Sforfetaldistress.30

AstudydoneinUSA,among953womenoveraperiodof 12months revealed an increased rateofC/S for fetaldistress in theoligohydramniosgroup (9.7% vs 5%). Of the women deliveredby C/S, those with oligohydramnios weremore likely to have fetal distress leading toC/S (47%) than thosewith anAFIgreater than5cm(20%).6InastudybyChauhanetal.overa time period in a tertiary hospital among 490patientswitholigohydramnios14%(70/490)hadcaesariansectionforpresumedfetaldistress.Theindicationsfortheseoperationswerebradycardiain29patients, recurrent latedecelerations in27,persistentseverevariabledecelerationsin6andbeattobeatvariabilityinthe9remainingcases.32The sum of theAFI in the upper quadrants wasgreaterthanthesumofAFIinthelowerquadrants,thepregnancywasatgreaterriskforpoorperinataloutcomeasdefinedbymeconiumstainedamnioticfluid,1minApgarscorelessthanseven,variablesdeceleration, latedeceleration,C/SforFD,umblicalarterialpH less than7.2.33 PregnancywithAFI lessthan5cmcomparedwithagroupofAFImorethan5cm,therewasincreasedriskofvariabledecelerationandC/SforFDbutnotmeconiumstainedamnioticfluid.34,35ChauhanetalhowevernotedthatanAFIlessthanorequalto5cmcomparedtoAFImorethan5cmwasapoorscreeningtesttoidentifypregnancy

thatareatriskforpresumedFDandApgarscore at 1 and5minlessthan7.32

Induction of labor with PGE2 at term in patientswithAFIequalorlessthan5cmisassociatedwithan increased risk of C/S for presumed FD.36Thetermisolatedoligohydramnios isusedtodescribeoligohydramnios in the absence of maternal orfetal risk factors, e.g. IUGR, spontaneous ruptureof membrane, diabetes, pre-eclampsia or severematernalsystemicdiseases.8InastudybyChhabra,DarganandBawaskar;theoveralllabourinductionduring thestudyperiodof the retrospectivecaseswas18.2%butinoligohydramnios,itwas66.1%.The C/S rate in the retrospective cases withspontaneous labor was 42.4% and with inducedlaboritwas38.5%.IntheretrospectivecaseswiththespontaneouslaborC/Sratewas50.4%andwithinducedlabor,itwas29.3%.FetaldistresswastheindicationforC/Sin79.9%retrospectiveand67.9%prospectivecases.11

Labor induction is the common response ofoligohydramniosintermgestationwhetherornototherriskfactorsarepresent.37-39Inastudy,183women underwent labor induction for isolatedoligohydramniosattermandtheywerematchedtoagroup183controls.Agreaterproportionofwomen in the oligohydramnios group than inthecontrolgroupunderwentC/S.40 Womenwitholigohydramnios were more likely than thosewith AFI > 5 cm to require cervical primingwithPGE2gel before inductionof labor.CaseswithAFI<5cmhadhigherrateofC/Sfornon-reassuring fetal heart tracing than before withAFI>5cm.41

ASSoCIATED MorBIDITy/MorTALITyPregnancies complicated by markedlydiminishedamnioticfluidvolumearefrequentlyassociated with adverse perinatal outcome.42 The mortality rate in oligohydramnios is high.The lack of amniotic fluid allows compressionofthefetalabdomen,whichlimitsmovementofitsdiaphragm.Inadditiontochestwallfixation,the lackofamnioticfluidflowinginandoutofthe fetal lung leads to pulmonary hypoplasia.43 Oligohydramnios is also associated withmeconium staining of the amniotic fluid, fetalheart conduction abnormalities, umbilical cord

KhatunTarannumetal.


compression,poortoleranceoflabor,lowerApgarscoresandfetalacidosis.7Incasesofintrauterinegrowth restriction (IUGR), the degree ofoligohydramniosisoftenproportionaltogrowthrestriction, is frequently reflectiveof the extentofplacentaldysfunction,andisassociatedwithacorrespondingincreaseinthePMR.22

A retrospective case control study done at theLiverpool Maternity Hospital found four foldriskoflowbirthweight(LBW)andhighrateofadmissiontoNICUincasesofoligohydramnios.31

Similar studies explored some associationwithstillbirth,nonreassuringfetalheartrate,admissiontotheneonatalintensivecarenursery,meconiumaspiration syndrome and neonatal death.64 a comparativestudydonebetween2groupsAFI<5cmandAFI5-8cmconcludedthat therewasnodifferencewithregardtomeconiumstainand1and5minApgarscore<7.6

Pregnant women with PPROM during 28-34gestational weeks having oligohydramnios hada high rates of cesarean section, Intra-amnioticinfection, fetal distress, neonatal asphyxia, early-onset neonatal sepsis and hypoxic-ischemicmyocardial injury.35 the risk increased by sevenfold if severe oligohydramnios is present.42 Witholigohydramnios, meconium stained liquor, fetalheartrateabnormalitiesanddepressedApgarscoresaremorefrequent;neonatalandfetalacidosisrateswere doubled compared with controls.25 cases of IUGR complicated with oligohydramnios hadsignificantlyhigherratesofperinatalmortalityandlow birth weight as compared to IUGR withoutoligohydramnios.44

Astudy found an inversebetween theAFI andnon-reactiveNST,fetalheartratedecelerations,meconiumstaining,C/S forFDand lowApgarscores.45 Sarno et al found that intrapartumoligohydramnios was associated with anincreasedriskofC/SforFD,anApgarscore<7at1minandabnormalfetalheartpatterns.30 Severalstudies explored no statistically significantdifferencebetweenthetwogroupsintheriskofthickmeconiumstainedamnioticfluid,variabledeceleration, late deceleration, caesarean section forFD,birthweight,Apgar score<7at5minandNICUadmission.40,46Changesintheamnioticfluidmeasurementsandfetaldopplervelocimetryinpatientswitholigohydramnioswereevaluated

for correlation with fetal outcome.47 Therewas no difference found in any ponderal indexbetween the oligohydramnios and the controlgroupwhenthefetalmiddlecerebral,renalandumbilical arterieswere examinedwithDopplervelocimetry.48

InaretrospectivestudybyShanksetalfrom1998to2008,studysubjectswereidentifiedbyAFI<5cmand<5thpercentileforgestationalage.Therewere 145NICUadmission among904patientswithAFI<5cmcomparedto235patientsamongthe1429patientswithAFIlessthan5thpercentilefor the gestational age. The sensitivity andspecificityforNICUadmissionofAFI<5cmwas10.9%and95.2%comparedto17%and92%.48

HyDrATIoN AND AMNIoINfuSIoNMaternal hydration with oral water and IVhypotonicsolutionshasshowntoincreaseAFV;oneisbyfetalurineproductionandanotherisbyimprovinguteroplacentalperfusion.So,itcanbetakenasanalternativetoimmediateinductionoflabourinwomenwithisolatedoligohydramniosat term pregnancy.6,31 It is found that maternalhydration using either oral or intravenousadministration of fluids increases the amnioticfluidvolume.39

Oligohydramnios may be responsible formalpresentation, umbilical cord compression,concentrationofmeconiuminliquoranddifficultor failed external cephalic version.5 Simplematernal hydration has been suggested as aneffective way of increasing AFV in order toreducesomeofthisproblems.39

Acute hypotonic oral rehydration in the thirdtrimesterdecreasesmaternalplasmaosmolality.11 The decrease in maternal plasma osmolalityafteroralhydrationcausesawatershiftfromthemothertofetus.22Asaconsequence,fetalplasmaosmolality decreases resulting in a fall in fetalargininevasopressinsecretion,whichcausesanincreaseinfetalurineproduction.4 Thispotentialrole of maternal hydration in the treatment ofoligohydramniosisalsofoundeffective.21

The ultrasonic visualization of fetal anatomy,particularly renalagenesis, isdifficult in severeoligohydramnios/anhydramnios. Intra-amnioticinstillation of normal salinemay help improve

OligohydramniosandFetalOutcome


ultrasonographic examination and lead to thediagnosis of fetal abnormalities like renalagenesis.49 However the use of amnioinfusionhas greatly diminished with the widespreadavailability of the use of color Doppler toidentify the renal arteries, being an accurateandanoninvasivewaytopredicttheabsenceofrenalfunctionasinrenalagenesisormuticysticdysplasticrenaldisease.50 Somereportshavealsoshownthatinpregnancieswithpretermprematureruptureofmembraneswitholigohydramniosat<26weeksofgestation,serialamnioinfusionimprovethe perinatal outcomewhen compared to thosewithpersistentoligohydramnios.51

An amnioinfusion test procedure to try andpreselectcasesofmidtrimesterpretermprematurerupture of membranes which may benefitfrom serial amnioinfusion.7 Prophylactic andtherapeutic amnioinfusion results in improvedoutcomeinoligohydramnios.Thereviewoftrailsfound that amnioinfusion for oligohydramnioshelps when the baby shows sign of distress.49 Severaltrialshaveshownthatbothprophylacticand therapeutic amnioinfusion are effectivein reducing the fetal heart rate decelerationand caesarian section. The findings of twostudies donotsupport theuseofamnioinfusionprophylactically for oligohydramnios rather itcan be used therapeutically when fetal heartratedecelerationor thickmeconiumstainingofamnioticfluidoccurs.49,50

Recent study suggest the effectiveness oftransabdominal amnioinfusion before inductionof labour in reducing the incidence of fetaldistress in pregnancies with oligohydramniosat term.52 The use of prophylactic intrapartumamnioinfusion in case of oligohydramnios hasbeendescribed tobeeffective in reducing theC/S rate for FD and improvement of the neonataloutcome.53 Amnioinfusion caused a significantincrease inAFIwith themedianvalue to6 cmbefore infusion to 11 cm after infusion. Thelatency period until delivery was longer inpatientswhounderwentamnioinfusion.54

In a retrospective case control study, FHRpatternsanduterineactivitybeforeandaftertheinstallation of intracervical PGE2 in the presence

orabsenceofoligohydramnioswascompared.55 Patients with oligohydramnios had more highamplitude contraction in the first hour beforedosingbut therewerenosignificantdifferencesinthefrequencyordurationofcontractionduringthe subsequent 5 hrs. Uterine hyperstimulationwasnotseenandtherewerenodifferencesinthefrequencyofvariableorlatedecelerations.55

CoNCLuSIoNIn conclusion, isolated oligohydramnios in termpregnancies is associatedwith an increased riskof obstetric intervention. The current literaturedoes not really provide further information inunderstandingthesignificanceofoligohydramniosat a particular gestational age, in terms of boththepathophysiology and themanagement.Earlydetectionofoligohydramniosanditsmanagementmayhelpinreductionofperinatalmorbidityandmortality in one side and decreased caesareandeliveriesontheotherside.

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14. Magann EF, Doherty DA, Chauhan SP, Busch FW,MecacciF,Morrison JC.Howwell do the amnioticfluidindexandsingledeepestpocketindices(belowthe 3rd and 5th and above 95th and 97th percentiles)predict oligohydramnios and hydramnios? Am JObstetGynecol.2004;190(1):164-69.

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23. HsiehTT,HungTH,ChenKC,HsiehCC,LoLM,ChiuTH. Perinatal outcome of oligohydramnios withoutassociatedprematureruptureofmembranesandfetalanomalies.GynecolObstetInvest.1998;45(4):232-36.

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30. Sarno AP Jr, Ahn MO, Phelan JP. Intrapartumamnioticfluidvolumeatterm.Associationofrupturedmembranes,oligohydramniosandincreasedfetalrisk.JReportedMed.1990;35(7):719-23.

31. Roberts D, Nwosu EC, Walkinshaw SA. The fetaloutcome in pregnancies with isolated reducedamnioticfluidvolumeinthethirdtrimester.JPerinatMed.1998;26(5):390-95.

32. ChauhanSP,HendrixNW,MorrisonJC,MagannEF,Devoe LD. Intrapartum oligohydramnios does notpredictadverseperipartumoutcomeamonghigh-riskparturient.AmJObstetGynecol.1997;176(6):1130-38.

33. MylesTD,StrassnerHT.Amnioticfluiddistributioninpredictingperinataloutcomeinpatientswithrupturedmembranes.ObstetandGynecol.1997;89(5):723-28.

34. Baron C, Morgan MA, Garite TJ. The impactof amniotic fluid volume assessed intrapartumand perinatal outcome. Am J Obstet Gynecol.1995;173(1):167-74.

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38. MiyamuraT,MasuzakiH,MiyamotoM,IshimaruT.Comparisonbetween thesingledeepesetpocketandamniotic fluid index in predicting fetal distress insmall-for-gestationalagefetuses.ActaObstetGynecolScand.1997;76(2):123-27.

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49. Cook V, Spinnato JA. Prophylactic versustherapeutic amnioinfusion. Am J Obstet Gynecol.1993;168(1):363.

50. Ogundipe OA, Spong CY, Ross MG. Prophylacticamnioinfusion for oligohydramnios: a reevaluation.ObstetGyneol.1994;84(4):544-48.

51. AkterMD,KabirN,ShahMS,IslamF,TasnimS.EffectofmaternaloralhydrationtherapyinOligohydramnios.MymensinghMedJ.2012;21(4):723-28.

52. GhafarnejadM,TehraniMB,AnarakiFB,MoodNI,NasehiL.Oralhydrationtherapyinoligohydramnios.JObstetGynaecolRes.2009;35(5):895-900.

53. Flack NJ, Sepulveda W, Bower S, Fisk NM.Acute maternal hydration in third–trimesteroligohydramnios: effect on amniotic fluid volume,uteroplacental perfusion, and fetal blood and urineoutput.AmJObstetGynaecol.1995;173(4):1186-91.

54. SarnoAP Jr,AhnMO, Brar HS, et al. IntrapartumDopplervelocimetry,amnioticfluidvolume,andfetalheart rate as predictors of subsequent fetal distress.AmJObstetGynecol1989;161(6):1508-14.

55. Vergani P,Ceruti P, StrobeltN,LocatelliA,D’OriaP, Mariani S. Transabdominal amnioinfusion inoligohydramnios at term before induction of laborwith intact membranes: a randomized clinical trial.AmJObstetGynecol.1996;175(2):465-70.

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Cite this article as:KhatunT,AnsariAA,HamidI,GuptaRS,AhmadMP.Oligohydramniosandfetal outcome: a Review.MEDPHOENIX2016;1(1):23-30


A Review on Pharmacological and Chemical Documentation of Euphorbia Hirta Linn (Asthama Herb)

rEVIEw

Ansari Akhtar Alam1, Khatun Tarannum2, Ahmad Md. Parwez1, Gupta Ravi Shankar3, Ansari Mukhtar1, Madhikarmi Nirjala Laxmi4

1DepartmentofPharmacology,NationalMedicalCollegeandTeachingHospital,Birgunj,Nepal2DepartmentofObstetrics&Gynaecology,NationalMedicalCollegeandTeachingHospital,Birgunj,Nepal3DepartmentofMicrobiology,NationalMedicalCollegeandTeachingHospital,Birgunj,Nepal4DepartmentofBio-chemistry,MBKediaDentalCollege,Birgunj,Nepal

ABSTrACTAyurveda, an orthodox aswell asmain streamsystem of medicine has been with a sourceof new concepts and products for healthcare.Euphorbia hirtaLinn.(EH)isanadvantageouslyauthenticated medicinal plant as observablefrom the literature. Rural and tribal people ofIndiausedifferentpartsofthisplanttosurmountvariousdiseases. Itsuse in traditionalmedicinehasbeenassuredonscientificbasisbystudyingpharmacology of the plant in vitro and in vivoInformation was collected from different databaseresources. Key words:Asthmaherb,Dudhia,Euphorbia hirta

Corresponding Author: Dr.AkhtarA.Ansari,Departmentof Pharmacology, National Medical College, Birgunj;E-mail:[email protected]

INTroDuCTIoNIn India, used of the different parts of severalmedicinal plants to cure specific diseaseshas been in practice from ancient times.The indigenous system of medicine namelyAyurveda, Siddha, and Unani, has been inexistence for several centuries. Some drugsfrom Ayurveda approaches modern diseases,had already reached the market place.1 In

modern medicines, plants occupy a veryimportant place as the raw material for someimportant drugs. Synthetic drugs are effectivein controlling different diseases but thesesynthetic drugs are out of reach of millionsof people and have various side effects. It isestimatedthataround70,000plantspecieshavebeen used for medicinal purposes. The herbsprovide the starting material for the synthesisof conventional drugs. India recognizes morethan2500plantspecieshavingmedicinalvalue,SriLankaaround1400andNepalaround700.2 This reviewintends toprovideanoverviewofthechemicalconstituentsandpharmacologicalactionsofEuphorbia hirta.

METHoDSFor literature survey we used various searchenginelikeGooglescholar,SciencedirectandPubmed. The present review gives completeinformation on the pharmacological activitiesand chemical constituents of this plantmentioned from the year as back as 1963 tothe latest December 2014 and it includes 37references.

Distribution and habitatEuphorbia hirtaisdistributedthroughoutthehotterpartsofIndiaandAustralia,frequentlyfoundinwasteplacesalongtheroadsides. 3


Plant DescriptionE. hirtaLinn.Syn-E. piluliferaLinn.Chamaesyce pilulifera Linn[4].Family:Euphorbiaceae VernacularNames Bengal-Barokhervi English-bearingspurge,asthmaherb,snakeweed Gujarat-Dudeli Hindi-DudhiNepali-Dudhia Indonesia-DaunbijikacangMalayalam-Nelapalai Malaysia-Ambinjanyan Marathi-Dudnali,govardhan Orissa-JhotikhuntiaSanskrit Tamil-Amumpatchaiyarissi Telagu-Reddinanabrolu

Morphology

Scientific classificationKingdom -Plantae

Angiosperms eudicots

RosidsOrder -MalpighialesFamily -EuphorbiaceaeGenus -EuphorbiaSpecies -E. hirtaBinomialname -Euphorbia hirta L.Synonyms -Chamaescyehirta(L.) Millsp

Itisaslender-stemmed,annualhairyplantwithmanybranchesfromthebasetotop,spreadingup

to40cminheight,reddishorpurplishincolor.Leaves are opposite, elliptic-oblong to oblong-lanceolate,acuteorsub-acute,darkgreenabove;palebeneath,1-2.5cmlong,blotchedwithpurpleinthemiddle,andtoothedattheedge.Thefruitsare yellow, three-celled, hairy, keeled capsules,1-2 mm in diameter, containing three brown,four-sided,angular,wrinkledseeds.1,2

Therapeutic applicationsE. hirtaisusedinthetreatmentofgastrointestinaldisorders (e.g. diarrhea, dysentery, intestinalparasitosis), bronchial and respiratory diseases(e.g. asthma, bronchitis, hay fever), and inconjunctivitis.HypotensiveandtonicpropertiesarealsoreportedinE. hirta.Theaqueousextractexhibits anxiolytic, analgesic, antipyretic, andanti-inflammatoryactivities.Thestemsapisusedinthetreatmentofeyelidstyesandaleafpoulticeisusedonswellingandboils.5

Extracts of E. hirta have been found to showanticanceractivity.TheaqueousextractoftheherbstronglyreducedthereleaseofprostaglandinsI2,e2, and D2.

5 The aqueous extract also inhibitsaflatoxin contamination in rice, wheat, maize,andmustardcrops.6Methanolicextractofleaveshave antifungal and antibacterial activities.The leaves poundedwith turmeric and coconutoil arewarmed and rubbed on itchy soles.Thelatex of E. hirta is applied on lower eyelids,likesurma tocureeyesores.Therootexudatesexhibitnematicidal activity against juvenilesofmeloidogyneincognita.7

Decoction of dry herbs is used for skin diseases.Decoctionoffreshherbsisusedasgargleforthetreatmentofthrush.Rootdecoctionisalsobeneficialfornursingmothersdeficientinmilk.Rootsarealsousedforsnakebites.1ThepolyphenolicextractofE. hirtahasantiamoebicandantispasmodicactivities.8 Quercitrin, a flavanoid glycoside, isolated fromthe herb showed an antidiarrheal activity.9 it is reportedtohavearelaxationeffectonrespiration.The alcoholic extract of whole plant showshypoglycemic activity in rats.5 It has a sedativeeffectonthegenito-urinarytract. 3

PHArMACoLogICAL ACTIVITIESHerbalmedicineshavebecomean integral partof standardhealthcare, basedon a combinationof time honored usage and outgoing scientific

AnsariAkhtarAlametal.


research.Burgeoninginterestinmedicinalherbshasincreasedscientificscrutinyoftheirtherapeuticpotentialandsafety.Numberof researchershasmade attempts to provide scientific backing tothetraditionalclaimsoftheplants.EHhasbeenfound to have tremendous pharmacologicalpotential. Researchers have tested this plantfor antibacterial, anti-inflammatory, analgesic,anti-pyretic, antihistaminic, anti-diabetic, anti-anemic, immuno-bioactivities and antioxidantactivitiesetc.

Antibacterial activityE. hirtaextractedusingthechloroform,methanol,acetone, and ethanol are used in saponificationprocedure. The efficacy of the extracts on theuropathogensweretestedbyagardiscdiffusionmethod in order to analyse the inhibitoryactivityofplantextracton themicroorganisms.Euphorbia hirta Linn. exhibitedhigh inhibitoryactivityagainstmostofthe11testedpathogens.Among the tested organisms, Pseudomonsas aeruginosa and Staphylococcus epidermidis were the most susceptible, and Serratia marcescens,Enterobacter cloaceae,Citrobacter koseri, andCitrobacter freundii were the leastinhibitedbymost of the extracts ofE. hirta. it isconcludedthatrevisedantibioticpoliciesandmore importantly the development of herbalmedicinesasanalternativemaybeincorporatedinurologicalpractice.10

Anti diabetic activityOral administration of E. hirta leaves extract(300mg/kgb.w./rat/day)foraperiodof30daysindicated the antidiabetic nature of the leavesextract.Onthebasisofdeterminationofthelipidperoxides, hydroperoxides, and both enzymaticand non-enzymatic antioxidants evidenced theantioxidantpotentialoftheleavesextract.11

Nephroprotective activityThe nephroprotective activity of the ethanolextract of E. hirta (400 mg/kg body weight)was studied in nitrobenzene-induced albinorats (1000 mg/kg body weight). The activitiesof antioxidant enzymes superoxide dismutase(SOD), catalase (CAT), glutathione peroxidase(GPx), glutathione-S-transferase (GST), andthe levels of reduced glutathione (GSH), totalthiolsandvitamin-C in thekidney tissuesweredetermined. Histopathologic investigation was

performed in the kidney tissue samples. TheresultsindicatethattheethanolextractofE. hirta amelioratesrenaldysfunctionandcouldbeusedas an effective protector against nitrobenzene-induced nephrotoxicity, primarily through itsantioxidantcapacity.12

Enzymes inhibition activityMethanolicextractswereusedfortheirGlutathione-s-transferase(GST),Acetylcholinesterase(AChE),Carboxylesterase (CES) and Xanthine Oxidase(XO)inhibitoryactivitiesatconcentrationof100µg/mL.13

Antioxidant activityThe leaves extract exhibited a maximumDPPH scavenging activity of (72.96±0.78) %followedbytheflowers,rootsandstemswhosescavenging activities were (52.45±0.66)%,(48.59±0.97)%,and(44.42±0.94)%respectively.The standard butylated hydroxytoluene (BHT)was (75.13±0.75) %. The IC (50) for leaves,flowers,roots,stemsandBHTwere0.803,0.972,0.989,1.358and0.794mg/mLrespectively.14

Immunomodulatory ActivityMethanolic extract of E. hirta showsimmunomodulatory activity, which has beenproved using simple techniques like themacrophage activity testing, carbon clearancetestandmastcelldegranulationassay.15

Molluscicides activityEuphorbia hirta Linnlatexpowderwereevaluatedagainst the freshwater snails Lymnaea (Radix)acuminata and Indoplanorbis exustus in pond.Thesecombinationsshowedsignificant timeanddosedependenteffectagainstboththesnails.16

Anti cytotoxicity activityThe alcoholic extract of E. hirta showsprotective effect of against antituberculardrug-induced cytotoxicity in freshly isolatedhepatocytes. It normalized the levels of aspartate aminotransferase (AST), alanineaminotransferase (ALT), alkaline phosphatase(ALP), lactate dehydrogenase (LDH),triacylglycerol(TAG),cholesterol,totalprotein,albumin, total and direct bilirubin, whichwerealteredduetoanti-tuberculardrugintoxication.17

Antidote activityFish poisoning (CFP) is an illness caused byeating tropical coral fish contaminated with

PharmacologicalandChemicalDocumentationofEuphorbia


ciguatoxins (CTXs). The clinical managementof patients with CFP is generally supportiveCiguatera and symptomatic in nature as noantidoteexists,methanolicextractof E. hirtahasshown protective effect against Ciguatera fishpoisoning.18

Antifungal activityMethanolic extracts ofEuphorbia hirta leaves,flowers,stemsandrootswereevaluatedagainstthe yeast using the agar disc diffusionmethod;one yeast (Candida albicans) was screened.Inhibitionzonesranged16-29mm.Leavesextractinhibitedthegrowthofyeastwithlargezonesofinhibition,followedbythatofflowersindicatingitsfungicidalactivity.19

Anthelmintic activityEthanolicextractsofE. hirta were assessed for theirinvitroanthelminticactivitybyusingthebovinefilarialparasiteOnchocerca ochengi andthefreelivingnematodeCaenorhabditis elegans,amodelorganismforresearchonnematodeparasites.Wormswereincubatedinthepresenceofdifferentconcentrationsofextractsandinhibitoryeffectsweremonitoredatdifferenttimepoints.EthanolicextractofE. hirtaaffectedthegrowthandsurvivalofC. elegansandO. ochengisignificantly.20

Anti anxiety activityHydro-alcoholic extract ofEuphorbia hirta (EH)shows anxiolytic property in chronically stressedrats subjected to elevated plus maze (EPM) andopen field test (OFT). Treatment with EH (200mg/kgorallyforsevendays)showedmarkedanti-anxiety activity in chronic immobilization stress.Incontrast,theforcedswimstress-inducedanxietywasonlypartiallydecreasedby EH.Co-treatmentof ratswithflumazenil at thedoseof 0.5mg/kg,Intraperitonally(i.p),bicuculline (1mg/kg, i.p.)orpicrotoxin (1mg/kg, i.p.) resulted ina significantreduction of anxiolytic effect of EH indicatingthat its actions are mediated through GABA(A)receptor-benzodiazepine receptor-Cl (-) channelcomplex.21

Anti inflammatory activityThe ethanolic extract of Euphorbia hirta L. (EH) shows anti-inflammatory action onlipopolysaccharide(LPS)inducedinflammation.The ethanolic extract of Euphorbia hirta L. (EH) and its active componentwere studied in

lipopolysaccharide (LPS)-activatedmacrophagecells(RAW264.7)asanestablishedinflammationmodel. After activation, nitric oxide (NO)production and expression of iNOS proteinand iNOS mRNA were measured by using acolorimetric assay (Griess reagent), westernblotting, and reverse transcription polymerasechain reaction (RT-PCR), respectively. ThealterationinthecontentofPGE(2),TNF-alpha,andIL-6wasconcurrentlymonitoredbyELISA.In results, we found that in the concentrationrangewithoutshowingcytotoxicity, EHproduceda remarkable anti-inflammatory effect via itsactive component of beta-amyrin and showeda dose-related inhibition of LPS-induced NOproduction.22

Antimutagenic activityAqueous and methanolic extracts of Euphorbia hirta shows anti mutagenic activity in theAmestest utilising the mutant Salmonella typhimurium TA98 and TA100 strains. Quercetin (25 microg/mL) was found to be strongly mutagenic inSalmonella typhimuriumTA98intheabsenceandpresence of S-9 metabolic activation. However,both the aqueous and methanol extracts did notdemonstrateanymutagenicpropertieswhentestedwith Salmonella typhimurium TA98 and TA100strainsatconcentrationsupto100microg/mLintheabsenceandpresenceofS-9metabolicactivation.23

Antiviral activityAqouesextractofE.hirtashowsantiviralactivitydirect effects of the aqueous extract onHIV-1,HIV-2 and SIV (mac251) reverse transcriptase(RT)activityweredetermined.Adose-dependentinhibition of RT activity was observed for allthreeviruses.24

Anti arthritics activityWater extracts of Euphorbia hirta at lowdoses shows beneficial in reducing cartilagedegenerationincasesofarthritis.25

Larvicidal activityEthylacetate,butanol,andpetroleumetherextractsEuphorbia hirta,weretestedagainsttheearlyfourthinstarslarvaeofAedes aegypti L.andCulex quinquefasciatus(Say).Thelarvalmortalitywasobservedafter24hofexposure.However,thehighestlarvalmortalitywasfoundinpetroleumetherextractagainstA. aegyptiandagainstC. quinquefasciatusofthe



variousratiostested.Thisisanidealecofriendlyapproachforthecontrolofthedenguevector,A. aegyptiandthelymphaticfilariasisvector,C. quinquefasciatus.26

Anti Helicobacter pylori activityMethanol plant extracts Euphorbia hirta tested demonstrated anti-Helicobacter pylori activitybyantimicrobialactivitywithzonediametersofinhibition ranging from 0-30mm.These extractshows very potent antibacterial activity on theisolatesH. pylori.27

Anti-anaphylactic activityEthanolicextract (EHA001)ofEuphorbia hirta wasfoundtopossessaprominentanti-anaphylacticactivity.A preventive effect of EH-A001 givenby oral route at dose from 100 to 1000 mg/kgwas observed against compound 48/80-inducedsystemicanaphylaxis.Atthesamerangeofdose,EH-A001inhibitedpassivecutaneousanaphylaxis(PCA)inratandactivepawanaphylaxisinmice.AsuppressiveeffectofEH-A001wasobservedonthereleaseofTNF-alphaandIL-6fromanti-DNP-HSAactivatedratperitonealmastcells.28

Anti microbial activityThe ethanolic extracts of the, aerial parts ofEuphorbia hirta were tested for antimicrobialactivity. This plant exhibited a broad spectrumof antimicrobial activity, particularly againstEscherichia coli(uropathogen),Proteus vulgaris,Pseudomonas aeruginosa and Staphylococcus aureus.29

Anti diarrheal activityThe aqueous leaf extract of Euphorbia hirta decreasedthegastrointestinalmotilityinnormalratsanddecreasedtheeffectofcastoroil-induceddiarrheainmice. 30

Anti malarial activityEthanolic extractsEuphorbia hirtawhole plantshowed anti-plasmodial activity which maybe related to thepresenceof terpenes, steroids,coumarins, flavonoids, phenolic acids, lignans,xanthonesandanthraquinones.EtOHandCH2Cl2 E. hirtaused inCongolese traditionalmedicinefor the treatment of malaria were submittedto a pharmacological test in order to evaluatetheir effect onP. falciparum growth invitrooftheseplantspecies,14(70%)extractsincludingEtOHandCH2Cl2 from Euphorbia hirtawhole

plantproducedmorethan60%inhibitionoftheparasite growth in vitro at a test concentrationof 6microg/ml.Extracts from E. hirta showedasignificantchemosuppressionofparasitaemiainmice infectedwithP. bergheiatorallygivendosesof100-400mg/kgperday. 30

Antifertility activityThe aqueous crude extracts of E. hirta were administered to thirty eight-week old sexuallymaturemale albino to determine the effects oftheseextractsonthemalereproductiveorgansoftheseanimals.TheresultsfromthisstudyrevealedthattheaqueouscrudeextractsofE. hirta caused varyingdegreesoftesticulardegenerationaswellas reduction in the mean somniferous tubulardiameter(STD)inthetreatedrats.Thus,itshowsthattheaqueouscrudeextractsofE. hirtahavepotentially deleterious effects on the testes andaccessoryorgansofrat’scause’sinfertility. 3

Anti amoebic activityThree major extracts from some traditionalpreparations, based on medicinal plants, used asantidiarrhoeal agents were investigated for theirputativeantiamoebicandspasmolyticactivitiesinvitro.Resultsindicatedthatbothbiologicalactivitiesareconcentratedinthepolyphenolicfraction,andnotinthesaponinoralkaloidcontainingfractions.ThemostactivepolyphenolicextractswerethosefromEuphorbia hirtawholeplantinhibitingEntamoeba histolytica growth withMAC < 10 micrograms/ml. The same extracts, at a concentration of 80micrograms/ml in an organ bath, also exhibitedmore than 70% inhibition of acetylcholine and/or KCl solution-induced contractions on isolatedguinea-pigileum. 7

Diuretics activityThewaterandethanolicextracts(50and100mg/kg)oftheplantproducedtime-dependentincreasein urine output. The water extract increased theurineexcretionofNa+,K+andHCO3

-.Incontrast,theethanolextractincreasedtheexcretionofHCO3

- decreased the loss of K+ and had little effect onrenalremovalofNa+.Acetazolamide,likethewaterextract, increased urine output and enhanced theexcretionofNa+,K+andHCO3

-.Thehigh-ceilingdiuretic,furosemide,increasedtherenalexcretionofNa+andCl-;buthadnoeffectonK+andHCO3

-loss.Thisstudysuggeststhattheactivecomponent(s)inthewaterextractofE. hirtaleafhadsimilardiureticspectrumtothatofacetazolamide.32



Analgesic and antipyretic activityLyophilized aqueous extract of Euphorbia hirta L. from thedosesof20,25mg/kg showsanalgesicactionagainstchemical(writhingtest)andthermic(hotplatetest)stimuliandantipyreticaction andantipyreticactivitywasobtainedatthesedativedosesof100and400mg/kg,ontheyeast-inducedhyperthermiainratandmice.2

Antiasthmatic activityE. hirta is reported to have an anti-asthmaticactivity due to the relaxation effect on thebronchial tubes and a depressant action onrespiration. 33

galactogenic activityThe powdered E. hirta showed a galactogenicactivity in guinea pigs before puberty byincreasing the secondary sexual organ andinductionofmilksecretion. 34

repellent and antifeedant activityTheethanoiclextractsofEuphorbia hirtapresentthe repellent and anti-feedant effect. The anti-feedantratesofdiamondbackmoth(DBM)Plutellaxylostellalarvaewereallmorethan80%.35

Immunostimulant activityThe present study was undertaken to improvethe immune power of Cyprinus carpio byusing Euphorbia hirta plant leaf extract usedas immunostimulants. The haematological,immunological and enzymatic studies wereconducted on the medicated fish infected withAeromonas hydrophila pathogen. The resultsobtained from the haematological studies showthattheRBCcount,WBCcountandhaemoglobincontent were increased in the infected fish athigher concentration of leaf extract. The feedswith leaf extract ofEuphorbia hirta were ableto stimulate the specific immune response byincreasingthetitrevalueofantibody.Itwasabletostimulate theantibodyproductiononlyup tothe5thday,whenfedwithhigherconcentrationsof(25gand50g)plantleafextract.36

Anticancer activityAnotherstudyreportedthechemicalcomposition,antioxidant, anti-inflammatory and anticanceractivities of Euphorbia hirta L. extract. Theantioxidant activities ofwholeE. hirta ethanolextract were determined by electron spin

resonance spectrophotometric analysis of1 ,1-diphenyl-2-picryl-hydrazyl(DPPH),hydroxyl,andalkylradicallevelsandbyusinganonlinehigh-performanceliquidchromatography(HPLC)-2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) assay. The E. hirta ethanolextract(0.5mg/mL)exhibitedDPPH-scavengingactivity of 61.19%±0.22%,while the positivecontrol (0.5mg/mLascorbic acid)had100%±0.22%activity.Theconcentrationof theextractrequiredtotrap50%ofDPPH(IC50)was0.205mg/mL. Online HPLC analysis of the extractalsoshowedstrongantioxidantactivity.Theanti-inflammatoryactivityoftheE. hirtaextractwasassessed in lipopolysaccharide-induced RAW264.7 macrophages. The anti-inflammatoryactivitywashighest in thepresenceof200µg/ml E. hirtaextract,andnitricoxideproductionwas decreased significantly (p < 0.05). Theextractalsoshowedselectiveanticanceractivityat a concentration of 100 µg/mL (p < 0.05).TheseresultsindicatedthatE. hirtamaywarrantfurther investigation for the development ofantioxidant, anti-inflammatory, and anticancerherbalmedications.37

PHyToCHEMISTryE. hirtahasbeenstudiedbyvariousworkersandanumberofactiveconstituentshavebeenisolated.Afzelin, quercitrin and myricitrin have beenisolatedfromthemethanolicextractofE. hirta.40 The chemical investigation ofE. hirta has ledtotheisolationofrutin,quercitrineuphorbin-A,euphorbin-B, euphorbin-C, euphorbin-D , 2, 4,6-tri-O-galloyl-β-d-glucose, 1, 3, 4, 6-tetra-O-galloyl-β-d-glucose,kaempferol,gallicacid,andprotocatechuic acid.13,14 E. hirta also containsβ-amyrin,24-methylenecycloartenol,β-sitosterol,heptacosane,1 shikmic acid, tinyatoxin, choline,camphol, and quercitol derivatives containingrhamnoseandchtolphenolicacid. 4

CoNCLuSIoNReview of the literature reveals that differentpartsof theplantEHhavevarietyofmedicinalapplicationswhichattracttheattentionofmanyscientiststoscreenthisplantonthescientificbasistodiscloseitasapotentmedicinalagent.Severalpharmacological studies have been carried outwith extract of the plant. Since the compounds



fromthenaturalsourcesaresafeforuse,theneedistoisolatepotentchemicalconstituentsfromtheplantandtocarryoutpharmacologicalscreeninginsearchofsafeandusefuldrug.

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33. Pretorius E, Ekpo OE, Smit E. Comparativeultrastructural analyses of platelets and fibrin

networks using themurinemodel of asthma. ExpToxicolPathol,2007,59(2),105-14.

34. Blanc P, Bertrand P, de Saqui-Sannes G, LescureR.GalactogenicpropertiesofplantsoftheAfricanflora: Sersalisia djalonensis and Euphorbia hirta. AnnBiolClin(Paris),1963,21:829.

35. H.Wei,Y.Hou,G.Yang,J.Fu,M.You.Repellentandantifeedanteffectofsecondarymetabolitesofnon-host plants on Plutella xylostella. YingYongShengTaiXueBao,2004,15(3):473-476.

36. Pratheepa V, Sukumaran N. Effect of Euphorbia hirtaplantleafextractonimmunostimulantresponseof Aeromonas hydrophilainfectedCyprinus carpio. PeerJ,2014,13:671

37. Sharma N, Samarakoon KW, Gyawali R, Park YH, Lee SJ, Oh SJ, et al. Evaluation of theantioxidant, anti-inflammatory, and anticanceractivities of Euphorbia hirta ethanolic extract.Molecules, 2014,19(9):14567-81

Cite this article as: AnsariAA,KhatunT,AhmadMP,GuptaRS,MadhikarmiNL.Areviewonpharmacologicalandchemical documentationofEuphorbia hirtaLinn. (Asthmaherb).MEDPHOENIX2016;1(1)48-57:AReview.MEDPHOENIX2016;1(1):31-38


Bhatt Pashupati NDepartmentofSurgery,NationalMedicalCollege,Birgunj

Acute Intestinal obstruction Due to AscarisCASE rEPorT

ABSTrACTAscaris lumbricoidsisthemostcommonhelminthesintropicalareasofdevelopingcountriesaroundtheworld.IntestinalobstructionduetoA. lumbricoids is awellknownandseriouscomplicationinchildren.We report a case of six yrs female child withintestinal obstruction,whounderwent laparotomyandenterotomytoremovethewormmasses.Earlydiagnosisandsurgerypreventedcomplicationsandsavedthelifeofpatient.

Keywords: Acute, Ascaris Lumbricoides,helminths,Intestinalobstruction

Corresponding Author: Dr. Pashupati NBhatt,DepartmentofSurgery,NationalMedicalCollege, Birgunj; E-mail: [email protected]

INTroDuCTIoNIntestinal obstruction is the most commoncomplication ofAscaris lumbricoids. The largestproportionofascariasisisfoundinschoolchildrenwhereas,ininfantandpre-schoolage,complicationsarefrequentlyseen.1Thehighestprevalenceisseenin tropical and sub-tropical areas.2Thosepatientswho report with ascaridial obstruction are veryalarming and challengeable for the surgeons dueto the fear of strangulation, intestinal gangreneandtheirmigratoryhabitandtendencytoexploreorifices and ducts leading to a variety of otherdangerouscomplicationsandpoorprognosis.

CASE DETAILSA six years old girl presented to emergencydepartment of National Medical CollegeTeachingHospital,Birgunjwithabdominalpainand not passing stool and flatus for two days.She had three episodes of vomiting and had

expelledascaristhroughmouththreedaysback. De-wormingwasdoneninedaysback.

Her general condition was fair with milddehydrationandpulserateof96/min,respirationrate 18/min. She was afebrile. Abdomen wassoft,distended,non-tenderwithintra-abdominaltubularmassextendingfromlefthypochondriumto right iliac fossa. The mass had ‘crepitativesound’ on deep palpation. Bowel sound washighpitched.Othersystemicexaminationswerenormal.Laboratoryparameterswerenormalwithhaematocrit 38%, leukocyte count 12000 cmm,Na+ 136 mmol and K+ 4.2 mmol. Distendedbowel loops were seen on abdomen x-ray.Ultrsonography (USG) of abdomen revealedmultipletubularloopsinsmallbowel.

Patientunderwentexploratorylaparotomy.Surgeryrevealeddistendedsegmentofileumapproximately25 cm, congested and densely packedwith adultascaris(Figure1).Proximalanddistalsegmentofileumwas collapsed. Enterotomywas performedtoremovetheknottedbowelofascaris(Figure2).Patienthaduneventfulrecoveryandwasdischargedon7thpostoperativeday.

figure 1: Smallbowelobstructedbyascariswithcollapsedproximalanddistalbowel


figure 2: Extracted adult live worms afterenterotomy

DISCuSSIoNIndevelopingcountrieslikeNepal,thereisaproblemofcleanportable,pipedwatersupplyandpeopleuseriver or lakes for washing, cleaning, ablution andevendrinking.Manypeopledonothaveaccesstotoilets,anduse‘openfield’whichcontaminatesfoodchainpredisposingtoinfestationwithhelminthes.

Ascariasisismorecommoninpediatricsagegroup(2-10years), in tropical and subtropical areas. 3,4 Approximatelytwomillionpeoplegetinfestedand20,000deathsoccureveryyearinendemicarea.2

Clinicalillnessdependsonparasiticloadandpatientsmaybeasymptomaticorpresentwithmalnutrition,chronicabdomenpain,nausea,vomiting,abdomendistension and acute intestinal obstruction.Manypatients have history of expulsion of ascaris perrectumormouth.2Thefactorthatleadstointestinalobstructionsaremassivenumberofworms,interlaceandcoiledupbehavior,secretionofneurotoxinandperistalticcontraction.5

Diagnosisofroundwormobstructionissuspectedonhistoryandsymptomsofintestinalobstruction,whichissupportedbyabdomenx-rayshowingairfluidlevelsandmultiplelinearimagesofAscaris lumbricoides indilated intestinal loops.USGisdiagnostic with hyperechoicmass of coiled upwormswithorwithoutacousticshadow.6

Majorityofthepatientswithoutsignandsymptomsofperitonitiscanbetreatedeffectivelybyconservativemanagement,withnilbymouth, intravenousfluid,nasogastrictubeaspiration,rectalenemasofglycerinplusliquidparaffinenemaandpiperazinesaltthroughnasogastrictubetogetherwithcloseclinicalandwithserialabdomenx-raymonitoring.2

Surgical procedures performed in intestinalobstruction causedbyAscaris lumbricoides are manual advancement, enterotomy, intestinalresection,appendectomy.1,8

CoNCLuSIoNRoundworminfestationiscommonindevelopingcountries,andmostoftheintestinalobstructioncausedbyitcanbemanagedconservatively,butsurgeryisnecessaryinselectedcases.

As prevention is most important aspect of thisdisease, the author would like to suggest someimportantrecommendationstohighlighttheissuesofpopulationsanitaryhygieniceducation(mainlytheriskgroups),Implementationofanthelminthicmedicationprogramandtoempowertheauthoritiesandleadersabouttheintestinalparasitism.

rEfErENCES1. OchoaB.Surgicalcomplicationsofascariasis.WorldJ

Surg.1991;15(2):222-7.2. Upadhyaya VD, Gangopadhyaya AN, Pandey A,

Gupta DK, Upadhyay A. Round worm intestinalobstruction:asinglecenterstudy.Theinternetjournalof surgery [Internet]. 2006 [cited2016 Jun6];12(2).Availablefrom:https://ispub.com/IJS/12/2/3965

3. Mir IA, Wani NA,AhangerAG, Shah OJ, SaleemK, Patnaik R. Surgical ascariasis in children. JK-Practioner.2003;10(1):17-21.

4. Mishra PK, AgrawalA, Joshi M, Sanghvi B, ShahH, ParelkarSV.Intestinalobstructioninchildrenduetoascariasis:Atertiaryhealthcentreexperience.AfrJPaediatrSurg. 2008;5(2):65-70.

5. GilJG,EsturoCL,AyalaRP. Intestinalobstructiondueto ascaris.TheInternetJournalofSurgery[Internet].2005[cited2016Jun6];8(2).Availablefrom:https://ispub.com/IJS/8/2/9955

6. MirIA,WaniNA,AhangarAG,PathnaikR,SaleemK.Sonographicappearanceinintestinalascariasis.JK-Practitioner.2002;9(4):234-5.

7. Hussain Z, Sheikh KA, Lone R,Arif S, RasoolA,MudassirS,etal.Smallbowelobstructioninchildren-Asurgicalchallenge.JK-Practitioner.2006;13(4):186-9.

8. WardhanH,GangopadhyayaAN,GopalSC,SinghalGD.Ascarislumbricoidscausingintestinalobstructionin children: a review of 33 cases. Paedtr surg int.1989;4(2):88-9.

BhattPashupatiN

Cite this article as:BhattPN.AcuteintestinalobstructionduetoAscaris.MEDPHOENIX2016;1(1):39-40


gupta ravi Shankar1*, Khatun Tarannum2, Ansari Akhtar Alam3, Shidiki Amrullah1, Bhargava Dipak1, gupta Bidhya4, Majumder Anirban5

1 DepartmentofMicrobiology,NationalMedicalCollege,Birgunj,Nepal2DepartmentofObstetrics&Gynecology,NationalMedicalCollege,Birgunj,Nepal3 DepartmentofPharmacology,NationalMedicalCollege,Birgunj,Nepal4 NationalMedicalCollegeNursingCampus,Birgunj,Nepal5 DepartmentofPhysiology,NationalMedicalCollege,Birgunj,Nepal

Problems and Solution to Diagnose Extrapulmonary Tuberculosis in Central region of Nepal

CASE rEPorT

ABSTrACT Extrapulmonary Tuberculosis is high,challenging the clinicians to make correctdiagnosis. Microscopy, culture and fine needleaspiration cytology have their limitations inregardtospecificityandsensitivity.Inthisreport,polymerasechainreactionisusedfordetectinganddistinguishing Extrapulmonary Tuberculosis. Acaseofretropharyngealabscesswasselectedfromwhichpuswascollectedwhichwasnegativeformicroscopyandcultureinroutinemicrobiologyas well as mycobacteriology. Cytopathologicalexaminationwasalsonegative.Polymerasechainreaction was applied to detect Mycobacterium tuberculosis specific IS6110 gene.The patientsrespondedwithanti-tuberculosistreatmentwell.Polymerase chain reaction was introduced fordiagnosisofExtrapulmonaryTuberculosissinceitcanbedonewithinhours,monitortherapyandalso differentiate Mycobacterium tuberculosis fromotherMycobacterialspecies.

Keywords:Extrapulmonary,Nepal,Polymerasechainreaction,Tuberculosis

Corresponding Author: Dr. Ravi ShankarGupta, Department of Microbiology, NationalMedicalCollege,Birgunj,Nepal;E-mail: [email protected]

INTroDuCTIoNTuberculosis(TB)isamajorcauseofmorbidityandmortalityglobally,withmostcasesoccurringin developing countries.1 In Nepal, about 45

percent of the total population is infectedwithtuberculosis, and 60 percent are in the adultgroup.Everyyear40,000peopledevelopactivetuberculosis, 20000 peoples result in infectiouspulmonary disease and 5000-7000 people dieeveryyear.2Tuberculosisaffectvarioussystemsintheinfectedbody.3

ExtrapulmonaryTuberculosis (EPTB)hasnotyet been ascertained in developing countriesbecause of the difficulty in conformationby clinical and laboratory diagnosis.4 Thediagnosis ofEPTBposes a special challenge,as it is often missed or misdiagnosed due toits atypical presentations.Thefirst step in itsdiagnosis is awareness and a high index ofsuspicionby thephysician.5 it isalsodifficultto isolate Mycobacterium tuberculosis due to the small number of organisms present atthesesites.4 Variousinternationalstudiesintheworldhavefocusedon theproblemofEPTB,reportingahighfrequency.4,5

Adiagnosisof tuberculosis isconfirmedby thepresenceofAcidFastBacilli(AFB)andisolationof M. tuberculosis onculture.Thepaucibacillarynatureof the specimens, the sensitivityofAFBsmear and cultural growth, only mycobacteriagrow in 39 to 80% of cases.6 conventionalmethodsandcytologicalinvestigationsareusedin combinationwith polymerase chain reaction(PCR)techniqueindetectionandcharacterizationof pathogenic mycobacteria associated withhumanlymphadenitis.7


The study evaluates the importance of PCRin combination with histo-cytopathologicalexamination in the diagnosis of tuberculousretropharyngealabscessincaseofextrapulmonarytuberculosis.

CASE DETAILSA17yearsoldmalewasadmittedinENTwardwith thecomplaintsof (i)a rapidlyprogressiveswellingoftheneckbelowthelowerjawinrightside,with difficulty in breathing and dysphasiafor solid food foraboutoneweek,and (ii) lowgrade irregular fever for about 10 days. Therewas no history of pain in neck, impaction offoreignbodies,infectioninear,dentalextraction,endoscopy or any other invasive procedures,bloodtransfusion,sexualexposure.Hehadapasthistory of tuberculosis three years back whichwasdiagnosedbyFNACforwhichhehadtakenAnti-tuberculardrugsforsixmonths.

On examination, the patient was mild anemic.Therewasnopedaledema,icterus,cyanosis,norclubbingoffingers.Pulseratewasregularwith80/min,BP130/70mmHg.Therewasapalpablemass of 7cm x 2cm in size projecting beneaththe anterior border of sternomastoid deep tothemuscleon therightside. Itwas irregular inoutline, cystic in consistency and non-tender.Cervical spine was devoid of bony tenderness.Similarly, there were no lymphadenopathy,splenomegalyandhepatomegaly.Examinationofcardiovascular, respiratoryandnervoussystemsdidnotshowanyabnormality.

Investigations revealed: Hb 9.5 g%, totalleucocytes10,600/cmm,DLC-N45,L55,E01andM01.Plateletcount258000/cmm,ESR19/1st hr,bloodsugar(R)70mg%,sodium129mmol/L,potassium3.6mmol/L,prothrombintime18sec,blood group-AB positive, total calcium 7.8mg/dL,urea19mg%andcreatinine0.7mg%.

FNAC repot was negative for tuberculous. ChestX-rayshowednoevidenceofpulmonarytuberculosis.CT scan of neck showed a huge retropharyngealabscesspushingthetracheaandesophagustotheleft.Findings by indirect laryngoscopywere -Fullnessof right pyriform fossa, larynx normal and rightpharyngealwallswollen.

Aspiration of pus was done from the bulgingsiteof the lesion.Pus from the retropharyngealabscess was negative for bacteriologicalculture,negative forAFBandpositivebyPCR for AFB (eppendorf, Germany) (Figure 1 and2), was to detect Mycobacterium tuberculosis specificIS6110geneincaseasExtrapulmonarytuberculosis. The patient was treated with fourantituberculosis drugs (e.g. HRZE) and wasdischarged.

figure 1: PCr machine

Figure 2: Bands showing Mycobacterium tuberculosis

DISCuSSIoNDefinitiveandrapiddiagnosisofextrapulmonarytuberculosisisverydifficultduetosmallsamplesizeandamounts;theapportioningofthesamplefor various diagnostic tests (histology/cytology,biochemical analysis, and microbiology),resulting in non-uniform distribution ofmicroorganisms; paucibacillary nature of thespecimens; and the lack of an efficient sampleprocessing technique universally applicable onalltypesofextrapulmonarysamples.5

Reteropharyngeal abscess where conventionaldiagnosis fails and where the provisional

NegativeControlBand

PositiveControlband

Patient’sDNAband

GuptaRaviShankaretal.


diagnosis of tuberculosis is made on the basisof clinical presentation and histology/cytologyexaminationwithoutevidenceofAFB.6

PCR was thus introduced for diagnosis EPTBsince it can be done within hours, monitortherapy and also differentiate Mycobacterium tuberculosisfromotherMycobacterialspecies.

CoNCLuSIoNThe application of this technology couldattain a particular relevance in developingcountries such as Nepal, where there is aburden of Extrapulmonary diseases. However,establishmentofthesetupisexpensive.

rEfErENCES1. MurrayCJ,StybloK,RouillonA.Tuberculosisinthe

developing countries: burden, intervention and cost.BullIntUnionTubercLungDis.1990;65(1):6-24.

2. AnnualreportofTuberculosiscontrolprogramNepal2002/03.BhaktapurNepal:Ministryofhealth,Nepal;2003.

3. MaherD,ChauletP,SpinaciS,HarriesA.Treatmentof tuberculosis: guidelines for national programs.Geneva:WHO;1997.

4. FanningA.Tuberculosis:6.Extrapulmonarydisease.CMAJ.1999;160(11):1597-603.

5. GermaF,FitzGeraldJM.Extrapulmonarytuberculosis:twocasereports.BCMedicalJournal.2002;44(1):27-9.

6. Prakash UB, Reiman HM. Comparison of needlebiopsywith cytologic analysis for the evaluation ofpleural effusion: analysis of 414 cases. Mayo ClinProc.1985;60(3):158-64.

7. Kidane D, Olobo JO, Habte A, Negesse Y, AseffaA, Abate G, et al. Identification of the causativeorganismoftuberculouslymphadenitisinEthiopiabyPCR.JClinMicrobiol.2002;40(11):4230-4.

Cite this article as:GuptaRS,KhatunT,AnsariAA,ShidikiA,BhargavaD. ProblemsandsolutiontodiagnoseExtrapulmonaryTuberculosisincentralregion of Nepal.MEDPHOENIX2016;1(1):41-43

ExtrapulmonaryTuberculosis


role of DNA fingerprinting in Disputed PaternityCASE rEPorT

Mishra Amarnath1* Sathyan Sukumaran2

1 DepartmentofForensicMedicine,NationalMedicalCollege,Birgunj,Nepal2DepartmentofBiologyandDNAFingerprintingUnit,CentralForensicScienceLaboratory,Hyderabad,India

ABSTrACTTheuseofDNAfingerprintinginsolvingcrimeisprovingtobeasrevolutionaryastheintroductionof fingerprint evidence in court more than acentury ago. It has emerged as one of themostpowerfultoolsavailableforsolvingmanymedicalaswellaslegalcomplexities.DNAfingerprintingplaysanimportantroleintheestablishment ofthepaternityofanindividual.Mostofcasesregardingdisputedpaternityariseinthecontextofaffiliationorders, divorce proceedings and questionedlegitimacy,mayalsobeusedtofindoutpaternityincasesofinheritance,guardianship,maintenance,legitimacy, adultery or fornication. The presentworkwasdonetosolvethepaternitydispute.

Keywords: Dispute,Forensicgenetics,Paternity

Corresponding Author:Dr.AmarnathMishra,Department of Forensic Medicine, NationalMedical College, Birgunj, Nepal; E-mail:[email protected]

INTroDuCTIoN TheuseofDNAfingerprintinginsolvingcrimeisasrevolutionaryastheintroductionoffingerprintevidence in court more than a century ago. Ithas emergedasoneof themostpowerful toolsavailable for solving many medical as well aslegalcomplexities.DNAfingerprintingisprovingtobeofgreatimportanceintheestablishment of thepaternityofanindividual.AlthoughforensicDNA analysis is commonly used to detect thecriminalactivitiessuchashomicideandrape,itcanplayvitalroleindisputedcasestoestablishthepaternityofdisputedoffspring.1,2

DNAfingerprintingisatechnique,inwhichvirtuallyunique sequence of bases in the DNA strands ofchromosomes are used to compare one biologicalsamplewithanothertoinvestigategeneticrelationship.Using these sequences of base pairs, every personcouldbeidentifiedsolelybythesequenceoftheirbasepairs.DNAfingerprintingtestsanyDNAcontainingbiological traceevidence.ThecompositionofDNAmoleculedoesnotvaryfromcelltocellthus;DNAinbloodisidenticaltothatinotherbiologicalmaterialsuchashair,semen,skin,andbonemarrow.3

In IndiaandNepal,DNAfingerprintinghasbeenaddedtotheroutineworkofdisputedpaternitycasesasareliabletoolofinvestigationinForensiccases.Theoldconventionalinvestigationbasedonbloodantigen systems like variable bloodgroups,HLAtissue typingwasnomoreused in such sensitivecases because of the limitationor invariability oflocianalyzed.4

Paternity, the state of being a father, canbe legallyestablished in severalways.When the parents of achildaremarried,paternity iscommonlypresumed.Todeterminewhetheramanisthefatherofachildbornoutofwedlock,alawsuitknownasa“paternityaction”mustbebrought.Insuchasuit,paternitymaybeestablishediftheallegedfatheradmitspaternity.5

Blood-groupstudies,whichcommonlyemploytheABO system, cannot establish paternity but canconclusivelyexcludeanallegedfatherfrombeingacandidate.Thisisthecasebecauseachildmustinheritbloodtypefromthemotherandfather;thus,ifthechild’sbloodtypediffersfromboth:themother


andtheallegedfather,themancouldnotpossiblybeaparentofson.Atypicalpopulationfrequencyforconventionalbloodtypingmightbeonein200,forDNAonein5,000,000.Thismeansthatonlyonein5,000,000peoplewouldhavethesameDNAprofile.AdequatesamplesforDNAtypingcanbecollectedfromblood,bloodstain&oral swabeasily.DNAtypingcomparesstrandsofgeneticmaterialbetweenthechildandallegedfathercomparingstrandsfromvarious locations of the genetic material allowsaccuracyratingsof99.9%.6DNAtypingallowsanallegedfathertobeexcludedwith100%certainty.7 Theaimofpresentworkistoperformananalysisofevidencetohelpthecourt inestablishingphysicalfactsofcriminalandcivildisputes.

CASE DETAILSThe study was conducted at Central ForensicScience Laboratory, Hyderabad, India afterethicalapprovalfromDirector,CFSL,Hyderabad.Samples were collected from Department ofBiology and DNA Fingerprinting Unit, CentralForensicScienceLaboratory,Hyderabadthroughlegalproceedings.

As per the court order in paternity dispute,the parents along with the child were sent tothe laboratory, for collection of blood samplesregardingthepaternitytest.Twomilliliterbloodfrom the alleged father, mother and child werecollectedandstoredforfurtheranalysistocarryoutDNAfingerprinting.

Bloodsampleoffather:ExhibitA,bloodsampleofmother:ExhibitBandbloodsampleofchild:ExhibitC.

IsolationofgoodqualityDNAisaprimarystepinDNAfingerprintingwhichwasdonebyorganicextraction method. To check the quantity ofDNA,itsquantificationwasdoneby1%agarosegel electrophoresis. Then, its amplification wascarried out by using PCR machine to increasethe quantity of the DNA.8 After amplification,denaturation of double strand was taken placefollowedbyDNAproofingwhichwasbasedonSTRs(shorttandemrepeats).AllelicdistributionofSTRlociofallexhibitsispresentedinFigure1(a)tofigure1(d),whicharerepresentedinTable1.

figure 1 (a): Allelic distribution of disputedpaternity case (STR loci: D8S1179, D21S11,D7S820andCSF1PO)

figure 1 (b): Allelic distribution of disputedpaternity case (STR loci: D3S1358, THO1,D13S317,D16S539andD2S1338)

figure 1 (c): Allelic distribution of disputedpaternitycase(STRloci:D19S433,VWA,TPOXandD18S51)

MishraAmarnathetal.


figure 1 (d): Allelic distribution of disputedpaternity case (STR loci: D5S818, FGA andaMelogeNiN.

Table 1: The results of autosomal genetic markers of CoDIS for the trios on the bases of DNA profile [from figure 1(a) to figure 1(d)]

Locus/marker father (Exh.-A)

Mother (Exh.-B)

Baby (Exh.-C)

D8S1179 10,15 14,16 10,14D21S11 28,29 29,31.2 28,31.2D7S820 11,12 11,12 11,11CSF1P0 11,11 10,12 10,11D3S1358 14,15 16,17 15,17THO1 8,9 9,9 8,9

D13S317 7,12 12,14 12,14D165539 9,12 8,10 9,10D2S1338 21,25 18,22 21,22D19S433 13,16.2 14,14.2 13,14vWA 17,18 17,18 18,18TPOX 9,10 10,11 9,10D18S51 13,15 14,16 14,15

Amelogenin X,Y X,X X,YD5S818 10,13 12,13 10,12

Fga 19,23 20,22 20,23

DISCuSSIoNLiquidbloodsample‘B’ofmotherhadoneofthealleles in the genotype profile, of the amplifiedidentifierSTRloci,aliketooneof theallelesinthe genotype profile of the liquid blood sampleof baby ‘C’.All the non-maternal alleles of theamplifiedidentifierSTRlociof the liquidbloodsampleoffather‘A’werealiketooneoftheallelesinthegenotypeprofileoftheliquidbloodsamplebaby‘C’[Figure1(a)toFigure1(d)].

Theaboveobservationrevealedthatperson‘A’isthebiologicalfatherofbaby‘C’.Theprobabilityof Exhibit ‘A’ contributing the non-maternalallelestothebabyis99.999%thananypersonatrandom(Table1).

CoNCLuSIoNDNA typing has an advantage over traditionalproteinanalysisanditismoreinformativeandcanbe analyzed inminute and/or degradedmaterialas DNA is physically much more resistant todegradationthatprotein.DNAanalysisprovidesthebestavenueforunequivocalexclusionoftheinnocent suspects. Due to all these impressiveapplications,DNAtesthasbecomethedarlingofthecriminalandciviljusticesystemglobally.

ACKNowLEDgEMENTTheauthorswouldliketothankDr.S.K.Shukla,Ex-Director;CentralForensicScienceLaboratory,Hyderabad,MinistryofHomeAffairs,GovertmentofIndiaforfinancialandtechnicalsupportatthetimeofthisstudy.

rEfErENCES1. Jeffreys AJ, Wilson V, Thein SL. Hypervariable

minisatellite regions in human DNA. Nature.1985;314(6023):67-73.

2. Jeffreys AJ, Wilson V, Thein SL. Individual-specific ‘fingerprints’ of human DNA. Nature.1985;316(6023):76-9.

3. Watson JD, Crick FH. The structure of DNA. ColdSpringHarbSympQuantBiol.1953;18:123-31.

4. VanOorschotRA,JonesMK.DNAfingerprintsfromfingerprints.Nature. 1997;387(6635):767.

5. PrimoracD,SchanfieldMS.Applificationof forensicDNA testing in the legal system. Croat Med J. 2000;41(1):32-46.

6. Lygo JE, Johnson PE, Holdaway DJ, Woodroffe S,WhitakerJP,ClaytonTM,etal.Thevalidationofshorttandemrepeat(STR)lociforuseinforensiccasework.IntJLegalMed.1994;107:77-89.

7. ButlerJM,ShenY,MccordBR.ForensicDNAtyping:biologyandtechnologybehindSTRmarkers.London:AcademicPress.2001.

8. MullisK,FaloonaF,ScharfS,SaikiR.AmplificationofDNAinvitro:thepolymerasechainreaction.ColdSpringHarbSympQuantBiol.1986;51(1):263-73.

Cite this article as:MishraA,SathyanS.RoleofDNAfingerprintingindisputedpaternity.MEDPHOENIX2016;1(1):44-46

dNaFingerprintinginDisputedPaternity


Can You Grow Grain in Jumla?

Shankar Pathiyil ravi

DepartmentofPharmacology,XavierUniversitySchoolofMedicine,Aruba,DutchCaribbean Corresponding Author: Dr.PRaviShankar,FAIMERFellowinHealthProfessionsEducation,ProfessorofPharmacologyandChairofthecurriculumCommittee,XavierUniversitySchoolofMedicine,#23,SantaHelenastraat,Oranjestad,Aruba,DutchCaribbean;E-mail: [email protected]

LETTEr To EDITor

Dear Editor,I was attending a review and disseminationsessionforethicalreviewcommittees/institutionalreviewboardsintheHimalayancountryofNepalorganizedbytheNepalHealthResearchCouncil(NHRC) in the nation’s capital, Kathmandu.Arespected clinician researcher and member oftheethicalreviewcommitteeoftheNHRCwerehighlighting the informed consent process. Hewas informing us how in developing countrieslikeNepalhealthresearchersoftenwerenotfullycognizantoftherightsofthegeneralpopulation(participants)while recruiting study participantsand obtaining informed consent. InmanyAsiancountriestheeducatedelitemayhaveanegativeand slightly condescending attitude towards thegeneral population (who may be from a lowersocioeconomic and/or caste/ethnic group). Thisattitudemaybeevidentwhenresearchersrecruitparticipants, obtain informed consent and dealwith the general community. This may also bepartlyresponsibleforethicalproblemsnotedwithclinicalandcommunitybasedresearch inNepalandtheSouthAsiaregion.

Thepresenterwasdescribingobtaininginformedconsent from a poor farmer in Jumla, animpoverished, remote and mountainous districtof Nepal. Poor rural people from Nepal’sremote districts may be looked down upon bytheeducatedelitewhoconductresearchstudies.In Nepal like in other South Asian countries,manuallaborisregardedasinferiortomentalandintellectualpursuits.Butthepresenterprovidedadifferentperspective.Aseducatedurbanpersons,

heenquired,willitbepossibleforustofarmthesteephillsides,growgrainandsurviveinJumla?Most probably we cannot. Sowhy dowe havea condescending attitude towards people livingand thriving in that environment?All skills areimportant and while doing research, recruitingparticipants and obtaining consent we shouldconsider the skills of the community and haverespectfortheperspectiveandviewsofthefarmerfromJumla.

Arecentarticlehadexaminedessentialelementsof informed consent and the author mentionsthatduring thefirst step the research teammustprovide full and transparent information aboutthe researchandparticipant’s rights inaneasilyunderstandablemanner.1Duringthesecondstep,the participant must clearly understand whatis expected of him or her. To ensure this theinformationmustbepresented inasimple,easyto understand manner. Preferably the consentshould be obtained in writing. Participatoryaction research is gaining increased attention incommunityandpublichealthresearch.Howevera number of ethical concerns can arise duringthe course of such research.2 Community basedparticipatoryresearch(CBPR)mustconsidertherights andwell being of communities.A recentsystematic review concluded that the reviewedarticles mentioned few measurable objectivesoruniformguidelinestoensurethathighethicalstandardshavebeenmet.3AuthorsofthepublishedpapersreviewedseemtoagreethatethicsinCBPRareemergentandaresituationspecific.Community based medical education is


becomingly increasingly important in theundergraduate medical curriculum.4, 5 In Nepal,medical students are posted in rural or semi-rural areas at different periods during theirundergraduatemedical course.Theystay in thecommunity, interactwith thepopulation,obtainhealthrelatedandotherinformation(whichmayattimesbesensitive),presentthefindingsoftheirstudytothecommunityandarriveatapossibleplan of action with community involvement.Appreciating and respecting the skills andknowledgeof the ruralcommunitymay lead toahealthierattitudetowardsandrespectforthemand thestudentmaybemore likely toconsiderthe community perspective while addressingproblemsandsuggestingsolutions.Itellmedicalstudents the exampleof the farmer from Jumlatomakethemconsidertheruralperspectiveanddeveloprespectforruralcommunities.

I also used to conduct a clinical researchmodule for Pharm D students of KathmanduUniversitywhereIdiscusstheorganizationandconduct of clinical trials in detail.6Obtaining aproper informedconsent is amajor issuewhilecoordinatingaclinicaltrial.Respectforthestudyparticipants and their autonomy is important. ItellPharmDstudents thescenarioof thefarmerfrom Jumla to highlight the importance of thecommunityperspective.

Since the last two years, I have been a facultyat a medical school in Aruba in the DutchCaribbean. We conduct a module on researchand critical appraisal of scientific literature forbasic science students.7 Informed consent is animportantissuetobeconsideredwhileanalyzingascientificpaperandaclinicaltrial.Withsomeclarificationandexplanation,Iusethecaseofthefarmer from Jumla to underline the importance

of understanding the patient/participant andcommunity perspective while conductingresearch.

I am impressed by the simplicity and possibleprofound impact of this scenario and its wideapplicabilityinavarietyofsettings!

rEfErENCES1. Bhutta ZA. Beyond informed consent. Bull World

HealthOrgan.2004;82(10):771-7.2. Khanlou N, Peter e. Participatory action

research: considerations for ethical review. Soc SciMed. 2005;60(10):2333-40.

3. Mikesell L, Bromley E, Khodyakov D. Ethical community-engaged research: a literature review. AmJPublicHealth.2013;103(12):e7-e14.doi:10.2105/AJPH.2013.301605.

4. Koju R. Community based medical education-necessity, realization and practice. KathmanduUnivMedJ.2013;11(43):189-90.

5. 5. ArtB,DeRooL,WillemsS,DeMaeseneerJ.Aninterdisciplinary community diagnosis experience inanundergraduatemedicalcurriculum:developmentatGhentUniversity.AcadMed.2008;83(7):675-83.doi:10.1097/ACM.0b013e31817829a6.

6. ShankarPR,ShakyaR.Knowledge,attitudeandskillsbeforeandafteraclinical researchmodule.NationalJournalofLaboratoryMedicine.2012:1(1):45-50.

7. ShankarPR,BhartiR,RamireddyR,BalasubramaniumR, Nuguri V. Students’ perception of the learningenvironmentatXavierUniversitySchoolofMedicine,Aruba: a follow up study. J Educ EvalHealth Prof.2014;11:9http://dx.doi.org/10.3352/jeehp.2014.11.9

Cite this article as:ShankarPR.CanyougrowgraininJumla?MEDPHOENIX2016;1(1):47-48

ShankarPathiyilRavi


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The limitations of the study are thosecharacteristics of design or methodology thatimpactedor influenced the interpretationof thefindingsfromyourresearch. Ifyoudoconnectyourstudy’slimitationstosuggestionsforfurtherresearch, be sure to explain theways inwhichthese unanswered questionsmay becomemorefocusedbecauseofyourstudy.Akeyobjectiveof the research process is not only discoveringnewknowledgebuttoalsoconfrontassumptionsandexplorewhatwedon’tknow.

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Articles in JournalsAnsariM,NeupaneD. Study on determinationof errors in prescription writing: A semielectronicperspective.KathmanduUnivMedJ.2009;7(3):238-41.

Books and other Monographse. g. Chapter in a bookMeltzer PS, Kallioniemi A, Trent JM.Chromosomealterationsinhumansolidtumors.In:VogelsteinB,KinzlerKW,editors.Thegenetic

basisofhumancancer.NewYork:McGraw-Hill;2002.p.93-113.

other Published Materiale. g. Newspaper articleTynanT.Medicalimprovementslowerhomiciderate: study sees drop in assault rate. TheWashingtonPost.2002Aug12;Sect.A:2(col.4).

Electronic MaterialJournal article on the Internet Abood S. Quality improvement initiative innursing homes: the ANA acts in an advisoryrole.AmJNurs[Internet].2002Jun[cited2002Aug 12];102(6):[about 1 p.]. Available from:http://www.nursingworld.org/AJN/2002/june/Wawatch.htmArticle

Fordetailaboutreferencingstyle,pleaserefertotheguidelinesat:http:/ /www.nlm.nih.gov/bsd/uniform_requirements.html or http://www.icmje.org orhttp://www.ncbi.nlm.nih.gov/books/bookres.fcgi/citmed/frontpage.html

7. TABLES/ILLuSTrATIoNSTables and figures or illustrations should beplaced at appropriate place along with text inresult section of the manuscript and should betypedinsinglespace.Tablesandfiguresshouldbeinblackandwhiteformatratherthancolored.Weencourageyoutokeepthenumberoftablesandfiguresasfewaspossible(e.g.2-3tablesandfigureseachinthecaseofResearchArticles).

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figures or illustrations should be drawnprofessionally with “low ink to data ratio”and without “3D” view. Photographs shouldbe sharp (contrast) andbe inblackandwhite(usually 5x7 inches). Letters, numbers andsymbols shouldbe clear and even throughout


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PrESENTATIoN AND forMATMargins:1incheachsidePage number:NumberedatbottomrightTitle:16ptTimesNewRoman,bold,centred.Author and co-authors: 12 pt Times NewRomancentred,bold-authorandallco-authorsnames in one line. The corresponding authorshouldincludeanasterisk*.Authors address: Each authors’ affiliation(i.e. Department/Organization /Address/ Place/Country/email).Author for Correspondence:Name,Department/Organization/Address/Place/Country/emailAbstract: Structured (Research/Review article& Short communication-250 words, Casereport-100words)

Text: Headings: ABSTrACT, Key words,INTRODUCTION, MATERIALS AND METHODS, RESULTS, DISCUSSION followed by Limitations, CoNCLuSIoNS and ACKNowLEDgEMENTS andrEfErENCES.

Tables/Illustrations: Should be incorporatedalongwiththetext.Headingshouldbeatthetop

(fortables)andatthebottom(forfigures/images)references: Vancouver style (http://www.nlm.nih.gov/bsd/uniform_requirements.html)

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youuse(orBritishbutdonotmixup)Abbreviationsspeltoutinfullforthe

firsttimeNumeralsfrom1to10speltoutNumeralsatthebeginningofthe

sentencespeltout

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intextActualnumbersfromwhichgraphs

drawn,providedFiguresnecessaryandofgoodquality

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MED PHoEnix : An Official Journal of NMC, Birgunj, Nepal, July 2016, Volume (1), Issue (1), ISSN: 2392 – 425X (Print)

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