infectious issn 1027-0299 recognised and … 1027-0299 recognised and registered with the ... case...

ISSN 1027-0299Recognised and registered with thePakistan Medical & Dental CouncilNO.PF.11-F-96 (Infectious Diseases) 2560College of Physicians & Surgeons, PakistanHigher Education Commission, PakistanIndexed - WHO EMRO

773

776

772

781

781

January - March 2015 Volume 24 Issue 01

INFECTIOUSDISEASESJOURNALPublished by the Medical Microbiology & Infectious Diseases Society of Pakistan

of Pakistan

IDJ

Jan - Mar 2015. 771Volume 24 Issue 01

CONTENTS PAGE #

Rights:No part of this issue or associated program may be reproduced, transmitted,transcribed, stored in a retrieval system or translated into language orcomputer language in any form or means, electronic, mechanical, magnetic,optical, chemical, manual or otherwise without the express permission ofthe editor/publisher and author(s) of IDJ.

Disclaimer:Statements and opinions expressed in the articles, news, letters to the editorsand any communications herein are those of the author(s), the editor and thepublisher disclaim any responsibility or liability for such material. Neitherthe editor nor publisher guarantee, warrant, or endorse any product orservice advertised in their publication, nor do they guarantee any claimmade by the manufacturers of such product or service.

Submission:Infectious Diseases Journal (IDJ) is published quarterly. Please submit manuscriptsat [email protected]. See author guidelines.

Designed & Printed by:Mediarc PublicationsA-452, Ground Floor, Block 7, K.A.E.C.H.S, Karachi.Tel:34555263, E-mail:[email protected]

Proprietor:Medical Microbiology & Infectious Diseases Society of Pakistan21 G /1, Block - 6, P.E.C.H.S., Shahrah-e-Faisal, Karachi. Ph: 0333-3977011E-mail: [email protected] Price: Rs. 100/-

Infectious Diseases Journal of PakistanOfficial Organ of the

Medical Microbiology & Infectious Diseases Society of Pakistan

President Aamer IkramDept of PathologyArmed Forces Institute of PathologyRawalpindi. Pakistan

Gen. Secretary Farah QamarDepartment of PaediatricsThe Aga Khan University, Karachi, Pakistan

Treasurer Seema IrfanDepartment of Pathology & Microbiology,Aga Khan University, Karachi, Pakistan

Editorial Office

Editors: Farah Naz QamarAli Faisal Saleem

Editorial Board: Aamer IkramNaseem SalahuddinAltaf AhmedEjaz A. KhanShehla BaqiLuqman SettiM. Asim BegNaila Baig AnsariRana Muzaffar

Business and CirculationNasir Hanook

Courtesy: Dr Ali Faisal Saleem, Aga Khan University, Karachi.

780

784

797

Place for front page picture

EDITORIAL

Adult Immunization and Pakistan. We are far behind.Ali Faisal Saleem

ORIGINAL ARTICLES

Frequency and Viral Load of Hepatitis C Virus in a Tertiary CareTeaching Hospital of D.I. KhanSajjad Ahmad, Hamzullah Khan, Sadia Anwar, Muhammad Tariq,Muhammad Ismail Khan, Habib Ahmad

Microbiological Analysis of Different Snack Foods as Public HealthSignificanceMuhammad Rizwan Saif-ullah, Khawar Mehboob, Mansur-ud- DinAhmad, Rehan Rafique, Muhammad Hassan Mushtaq, Fozia Sharif,Shahida Parveen

Frequency and Antimicrobial Susceptibility Pattern of EscherichiaColi Isolated from Urine Specimens at a Tertiary Care DiagnosticCenterAamir Hussain, Nauman Akbar, Irfan Ali Mirza, Shamshad Ali,Muhammad Fayyaz , Tahir Ghafoor

GUIDELINES

Guidelines for the Initial Management of Adults with Sepsis/SevereSepsis/Septic Shock: 2015Erfan Hussain, Bushra Jamil, Naseem Salahuddin

CASE REPORT

Autoimmune hemolytic anemia in a child with AIDSSeema Sakina, Ayesha Junaid, Ejaz Ahmed Khan

NEWS & VIEWS

INSTRUCTIONS FOR AUTHORS

15 year old boy presented with the complaints of headache, vomitting andwalking problem for one month duration. His MRI brain showed posteriorfossa mass located in the right cerebella hemisphere with obstructivehydocephalus. Ventriculoperitoneal shunt was placed and biopsy of the masstaken. Histopathology of the biopsy showed chronic granulomatous lesionswith caseous necrosis consistant with Tuberculoma.

EDITORIAL

772 . Infectious Diseases Journal of Pakistan

Adult Immunization and Pakistan. We are far behind.

Vaccines have been one of the most equitable low-cost, high-impact public health measures that save millions of livesannually. Globally the major focus is children under-5; howevervaccines of adults is very important given that >25% of mortalityare due to infectious diseases.1 WHO estimates high mortalityand disability-adjusted life years (DALY) associated withvaccine preventable diseases.2 Childhood vaccination is deliveredto community through expanded programme of immunization(EPI) free of cost in developing countries; however there is noisolated adult vaccination programme except maternal tetanusvaccination. Therefore the concept of adult immunization isnew in developing countries. Adult vaccinations arerecommended on the basis of age, prior vaccination conditions,health condition, lifestyle, occupation, travel andcommunicability of the disease. The adult population is increasedrisk of getting communicable diseases owing to urbanization,globalization, and increasing international travel.1,3 Certainvaccines (i.e., Hepatitis B, pneumococcal, influenza, HPV)have already been recommended globally but their acceptabilityand availability remains a challenge for developed countries.However this has not much studied for developing countries.

Possible barriers to adult immunization may be at several levellike individual (i.e., preferences, financial, lack of awarenessof vaccine availability and risks of contracting VPDs, notadvised by physician), community (i.e., infrastructure, lack ofdemand, and lack of awareness), health care workers (HCW)level (physician knowledge about adult vaccinationrecommendations, reliance on guidelines, patients regular well-care visits and lack of an effective reminder system) and others(i.e., reimbursement issues for medical practices; failure toupdate vaccinations during office visits; logistics issues at theoffice level (e.g., limited refrigerator/freezer space and up-frontvaccine costs); and a variable supply of vaccines.4,5 It is importantthat efforts should be targeted to improve HCWs compliancewith adult immunization guidelines so this eventually influencesand enhance knowledge and attitudes of the community. Thereis evidence on interventions to improve vaccine uptake amongHCWs in developed countries where education/promotioncampaigns and improved access to vaccines, had significantpositive effects in vaccine uptake among HCWs.6 Evidencefrom two observational studies from the USA suggests mandatoryvaccine policies are more successful in improving rates ofvaccination above 95% than relying on enabling approaches,mainly HCW approach.6

In Pakistani context there is as such no adult immunizationpolicy. The Maternal and Neonatal tetanus (MNT) is largely

preventable through Tetanus Toxoid (TT) vaccine that is alreadyavailable free-of-cost in the national expanded program onimmunization (EPI) in Pakistan. The recent most officialcoverage estimates available for maternal two doses of TTvaccine revealed 62% in Pakistan, ranged from 60% to 74%over the last decade. Amongst women of child bearing agetetanus coverage is very poor. A recent study concluded only43% women received at least one doss of tetanus. This coverageis markedly low among unmarried where only 4% received atleast one dose of tetanus vaccine. (Unpublished, Saleem et al.2015) Because of this low immunization coverage the burdenof maternal and neonatal tetanus incidence is high. Communityknowledge about TTV (tetanus toxoid vaccine) was found tobe very poor.

Another important and potential adolescent vaccine is HumanPapilloma Virus (HPV). This vaccine is very effective and helpin preventing cervical cancer which is third leading cause ofwomen cancer worldwide.

Importance and effectiveness of pneumococcal polysaccharidevaccine cannot be underestimated. Its use helps and preventpneumonia in elderly and also produces herd effect leading toprevention of pneumonia in the young members of the family.

Adult immunization is important and effective. There are certainbarriers particularly physicians and community knowledge,financial and socio-cultural. All stake holders includinggovernment, private sector, physicians and health workers haveto work together to combat them.

Refrences1. Verma R, Khanna P, Chawla S. Adult immunization in India: Importance

and recommendations. Hum Vaccin Immunother. Jun 18;10(9).2. 2004. The World Health Report 2004—Changing History. Geneva: WHO.3. Asha N. Adult immunization. Immunology. Available from:

http://www.apiindia.org /medicine_update_2013/chap101.pdf.4. Johnson DR, Nichol KL, Lipczynski K. Barriers to adult immunization.

Am J Med 2008 Jul;121(7 Suppl 2):S28-35.5. Shefer A, Briss P, Rodewald L, Bernier R, Strikas R, Yusuf H, et al.

Improving immunization coverage rates: an evidence-based review ofthe literature. Epidemiol Rev 1999;21(1):96-142.

6. ECDC Technical report. Review of the scientific literature on drivers andbarriers of seasonal influenza converge in the EU/EEA, 2013. EuropeanCentre for Disease Prevention and Control, 2013.

Ali Faisal SaleemAssistant Professor, Paediatric Infectious Diseases,Department of Paediatrics and Child Health,Aga Khan University, Karachi. Pakistan.

Jan - Mar 2015. 773Volume 24 Issue 01

ORIGINAL ARTICLE

Corresponding Author: Sajjad AhmadAssistant professor, Histopathology, Department of PathologyGomal Medical College, D.I.KhanEmail: [email protected], [email protected]

Frequency and Viral Load of Hepatitis C Virus in a Tertiary Care Teaching Hospital of D.I. Khan

a very high 57% prevalence was observed in injecting drugusers and 48.67% in a multi-transfused population.2

Because of the use of viral kinetics during interferon-ribavirintherapy and the development of specific new anti-Hepatitis Cvirus (anti-HCV) drugs, assessment of the efficacy of anti-HCV drugs needs to be based not on end-point PCR assays buton real-time PCR.3

Based on pivotal trials in large multicenter studies, positiveand negative predictions of sustained virological response(SVR) using viral load kinetics have been established and arenow used for recommendations on antiviral therapy managementby the American and European international consensusconference.3,4 The data for the proposed algorithm for themanagement of antiviral therapy in patients with chronicHepatitis C were mainly based on measurements of viral loadsassessed by end-point PCR assays in centralized laboratories.It has its importance in the prognosis and management of thedisease.5

The present study was therefore conducted to determine thefrequency and viral load o f Hepatitis C virus in Southern areasof KPK

Material and MethodsThis descriptive cross sectional study was conducted in theDepartment of Pathology Mufti Mehmood teaching hospitalDera Ismail Khan from Jan 2013-dec 2013. A total of 224patients were selected randomly as referred to the departmentfor real time Quantitative PCR for Hepatitis C virus.

This was confirmed by using Method Cepheid SmartCycler@||RTPCR. HCV RNA was extracted from patient serumand performed by Real-time PCR with in a single tube. SmartCycler carried out amplification and detection simultaneously@||using Taqman probes and detected through fluorescent reporterdye specific for HCV. The result was calculated in ExponentialPhase, which is more sensitive and specific. This methodallowed us to screen plasma and serum samples over a rangebetween 100 and 10*107 copies of HCV RNA per milliliter(Cps/ml).

Relevant information’s were recorded on a pre-designed

Abstract

BackgroundHepatitis C infection is becoming a major health problem ofdeveloping countries, including Pakistan that has the secondhighest prevalence rate of hepatitis C ranging from 4.5% to 8%.

ObjectivesTo determine the frequency and viral load o f Hepatitis C virusin Southern areas of Khyber Pukhtunkhwa (KPK).

Material and MethodsThis cross sectional study was conducted in, the Departmentof Pathology, Mufti Mehmood Teaching Hospital Dera IsmailKhan from January to December 2013. A total of 224 patientsthat had positive Anti-HCV antibodies by ICT and ELISA werereferred by their physicians for real time Quantitative PCR forHepatitis C virus were selected.

ResultsTotal of 224 patients including 100 females (45%) and 124males (55.4%) were selected. The age range was 68 years withminimum of 7 year and maximum of 75 years. Median for agewas 35 years. 119(55.1%) resulted to be positive for HCV viruson. In positive female patients the mean viral load was 71388.2while in males it was 139575.3.

ConclusionThere is a high prevalence of HCV in KPK with males beingmore commonly infected. Viral load was found more in infectedmales as compared to females at the time of diagnosis beforeany intervention.

Key wordsHepatitis C Virus, Frequency, Viral Load

IntroductionIn Pakistan 10 million people are presumed to be infected withHCV.1 In Pakistan Percentage prevalence of HCV is 4.95% thegeneral adult population, 1.72% in the pediatric population and3.64% in a young population applying for recruitment, whereas

Sajjad Ahmad*, Hamzullah Khan*, Sadia Anwar**, Muhammad Tariq***, Muhammad Ismail Khan***,Habib Ahmad***

*Department of Pathology, Gomal Medical College,D.I.Khan, **Department of Obs & Gynae, Gomal MedicalCollege, D.I.Khan, ***Department of Genetics, Hazara University , Mansehra, Pakistan


proforma. Written informed consent was taken andconfidentiality of participants was ensured.

Demographic variables were age, gender and address andresearch variables were frequency of Hepatitis C cases, viralload and cycle values.

Data was descriptively analyzed in SPSS Version 12.0. Mean and standard deviation are reported.

ResultsA total of 224 patients including 100 females (44.6%) and 124males (55.4%) were selected. Gender distribution of patientsof patient is given in Table 1.

The age range was 68 years with minimum of 7 year and maximumof 75 years. Median for age was 35 years. Out of total screened,119 (55.1%) resulted to be positive for HCV virus on PCR and105 (46.9%) were negative for the virus studies (Table 1). Inpositive female patients the mean viral load was 71388.2 whilein males it was 139575.3 (Table 2).

DiscussionHepatitis C virus (HCV) infection is one of the most importantFlaviviridae infections with significant clinical problemsthroughout the world in humans and it is responsible for the

second most common cause of viral hepatitis.6 Frequency ofHepatitis C is quite high in our population mainly in youngadults. We need to adopt organized preventive strategies toovercome this problem.7 We found 119 (55.1%) cases resultedpositive for HCV virus on PCR. Of those positive cases, 43(36.13%) were females and 76 (63.86%) were males. Anotherstudy from Khyber Pukhtunkhwa reported that the frequencyof Hepatitis C was higher among the male, 409/751 (54.46%)as compared to female, 342/751(45.53%).8

The diagnosis of acute infection in the HCV-seropositive patientis strengthened by the use of virologic parameters that areuncommon in chronic disease. Viral load fluctuations and lowlevels of HCV-RNA should be incorporated into standarddiagnostic criteria.9 We tried to determine the viral load frequencyby gender, that if there is any sort immunity which neutralizesor protects females more than males. As female gender confermore resistance to HCV load than male gender.

Based on categorization of the viral load into three levels, low(< 60, 0000 IU/mL), intermediate (60,0000-80,0000 IU/mL)and high (> 80,0000 IU/mL), majority of the subjects in ourstudy had an intermediate load.10 In the referenced study viralload distribution was also categorized sex wise; for males itwas 58 (32.76%), 26 (14.68%) and 93 (52.54%) whereas forfemales it was 40 (31.25%), 34 (26.56%) and 54 (42.18%) forlow, intermediate and high viral load respectively.11

ConclusionHepatitis C infection is common in the southern areas of theprovince of KPK. This disease is common in males this couldbe because of the gender in-equality, as more males are referredto the health care centers, or it could be possibly due to someimmune protection in females against HCV virus. Viral loadwas found more in infected males as compared to females atthe time of diagnosis before any intervention.

There is need for further studies to discuss why females aremore resistant with low viral load as compared to males.

Refrences1. Hamid S, Umar M, Alam A, Siddiqui A, Qureshi H, Butt

J. PSG consensus statement on management of HepatitisC virus infection--2003. J Pak Med Assoc 2004; 54:146–50.

2. Waheed Y, Shafi T, Safi SZ, Qadri I., Hepatitis C virus in Pakistan: Asystematic review of prevalence, genotypes and risk factors. World JGastroenterol 2009; 15(45): 5647–53.

3. Davis, G. L., J. B. Wong, J. G. McHutchison, M. P. Manns,J. Harvey, and J. Albrecht. 2003. Early virologic responseto treatment with peginterferon alfa-2b plus ribavirin inpatients with chronic Hepatitis C. Hepatology 38:645-52.

4. European Association for the Study of the Liver. 1999.EASL International Consensus Conference on Hepatit isC. Paris, 26-28, February 1999, consensus statement. J.Hepatol 30:956-61.

5. Fried, M. W., M. L. Shiffman, K. R. Reddy, C. Smith, G.Marinos, et al. Peginterferon alfa-2a plus ribavirin for chronic Hepatitis

Table 1. Gender, age range and PCR findings.

Gender Frequency Percent

Female 100 44.6Male 124 55.4

Age 7-16 4 1.7917-26 54 24.1127-36 68 30.3637-46 48 21.4347-56 36 16.0757-66 12 5.3667-76 2 0.89

PCR ResultNegative 105 46.9Positive 119 53.1

Table 2. Viral Load statistics by gender

Female Male

Viral load (mean) 71388.29 139575.33Cycle value (mean) 12.58 17.39

Volume 24 Issue 01

C virus infection. N. Engl. J. Med 2002; 347:975-82.6. Leiveven J: Pegasys/RBV Improves Fibrosis in Responders, relapsers

& Nonresponders with Advanced Fibrosis. 55th Annual Meeting of theAmerican Association for the Study of Liver Disease: 2004 October 29– November 2: Boston, MA, USA.

7. Saleem M, Ahmad W, Sarwar J, Jamshed F, Gul N, Idrees M. Frequencyof Hepatitis C in asymptomatic patients in district headquarters hospitalKotli, Azad Kashmir. J Ayub Med Coll Abbottabad 2011;23(2):59-62.8.

8. Muhammad N, Jan MA . Frequency of hepatitis "C" in Buner, NWFP.JCPSP 2005; 15(1):11-4.

9. McGovern BH, Birch CE, Bowen MJ, Reyor LL, Nagami EH, ChungRT, et al. Improving the diagnosis of acute hepatitis C virus infectionwith expanded viral load criteria. Clinical Infectious Diseases2009;49(7):1051-60.

10. Afridi SQ1, Ali MM, Awan F, Zahid MN, Afridi IQ, Afridi SQ, YaqubT. Molecular epidemiology and viral load of HCV in different regionsof Punjab, Pakistan. Virol J 2014: 11-24.

11. Ali A, Nisar M, Ahmad H, Saif N, Idrees M, Bajwa MA. Determinationof HCV genotypes and viral loads in chronic HCV infected patients ofHazara Pakistan. Virol J 2011;8:466.

Jan - Mar 2015. 775


Corresponding Author: Rehan Rafique,Cell Culture Section, Veterinary Research Institute, ZarrarShaheed Road, Lahore Cantt. PakistanE-mail: [email protected]

ORIGINAL ARTICLE

Microbiological Analysis of Different Snack Foods as Public Health Significance

Abstract

BackgroundFood borne illnesses are considered as a an important challengeto the public health and significantly contribute to the cost ofhealth. Each year millions of illnesses in the world can beattributed to the contaminated foods. Hence a preliminary studywas conducted to estimate the qualitative and microbiologicalanalysis of different snack foods and its evaluation regardingpublic health significance.

MethodsSnack food samples (sandwiches, burgers and pizzas) werecollected from different retail outlets located at Lahore city andfurther processed for microbiological quality assays including;total aerobic plate counts, coliform count and enumeration ofStaphylococcus aureus and detection of Salmonella.

ResultsResults showed that sandwiches had the highest geometricmean of aerobic plate counts followed by pizzas and burgersrespectively. In total 73% of the snack foods were contaminatedwith coliforms. Staphylococcal contamination was higher ascompared to coliform contamination. The contamination levelwas above permissible level in 50% of the sandwiches, 27%of burgers and 45% of the pizza as per guidelines for gradingof ready to eat foods in Hong Kong and U.K. HoweverSalmonella was not detected in any food sample.

ConclusionSnack foods showed detectable levels of microorganisms ofpublic health significance. These foods are contaminated dueto poor hygiene practices. Necessary hygienic measures shouldbe recommended to reduce the ontamination level.

Key wordsSnack foods, Microbiological analysis, Food safety, Publichealth, Hygiene Measures

IntroductionFood borne diseases remain a persistent challenge to publichealth and the problems of food safety in the developed countriesdiffer from those faced by developing countries like Pakistan.The contamination of food products with infectious organismsand their persistence, growth, multiplication and/or toxinproduction is an important public health concern. With increasein urbanization throughout the world, the consumption of streetfood/ready to eat foods is becoming common in almost allcountries and the responsibility of food safety is shifting fromfinal cook or consumer to the entire food chain.1

Lahore is the historical city of Pakistan, famous for its traditionalfoods, and a large proportion of the ready-to-eat food is soldby the street venders. Keeping in view the prevailing conditions,a preliminary study was designed to take a snapshot ofcontamination level of the snack foods and to set a model forconducting similar studies of public health significance. Hygienicmeasures, necessary to reduce or prevent the contamination arealso suggested for improvement of snack food hygiene.

Material and Methods

Types of food samples & Sampling locationTotal number of 60 snacks (ready-to-eat) were randomly obtainedfrom different restaurants and snack bars located in Lahore.Samples were collected as in the form of consumable item(ready-to-eat) in sterile plastic bags and transported to laboratorymaintaining the temperature at 4ºC. The respective number ofsamples collected is given in Table 2.

Microbiological analysisFor microbiological analysis, 25 grams of sample was weighedand blended in 225 ml (0.1%) sterile peptone water. Ahomogenate was prepared (10% w/v) under sterile conditionsand was further diluted in tenfold serial dilutions.2

For aerobic plate count, one ml of sample homogenate fromeach dilution was transferred to Petri dishes and 15-20 ml ofautoclaved molten agar was added. Plates were gently swirledfor mixing and allowed to solidify at room temperature andincubated at 35ºC for 48 hrs.3 For coliform counts, one ml ofthe sample representing each dilution was added to 25 ml of

Muhammad Rizwan Saif-ullah*, Khawar Mehboob**, Mansur-ud- Din Ahmad*, Rehan Rafique**, MuhammadHassan Mushtaq*, Fozia Sharif***, Shahida Parveen**

*Department of Epidemiology & Public Health, University of Veterinary and Animal Sciences, Lahore Pakistan**Veterinary Research Institute, Zarrar Shaheed Road, Lahore Cantt. Pakistan***Services Institute of Medical Sciences, Lahore Pakistan

Volume 24 Issue 01

Violet red bile salt agar, maintained as liquid at 45ºC. Mediumwas allowed to solidify in petri dish and then overlaid with 3to 4ml of additional Violet red bile salt agar. After incubation,all the purplish red colonies surrounded by reddish zone ofprecipitated bile acids were counted as coliform colony.4 Forpre-enrichment of Salmonella, 25 gm sample was added to 225ml lactose broth (0.5%) and blended to prepare a homogenatewhich was then incubated for 24 hrs at 37°C. One ml ofhomogenate was transferred to 10 ml tetrathionate broth withIodine and incubated. After incubation, a loopful of growthwas streaked on three selective media i.e. Brilliant Green agar,Salmonella Shigella agar and MacConkey agar. The growingorganisms were further identified on the basis of colonycharacteristics; Gram’s staining and biochemical reactions.5

For enumeration of Staphylococcus, one ml from each dilutionwas cultured on Baird-Parker agar in triplicate and incubatedfor 48 hrs. The number of colonies appearing as distinct black,convex, with a diameter of 1.0-1.5mm was counted to calculatecfu. For identification of coagulase positive Staphylococcus,Coagulase test was performed by transferring 0.2 ml rabbitplasma in a sterile test tube and 0.8 ml of test broth culture wasmixed followed by incubation for 24 hrs.6-7

Grading of Ready-to-Eat FoodsThe microbiological quality of snack food samples was comparedwith guidelines for the microbiological quality of ready to eatfoods published by the Central Public Health Laboratory Services(PHLS) United Kingdom and Hong Kong (Table 1).8

ResultsIn the present study, various microbiological assays of 60 snackfood samples were carried out from food service establishmentslocated at suburbs of Lahore and from of outlets of internationalfood chains.

The aerobic plate count (APC) of snack food samples rangedfrom <10 to 109 cfu/g (Table 2). The APC range for sandwicheswas 103 to109, for burgers 10 to 109 and for pizza 10 to 109.Sandwiches had the highest geometric mean followed by thepizza and burger. Results for “sandwiches with salad” showed

that 6 samples were unsatisfactory with the range of 107(Table 2). This might be due to the reason that the salad usedin the sandwiches was brought from the suburban areas of thecity, irrigated by unclean water and not properly washed at thetime of use. Aerobic plate count for “sandwich samples withoutsalad”, showed that 3 samples were of unsatisfactory levelwhich might be due to contamination of chicken used or dueto the poor holding temperature. Snack foods like sandwicheswere usually kept at air conditioning temperature (18ºC to26ºC) in most of the places from where samples were collected.From 26 burger samples, 14 were in permissible range of <104

cfu/g while remaining 12 samples were in the range of 106

to109 cfu/g. As burger material is heated before preparation,the only chance of contamination is during the processing of beefor chicken. Aerobic plate counts in pizza samples were higher ascompare to sandwiches and burgers. In pizza 6 out of 20 sampleswere in permissible range of <104 cfu /g. Remaining 14 sampleswere found in the range of 105 to 109 cfu/g, which might be dueto the reason that pizzas are prepared well before time of use andnot properly heated at the time of sale.

Coliform counts for all the samples ranged from <10 to 106cfu/g. In sandwiches 7 samples (50%) contained coliform <10cfu/g. Pizza had highest counts ranging from <10 to 106cfu/g,where as in burger samples it was <10 to104cfu/g. Howeverin sandwiches highest geometric mean count was recordedfollowed by the burger and pizza (Table 2).

Coliform counts must be in the limit of 102 or less per gram ina food.8 According to criteria (Table 1), 65% sandwich sampleswere found in satisfactory and acceptable level, 77% burgersamples were of satisfactory quality as they had <10 cfu /g ofcoliform (Table 2). In case of pizza, 85% of the samples wereof satisfactory quality and their bacterial count was <10 cfu/g.Coliform counts are still considered as indicator for assessinggeneral hygienic status of food contact surfaces and theattachment of bacteria to contact surfaces increases the chancesof cross contamination to foods which come in contact withthese surfaces.9

The Coagulase positive Staph. aureus counts ranged from <10

Jan - Mar 2015. 777

Table 1. Type and category of food with description of unsatisfactory level

Type and category of food Description of unsatisfactory level of different microbes (cfu*/g)

Sandwich with salad, Category 4 Unsatisfactory APC level = 107

Sandwich without salad, Category 3 Unsatisfactory APC level = 106

Pizza, Category 2 Unsatisfactory APC level is = 105

Burger, Category 1 Unsatisfactory APC level is = 104

For all foods Coliform count unsatisfactory level is = 100For all foods Staphylococcal count unsatisfactory level is = 104 cfu/g.

*colony forming unit


to 107cfu/g. The coagulase positive Staph. aureus counts ofburgers and sandwiches were in the range of <10 to106, whilepizza had counts in the range of <10 to 107cfu/g. Sandwichesshowed the highest geometric mean count followed by the pizzaand burger as shown in Table 2.

Presence of Staph. aureus in snack food is a serious healththreat.10 According to our results 14.2% sandwiches hadacceptable level of Staph. aureus <102 cfu/g while 85.8%sandwich samples were of unsatisfactory quality, which iscomparable with another report,11 that Staphylococcal countsin chicken sandwiches without salad was ≤102 cfu/g. Out oftotal burger samples, 62% were of satisfactory and acceptablequality having counts <102 cfu/g, and 19% samples were of

unacceptable quality with microbial load in the range of 104 to106 cfu/g (Table 2). In another study researchers examined intheir study that burgers had Staph. aureus count in the rangeof 10-102 cfu/g. They performed Coagulase test on 45 snackfood samples, out of which 34(76%) samples were foundpositive for Staph. aureus.12 While in present study 25% of thepizza samples were of satisfactory quality with counts of <20cfu/g, while 5% samples were of acceptable quality havingmicrobial counts <102 cfu/g and remaining 40% samples wereof unsatisfactory quality (102 to 104 cfu/g).

All the food samples were also processed for the detection ofSalmonella. However Salmonella could not be found in anyone of the samples.11,13

Table 2: Total bacterial count of different ready to eat snack foods

Aerobic Plate Count

Pizza (n=20)Range (cfu/g) Sandwiches (n=14) Burgers (n=26)

Without Salad (n=7)With Salad (n=7)

<10 - - 4 (15.4%)* 1 (5%)*

102 to 103 - - 6 (30%)*

103 to 104 - 1(14.3%)* 10 (38.5%)** -104 to 105 - - - -105 to 106 - - - 1 (5%)†

106 to 107 1 (14.3%)* 3 (42.8%)* 1 (3.8%)† 7 (35%)†

107 to 108 5 (71.4%)** 3 (42.8%)** 3 (11.5%)† 3 (15%)†

108 to 109 1 (14.3%)† - 8 (30.8%)† 2 (10%)†

Geometric mean 10000000 1000000 278927.39 1717915

Coliform Counts

<10 7 (50%)* 20 (77%)* 17 (85%)*

101 to 102 - - 1 (5%)*

102 to 103 3 (21.4%)* 5 (19.2%)* 1 (5%)*

103 to 104 3 (21.4%)** (3.8%)** -104 to 105 1 (7.2%)† - -105 to 106 - - 1 (5%)†

Geometric mean 138.94 38.01 29.7

Staphylococcal Counts

<10 1 (7.1%)* 9 (34.6%)* 5 (25%)*

101 to 102 1 (7.1%)** 7 (27%)** 1 (5%)**

102 to 103 5 (35.8%)** 3 (11.5%)** 5 (25%)**

103 to 104 1 (7.1%)** 2 (7.7%)** 3 (15%)**

104 to 105 5 (35.8%)† 3 (11.5%)† 2 (10%)†

105 to 106 1 (7.1%)† 2 (7.7%)† 3 (15%)†

106 to 107 - - 1 (5%)†

Geometric mean 719.6 131.8 1025

Satisfactory* Acceptable** Unsatisfactory†

Volume 24 Issue 01

DiscussionCentre for Disease Control (CDC) USA reports food bornediseases are among the most important health problems in bothdeveloped and developing countries.14 Higher prevalence offood borne diseases has been reported number of times bymedia in Pakistan. There is a great relation between microbialquality of food and food borne diseases. Microbial contaminationof food items may occur during various production andpreparations steps involved.15 Strict hygienic measures need tobe taken care of at various steps in production, preparation andstorage of food to improve the shelf life of the product andpublic health concerns. Snack food are prepared using a varietyof food items from animal sources such as eggs, chicken, meat,vegetables and sauces.16 All of these food components couldbe the potential source of food poisoning or food borne illnesses.Moreover, improper cooking, storage and poor sanitary conditionsof the kitchen further deteriorate the quality of the food. Equipment,food handlers, raw product, dust or prolonged refrigeration arethe major sources of contamination.17 Microbiological standardsof food have been defined by various agencies and authoritiesto implement these in their region of control.

Comparison of Microbiological Analysis of Snack FoodIn this study a total number of 60 snack food (pizzas, burgerand sandwiches) were analyzed to evaluate their microbiologicalquality.

Aerobic Plate CountAerobic plate count is a useful tool for estimating microbialpopulation.18 According to our results aerobic plate count forsandwiches with salad; only 1 sample was in acceptable levelof < 106 while remaining 6 samples were found in unsatisfactorylevel in the range of 107. In sandwich samples without salad;4 samples were in acceptable level of < 105 and 3 were foundin unsatisfactory level. Aerobic plate count for burger samples,14 samples were in permissible range of < 104 cfu/g whileremaining 12 samples were in the range of 106 to109 cfu/g.Aerobic plate counts in pizza samples were higher as compareto sandwiches and burgers. In pizza 6 out of 20 samples werein permissible range of < 104 cfu /g. Remaining 14 sampleswere found in the range of 105 to 109 cfu/g.

A high aerobic plate count does not make food unsafe but itdoes suggest non hygienic handling, poor storage and inadequategeneral hygiene during processing and or poor quality rawmaterials.19

Salek conducted an assessment on 100 samples taken frommeat foods offered in clinical centers of Shahid BeheshtiUniversity of Medical Sciences Iran reported. Mean totalbacterial count in samples of grilled ground meat, grilledchicken, chicken and hamburger as were 2.04×105

, 2.16×102,

2.45×104, and 2.25×104 respectively.20 In this study total bacterial

count was higher as compare to Salek study.

Coliforms CountColiform counts must be in the limit of 102 or less per gram ina food.18 Studies showed that this is possible for the cookedfoods to be contaminated with coliforms.13 High microbial loadof coliforms indicates the possibility of fecal contamination.21

In our study coliforms count for three type’s snack food(sandwich, burgers and pizza) were done. As criteria set by thefood and environmental hygiene department reference guidelinesof public health laboratory services of the UK and Hong Kong,65% sandwiches samples were found in satisfactory andacceptable level and 35% sandwiches were of unsatisfactoryquality in the range of 102 to 105 cfu/g. 77% of burger sampleswere of satisfactory quality. They were found in the range of< 20 cfu /g. 23% samples were of unsatisfactory quality andthey were in the range of 102 to 104 cfu/g. Where as in case ofpizza 85% of pizza samples were of satisfactory quality andtheir bacterial count were in the range of < 20 cfu/g, 5% sampleswere of acceptable quality (<102 cfu/g). Remaining 10% sampleswere of unsatisfactory quality; their microbial counts were inthe range of 102 to 105 cfu/g. Coliform counts of pizza wereless as compare to burger and sandwiches.

Tavakoli evaluated 216 samples of consumed foods in sixclinical and educational centers and reported coliform countsin grilled ground meat were in the range of 1.98 × 102 cfu/grespectively.13 Same type of work is done by Tessi evaluated101 samples of cooked and prepared foods in university centersof Argentina. The mean total bacterial contamination reportedwas 3.3 × 104 and mean coliform contamination was 1.90 × 102

cfu/g.22 Coliform counts in our study were higher as comparedto Tavakoli and Tessi work.

Staphylococcal countThe permissible levels for Staphylococcal count reported are≤104 per gram.18 Presence of Staphylococcus aureus in snackfood is a serious health threat.10 The staphylococcal countsrecorded in the sandwiches and burgers were in the range of<10 to106 cfu/g. For pizza sample it was found in the range of<10 to104 cfu/g and in class 2 samples it was in the range of<10 to107 cfu/g. According to food and environmental hygienedepartment reference guidelines of public health laboratoryservices of the UK and Hong Kong, Staphylococcalcontamination was more in sandwiches as compare to burgerand pizza. Only 14% samples were found in acceptable levelwhere as 36% samples were of unsatisfactory quality, theirmicrobial counts were in the range of 102 to 103 cfu/g. 43%sandwiches were of unacceptable quality and their microbialload were in the range of 104 to 106 cfu/g. 62% burger sampleswere of satisfactory and acceptable quality, 11% were ofunsatisfactory quality and their microbial load were in the rangeof 102 to 104 cfu/g and remaining 19% samples were ofunacceptable quality due to having microbial load in the rangeof 104 to 106 cfu/g.

Our study showed that 25% pizza samples were of satisfactory

Jan - Mar 2015. 779


quality having counts < 20 cfu/g, 5% samples were of acceptablequality having microbial counts in the range of < 102 cfu/g andremaining 40% samples were of unsatisfactory quality (102 to104 cfu/g). 30% samples were of unacceptable quality havingmicrobial counts in the range of > 104 cfu/g.

A study was conducted on 108 shawarma samples and foundstaphylococcus aureus counts in the range of <10 to 105 cfu/gin beef, chick and lamb shawarma.23 Microbiology of kwek-kwek (a Philippine emerging street food made from boiled quaileggs, which have been battered and fried) was studied andmicrobial profile of ready-to-eat kwek-kwek during vendingwas: 104-106 cfu/g TPC; ≤102 cfu/g coliform count; ≤104 cfu/gcoagulase-positive staphylococci count.24 In our studystaphylococcal counts were more as compare to other studies.

SalmonellaForm 60 snack food samples incidence of salmonella was zero.All samples were from the presence of salmonella. It iscomparable with the findings of a study in which there wasalso no contamination of I.13 In contrast Salmonella agona hasbeen reported to cause international outbreak from a ready toeat savoury snack in England, Wales and United States duringyears 1994 and 1995.25

ConclusionThe microbiological and qualitative analysis of snack foodsconcluded detectable levels of different microorganisms ofpublic health significance. These types of foods are contaminateddue to poor hygiene practices and because of favorableenvironment for the growth of bacteria due to use of cream,cheese, sauces and meat as common ingredient. In this contextthe hygienic manipulation practices, such as (i) washing ofhands, utensils and dishes (ii) disinfection of usable things (ii)protection of food from insects, flies and rodents (iv) hygienicmeat with proper refrigeration, are recommended to decreasethe contamination level.

SuggestionFurthermore this preliminary study will help other scientists toaddress food borne diseases which pose important healthproblem especially in developing countries like Pakistan. Toour knowledge there are hardly few reports of snack foodcontamination from Pakistan, therefore continuous monitoringis needed for collection of baseline data to implement policiesfor its control by public health authorities.

References1. Tauxe RV. Surveillance and investigation of foodborne diseases; roles

for public health in meeting objectives for food safety. Food Control2002; 13: 363-369.

2. Sachindra NM, Sakhare PZ, Rao DN. Reduction in microbial load onbuffalo meat by hot water dip treatment. Meat Science 1998; 48(2): 149-157.

3. Robinson RK. Dairy Microbiology Handbook, Wiley-Interscience, New

York, 2002.4. Marshall RT. Standard method for the examination of dairy products,

American Public Health Association, Washington, DC, 1993.5. Saroj SD, Shashidhar R, Karani M, Bandekar JR. Rapid, sensitive, and

validated method for detection of Salmonella in food by an enrichmentbroth culture - nested PCR combination assay. Molecular Cell Probes2008; 22(3): 201-206.

6. James GC, Sherman N. Microbiology; A laboratory Manual, 7th edn.Benjamin Cumming Publisher, 384; 2004.

7. Stepanovic S, Dakic I, Hauschild T, Vukovic D, Morrison D, Jezek P,Cirkovic I, Petras P. Supplementary biochemical tests useful for thedifferentiation of oxidase positive Staphylococci. Systemic AppliedMicrobiology 2007; 30(4): 316-318.

8. Gilbert RJ. Provincial microbiological guidelines for some ready to eatfood sampled at point of sale: notes of PHLS Advisory Committee forFood and Dairy Products. Communicable Diseases and Public Health1992; 3: 163-167.

9. Christison CA, Lindsay D, Holy-von A. Microbiological survey of readyto eat foods and associated preparation surface in retail delicatessens,Johannesburg, South Africa. Food Control 2008; 19: 727-733.

10. Chomvarin C, Chantarasuk Y, Srigulbutr S, Chareonsudjai S, ChaicumparK. Enteropathogenic bacteria and enterotoxin-producing Staphylococcusaureus isolated from ready-to-eat foods in Khon Kaen Thailand. SoutheastAsian Journal of Tropical Medicine and Public Health 2006; 37(5):983-90.

11. Little CL, Mitchell RT, Barnes J. Joint Hospitality Industry Congress(JHIC); Memorandum submitted by the Joint Hospitality Industry Congress(c85). In: House of Commons Agriculture Committee Fourth Report,Food Safety. The Stationary Office, London. 2: 1998.

12. Essa HH, Makar NH. Bacteriological quality of beef burgers in Assuitcity. Assuit Veterinary Medicine Journal 2005; 55(2): 126-135.

13. Tavakoli HR, Razipour M. Microbial quality of cooked foods in TehranUniversities Restaurants. Pakistan Journal of Medical Science 2008;24(4): 595-599.

14. Jay IM. Modren food microbiology. 6thedn. Chpman and Hallpublication, New York, 2002; 157-159.

15. Harrigan FW. McCance ME. Laboratory methods in food microbiology.London Academy Press, 1968.

16. Graffham A, Zulu R, Chibanda D. Improving the safety of street vendedfoods in Southern Africa. Final Report, CPHP project R8272, 2005.

17. Adams MR, Moss MO. Food Microbiology. Royal Society of theChemistry, Cambridge, UK. 2000; 479.

18. Gilbert RJ, de Louvois J, Donovan T, Little C, Nye K, Ribeiro CD,Richards J, Roberts D, Bolton FJ. PHLS Advisory Committee for Foodand Dairy Products. Commun Dis Public Health 2000; 3: 163-167.

19. Gillespie, Giannella RA, Formal SB, Dammin GJ, Collins HJ. Pathogenesisof salmonellosis; Studies of fluid secretion, mucosal invasion, andmorphologic reaction in the rabbit ileum. Journal of Clinical Investigation2000; 52: 441-453.

20. Salek M. Microbial control of cooked meat foods ad lettuces served inBeheshti Medical Sciences University restaurants. PhD Thesis, 2000;63-69.

21. Ayulo AM, Machado RA, Scussalo VM. Enterotoxigenic Escheria coliand Staphylococcus aureus in fish and sea food from the southern regionof Brazil. Int J Food Microbiol 1994; 24: 171-178.

22. Tessi MA, Aringoli EE, Vincenzini AZ, Sabbag NG, Costa SC. Microbialquality and safety of ready to eat cooked foods from a centralized schoolkitchen in Argentina. J Food Port 2002; 65(4).

23. Ayaz M, Othman FA, Bahareth TO, Al-Sogair AM, Sawaya WN.Microbiological Quality of Shawarma in Saudi Arabia. J Food Prot1985; 48(9): 11-14.

24. Patricia, Sammarco ML, Ripabelli G, Ruberto A, Iannitto G, Grasso GM.Prevalence of Salmonellae, Listeriae and Yersiniae in the slaughterhouseenvironment and on work surfaces, equipment and workers. J Food Prot2004; 60: 367-371.

25. Killalea D, Ward LR, Roberts D, de Louvois J, Sufi F, Stuart JM, WallPG, Susman M, Schwieger M, Sanderson PJ, Fisher IS, Mead PS, GillON, Bartlett CL, Rowe B. International epidemiological andmicrobiological study of outbreak of Salmonella agona infection froma ready to eat savoury snack: England and Wales and the United States.British Medical Journal 1996; 313(7065): 1105-1107.

ORIGINAL ARTICLE

Volume 24 Issue 01

Corresponding Author: Aamir Hussain,Department of Pathology. Combined Military Hospital,Peshawar Cantt.Email: [email protected]

Frequency and Antimicrobial Susceptibility Pattern of Escherichia Coli Isolated from Urine Specimensat a Tertiary Care Diagnostic Center

Jan - Mar 2015. 781

Aamir Hussain, Nauman Akbar, Irfan Ali Mirza, Shamshad Ali, Muhammad Fayyaz , Tahir Ghafoor

Department of Microbiology, Armed Forces Institute of Pathology, Rawalpindi, Pakistan

Abstract

ObjectiveTo determine the frequency and antimicrobial susceptibility ofEscherichia coli isolated from urine specimens at a tertiary carediagnostic center.

MethodologyThis descriptive study was done at department of microbiology,Armed Forces Institute of Pathology, Rawalpindi from Januaryto December 2012. Semi-quantitative culture were done usingstrip method and only those urine specimens, which revealedthe growth of Escherichia coli were included in the study.Antimicrobial susceptibility pattern of these isolates was notedagainst routinely used antibiotics (nitrofurantoin, meropenem,imipenem, amikacin, piperacillin-tazobactum, cefoperazone-sulbactum, piperacillin-sulbactum, gentamicin, ceftriaxone,ampicillin, ciprofloxacin, trimethoprim-sulphamethoxazole,amoxycillin-clavualanic acid and pipemadic acid).

ResultsA total of 10831 urine specimens were submitted for cultureand drug susceptibility testing during the period of study. Outof these, 1904 specimens showed growth of different bacteria.Out of 1904 positive cultures, 990 (52%) were identified asEscherichia coli. Imepenem (96.3 %), Meropenem (92.9 %),Amikacin (83.7 %) and Nitrofurantoin (82.3 %) were the mostsensitive, whereas trimethoprim-sulphamethoxazole (24%sensitivity), ciprofloxacin (29% sensitivity) and amoxycillin-clavualanic acid (26% sensitivity) were the least sensitiveantibiotics against E.coli.

ConclusionE.coli was the most common isolated organism from urinecultures.In vitro susceptibility to imepenem, meropenem andnitrofurantoinwas found to be higher compared to otherantimicrobials. On the contrary in vitro susceptibilityoftrimethoprim-sulphamethoxazole, ciprofloxacin andamoxicillin-clavulanic acid was found to be extremely low.

Key WordsEscherichia coli, Susceptibility, urine culture.

IntroductionUrinary tract infections (UTIs) are among the most commonbacterial infectious diseases encountered in clinical practiceand accounts for significant morbidity and high medical costs.Escherichia coli dominate the list of pathogens, accounting for80-90% of community-acquired UTIs and 30-50% ofnosocomially-acquired UTIs.1 Escherichia coli, the mostcommon gram negative rod causing UTI, is isolated morefrequently from women than men.2, 3, 4 Acute urinary infectioncan be associated with severe morbidity and sometimes it maybe one of the early indicator of systemic infection such as septicshock.5, 6

Owing to infrequent but severe complications of UTI, empiricantibiotic treatment is commonly adopted. However, there aresignificant local differences in susceptibility of E.coli to differenturinary agents.7 Due to the increasing antimicrobial resistance,periodic evaluation of pathogens’ susceptibility pattern isrecommended. This study was thus undertaken with the objectiveof determining the antimicrobial susceptibility pattern of E.coliwith a view to formulate guidelines for empirical of urinaryantibiotic in our set up.

MethodologyClean catch, mid-stream urine specimens were collected fromthe patients in wide mouth, sterile containers. Urine specimenswere inoculated on cysteine lactose electrolyte deficient (CLED)agar, using semi-quantitative strip method (Abtek BiologicalsLtd, Liverpool), applying 0.2 µl per strip. Innoculated agarplates were incubated aerobically at 37ºC for 18 to 24 hours.Gram negative rods (more than 20 colonies, which indicates106 organisms / ml of urine, were considered significant), wereprovisionally identified by colony morphology and Gram'sstaining. Biochemical reactions were used for the finalidentification of Escherichia coli.

For antimicrobial susceptibility testing, Mueller-Hinton agar(Oxoid, UK) was inoculated with the test organism (0.5McFarland standard) to give a semi-confluent growth.Appropriate antibiotic discs were applied on this MH Agar.

Following overnight incubation in air at 35oC ± 2, zone diameterswere measured and interpreted as per Clinical and LaboratoryStandards Institute (CLSI) guidelines.8


The antibiotic discs used and the concentration used werenitrofurantoin 300 µg, meropenem 10 µg, imipenem 10 µg,amikacin 30 µg, piperacillin-tazobactum 100/10 µg,cefoperazone-sulbactum 30/10 µg, piperacillin-sulbactum100/10 µg, gentamicin 30 µg, ceftriaxone 30 µg, ampicillin 25µg, ciprofloxacin 5 µg, trimethoprim-sulphamethoxazole 25µg, amoxacillin-clauvalanic acid 30/10 µg and Pipemadic acid20µg.

Isolates were classified as either resistant, intermediate sensitiveor sensitive based on CLSI8. E.coli (ATCC25922) was used ascontrol strain.

ResultsOut of total of 10831 urine specimens submitted for culture anddrug susceptibility, 1904 specimens yielded significant bacterialgrowth. Out of 1904 positive urine cultures, 990 (52 %) wereidentified as Escherichia coli (Figure I).

A total of 589(59.5 %) culture positive specimens were frommale patients while 401(40.5 %) were from female patients.

Susceptibility pattern of E.coli revealed that 96.3 % (953)isolates were sensitive to Imipenem, 92.9% (920) to meropenem,83.7 %(829) to Amikacin and 82.3 %(815) to Nitrofurantoin.The susceptibility percentages of E.coli against variousantimicrobials tested is shown in Table 1.

DiscussionDevelopment of resistance to antibiotics is a serious health careconcern. Resistant bacteria are not only a cause of treatmentfailure but are also more expensive to treat. The results of ourstudy have revealed that 52 % of all the culture positive isolateswere identified as E.coli. Literature has shown that frequencyof isolation of E.coli as uropathogens varies in different regions.It has been reported to be as low as 25 % in some regions ofthe world to as high as 81 % in other regions.9,10,11,12,13, 14, 15, 16,

Our study has revealed that meropenem; imipenem, amikacinand nitrofurantoin had greatest in vitro sensitivities againstE.coli, as more than 80 % of the isolates were sensitive to thesefour antibiotics. Nitrofurantoin has shown similar susceptibilitypattern in two previous studies conducted in our institute in2004 and 2010. According to these two studies 88 % and 87% of E.coli were susceptible to nitrofurantoin respectively.14, 17

This antimicrobial has also been considered as an option forempirical therapy by many authors in Latin America and UnitedStates.18-20

On the contrary, in vitro susceptibility results of E.coli againstampicillin, trimethoprim-sulphamethoxazole, amoxicillin-clavulanic acid and ciprofloxacin were quite low, as less than30 % of all the isolates were sensitive to these groups ofantibiotics. Despite the fact that trimethoprim-sulphamethoxazoleis often empirically recommended as one of the antibiotic forthe treatment of uncomplicated UTI, our study has revealedthat quite a high percentage of E.coli isolates are resistant tothis antimicrobial.21 Studies conducted in Palestine and Brazilhave also shown that 64 % and 54 % of E.coli isolated frompositive urine culture were resistant to this antibioticrespectively.22,23 As regards the susceptibility results forciprofloxacin, our results were in concordance with anotherstudy conducted in Peshawar in 2005/2006 where only 28 %of all the E.coli isolated were susceptible to this compound.24

Our isolates have shown more resistance to ciprofloxacin ascompared to the studies reported from Palestine, Canada, USAand Turkey.22, 25-27 The probable reason for this increasedresistance may be widespread usage and over the counteravailability of this antibiotic.

12000

10000

8000

6000

4000

2000

0All Specimens Culture Positive E.coli

Fig 1. Frequency of E.coli in urine culture specimens (n=990)

Table 1: Antimicrobial susceptibility profile of E.coli (n=990)

Antibiotic Sensitive Resistantn (% sensitive) (n)

Imepenem 953 (96.3) 37Meropenem 920 (92.9) 70Amikacin 829 (83.7) 161Nitrofurantoin 815 (82.3) 175Sulbactam- Cefaperazone 795 (80.3) 195Tazobactum-Piperacillin 788 (79.6) 202Sulbactam-piperacilin 621 (62.7) 369Gentamicin 532 (53.7) 548Pipemidic Acid 366 (37.0) 624Ceftriaxone 354 (35.8) 636Ciprofloxacin 290 (29.3) 700Amoxicillin-clavulanic acid 236 (23.8) 764Trimethoprim-sulphametho-xazole 234 (23.6) 766Ampicillin 80 (8.1) 910

Volume 24 Issue 01 Jan - Mar 2015. 783

Sensitivity pattern of all other tested antibiotics ranged between30 to 80 %. Similar sensitivity profile was also reported froma study conducted in India, except that in their study majorityof E.coli isolates were susceptible tocefaperazone-sulbactamandpiperacillin-tazobactam(93 % and 91 % respectively).12

Appropriate clinical information was not available with thespecimens submitted, so clinical correlation could not be done.Other centers should also publish their data regarding urinarypathogens which will help clinicians in recommending empiricaltreatment for urinary tract infections.

ConclusionE.coli was the most common isolated organism from urinecultures.Although resistance has developed against routinelyused antibiotics like ampicillin, amoxicillin-clavulanic acid,ce f t r i axone , c ip ro f loxac in and t r ime thop r im-sulphamethoxazolebut antibiotics like nitrofurantoin andcarbapenems (meropenem and imipenem) were found to beactive in vitroas more than 80 % of the isolates were susceptibleto these antimicrobials.

References1. Ejrnæs K. Bacterial characteristics of importance for recurrent urinary

tract infections caused by Escherichia coli. Dan Med Bull2011;58(4):B4187.

2. Nicolle LE. Asymptomatic bacteriuria in the elderly. Infect Dis ClinNorth Am 1997;11:647-67.

3. Bakke A, Digranes A. Bacteriuria in patients treated with clean intermittentcatheterization. Scand J Infect Dis 1991; 23:577-82.

4. Noor N, Ajaz M, Rasool SA, Pirzada ZA. Urinary tract infectionsassociated with Multidrug resistant enteric bacilli: characterization andgeneticalstudies. Pak J Pharm Sci 2004; 17(2):115-23.

5. Dembry LM, Andriole VT. Renal and perirenal abscesses. Infect DisClin North Am 1997; 11:663-80.

6. Patterson JE, Andriole VT. Bacterial urinary tract infections in diabetes.Infect Dis Clin North Am 1997; 11:735-50.

7. Livermore DM, Pearson A. Antibiotic resistance: location, location,location. Clinical Microbiology and Infection 2007;13(2):7–16.

8. Clinical and Laboratory Standards institute. Performance Standard forAntimicrobial Susceptibility Testing. Twenty-First informationalsupplement. Wayne, PA, USA 2011; 46: M100 –S21.

9. Totsika M, Moriel DG, Idris A, Rogers BA, Wurpel DJ, Phan MD,Paterson DL, Schembri MA. Uropathogenic Escherichia coli mediated urinarytract infection.Curr Drug Targets 2012 May 28. [Epub ahead of print]

10. Magliano E, Grazioli V, Deflorio L, Leuci AI, Mattina R, RomanoP,et al. Gender and Age-Dependent Etiology of Community-AcquiredUrinary Tract Infections. ScientificWorld Journal 2012; 2012: 349597.

11. Okesola AO, Aroundegbe TI. Antibiotic resistance pattern of uropathogenicEscherichia coli in South West Nigeria. Afr J Med Sci 2011;40(3):235-8.

12. Baral P, Neupane S, Marasini BP, Ghimire KR, B and Shrestha B. High

prevalence of multidrug resistance in bacterial uropathogens fromKathmandu, Nepal. BMC Res Notes 2012; 5: 38.

13. GandapurAJ, Hameed A, Asghar AH. Etiology and clinical profile ofUTI in children J Med Sci 2003;11(1):59-61.

14. Sood S, Gupta R. Antibiotic Resistance Pattern of Community AcquiredUropathogens at a Tertiary Care Hospital in Jaipur, Rajasthan. IndianJ Community Med 2012; 37(1): 39–44.

15. Khan MT and Shah SH. Experience with Gram Negative Bacilli isolatedfrom 400 cases of Urinary Tract Infection (UTI) in Abbottabad.J Ayub Med Coll Abottabad 2000; 12(4):21-3.

16. Amjad A, Mirza IA, Abbasi SA, Farwa U, Sattar A, Qureshi ZA. Spectrumand antimicrobial susceptibility pattern of pathogens causing urinary tractinfection-experience in a tertiary care setting. IDJP 2011; 20 (2):297-301.

17. Patel MH, Trivedi GR, Patel SM, Vegad MM. Antibiotic susceptibilitypattern in urinary isolates of gram negative bacilli with special relationto Amp C beta-lactamase in a tertiary care hospital. Urol Ann 2010; 2:7-11.

18. Abubaker ME. Antimicrobial susceptibility pattern of pathogenic bacteriacausing urinary tract infections at the specialist Hospital, YolaAdamawstate, Nigeria. J Clin Med Res 2009; 1(1): 1-8.

19. Butt T, Leghari MJ, Mahmood A. In vitro activity of nitrofurantoin inEnterococcus urinary tract infection. JPMA 2004; 54: 466.

20. Cales AC, Sader HS, Jones RN. Urinary tract infection trends in LatinAmerican hospitals: report from the SENTRY antimicrobial surveillanceprogram (1997-2000). Diag Microb Infect Dis 2000; 44: 289-99.

21. Karlowsky JA, Kely LJ, Thonsberry C, Sahm D F. Trends in antimicrobialresistance among urinary tract infection isolates of Escherichia coli fromfemale outpatients in the United States. Antimicrob Agents Chemother2002; 46:2540-5.

22. McOsker CC, Fitzpatrick PM. Nitrofurantoin mechanisms of action andimplication for resistance development in common uropathogens.J Antimicrob Chemother 1994; 33: 23-30.

23. Blondeau JM. Current issues in the management of urinary tract infections:extended-release ciprofloxacin as a novel treatment option. Drugs2004;64(6):611-28.

24. Astal EZ. Increasing ciprofloxacin resistance among prevalent urinarytract bacterial isolates in Gaza strip, Palestine. J Biomed Biotechnol2005; 3:238-41.

25. Santo E, Salvador MM, Marin JM. Multidrug-resistant urinary tractisolates of Escherichia coli from Ribeirao Preto, Sao Paulo, Brazil. BJID2007; 11(6): 575-8.

26. Ullah F, Malik SA, Ahmen J. Antibiotic susceptibility pattern and ESBLprevalence in nosocomial Escherichia coli from urninary tract infectionsin Pakistan. Afr J Biotechnol 2009; 8(16): 3921-6.

27. Mazzulli T, Skulnick M, Small G, Marshall W, Hoban D J, Zhanel GG,et al. Susceptibility of community Gram-negative urinary tract isolatesto mecillinam and other oral agents. Can J Infect Dis 2001; 12(5): 289-

92.28. Sahm DF, Thornsberry C, Mayfield DC, Jones ME, Darlowsky JA.

Multidrug resistant urinary tract isolates of Escherichia coli: prevalenceand patient demographics in the United States in 2000. Antimicrob AgentsChemother 2001; 45(5): 1402-6.

29. Yukse S, Ozturk B, Kavaz A, Ozcakar Z B, A car B, Guriz H, et al.Antibiotic resistance of urinary tract pathogens and evaluation of empiricaltreatment in Turkish children with urinary tract infections. Int J AntimicrobAgents 2006; 28(5): 413-6.


GUIDELINES

Erfan Hussain*, Bushra Jamil**, Naseem Salahuddin***

*Section of Pulmonary and Critical Care Medicine, Department of Medicine, **Adult Infectious Diseases,The Aga Khan University, Karachi, Pakistan.*** Indus Hospital, Karachi, Pakistan

The “3 Hour” Bundle

IntroductionSepsis is the leading cause of morbidity and mortality. It isestimated that 60-80% of deaths in low and in low to middleincome countries occurs due to sepsis.1,2 Data from Pakistanis scanty. Sepsis accounts for about 1.3% of all admissions atthe Aga Khan University Hospital, Karachi, Pakistan, and hasmortality approaching 37%, which is significantly associatedwith the presence of septic shock.3

With the publication of both the Protocol Based Care for EarlySeptic Shock (ProCESS) and the Australasian Resuscitation inSepsis Evaluation (ARISE) trials in 2014, the importance ofadherence to the 3 Hour Bundle of initial sepsis managementto significantly impact on the morbidity and mortality of patientsalong the sepsis spectrum, was recognized.4,5,6

Severe sepsis and septic shock can be managed effectivelyusing our locally-adapted sepsis guidelines, which delineatesimple interventions to be instituted in a timely manner. Thisis expected to have a significant impact on sepsis outcomes inour patients.

The 3 Hour Bundle Management Protocol (Appendix 1)There are 4 main components to the 3 Hour Bundle. These are:

1. Early recognition of Sepsis/Severe Sepsis/Septic Shock2. Diagnostic work up3. Antibiotic administration4. Fluid and vasopressor management

Early RecognitionEarly recognition of the patient presenting along the sepsisspectrum remains critical to the effective early implementationof the 3 Hour Bundle. Two main areas to the successfuldevelopment of early recognition are education and the use ofscreening tools.

1. EducationEducation remains a cornerstone in the successfulimplementation of guidelines into practice. It is hoped that withthe development and acceptance of these guidelines a local,provincial and national dialogue can be started as to how tocollect the necessary epidemiological data for sepsis as well as

as well as plan for dissemination of these guidelines to thehealthcare professionals and our patients

2. Screening ToolsThe use of a screening tool can both help educate and providerapid identification of the patient with sepsis by first responderssuch as: ER Triage nurses and physicians, Rapid Response andCardiac Arrest Teams, and the bedside and/or OPD nurse andphysician. An example of a sepsis screening tool is providedin Appendix 2.

Diagnostic Work UpWhen possible a complete diagnostic panel for patients withsepsis should be sent including:

Complete blood count and PlateletsBasic Electrolytes (Sodium, Potassium, Chloride,Bicarbonate, Glucose, Bun and Creatinine)PT/PTT and INRUrine DRCultures: Blood, Urine and SputumLactic Acid

Radiological studies (x-rays, ultrasound, CT scans) should beordered guided by the history and physical exam. If an infectivesource is identified that is amenable to surgical correction asurgical consult should be called early for appropriate sourcecontrol.

Special Consideration of the following is recommended:Blood Cultures. Please see Appendix 3 for proper samplingand handling of blood culturesBiomarkers of Sepsis: Biomarkers of sepsis such as ESRand CRP have no role in the diagnosis, prognosticationand/or therapy guidance of sepsis. At present onlyProcalcitonin has any significant sensitivity and specificityfor diagnosis, prognostication and therapy guidance forsepsis. Having said that the sensitivity and specificity forany of the above tests (including Procalcitonin) remainsless than optimal and therefore its routine use in sepsiscannot be recommended.7,8

Lactate: The last decade has seen resurgence in thediagnostic, prognostic and therapeutic guidance of lactatefor the patient with sepsis, severe sepsis septic shock.9,10,11,12

It is important to emphasize that the patient who meets the

Guidelines for the Initial Management of Adults with Sepsis/Severe Sepsis/Septic Shock: 2015


definition of sepsis and yet has no other signs, symptomsor laboratory evidence suggestive of severe sepsis can stillmeet the new operational definition of severe sepsis withan isolated lactate of 4 mmol/L or higher (Glossary ofTerms).Base Deficit: In view of the resource limitations tohealthcare delivery in Pakistan it is understood that a bloodlactate test may not be available. It is therefore suggestedthat the base deficit in an arterial blood gas sample may beused to detect acidosis in the patient with sepsis.13, 14 Thisrecommendation is made with the following caveats:

o To date there has been no study to evaluate the utility ofbase deficit in lieu of a serum lactate for the diagnosis ofsevere sepsis or as a marker for resuscitation in severesepsis/septic shock.

o Base deficit cannot be used as a surrogate marker for lactateif there is hyperchloremic acidosis from normal salineadministration, renal failure or diabetic ketoacidosis.

o Given the varied reasons for an elevated base deficit, it ishas limitations as an endpoint of resuscitation for sepsis.

AntibioticsThe importance of early and appropriate antibiotic administrationcannot be overemphasized in in managing sepsis. The study byKumar et al demonstrated that for every hour delay inadministration of antibiotics to the patient with sepsis inducedhypotension or shock resulted in 12% decreased probability ofsurvival.15

The factors which are clearly associated with sepsis outcomesinclude the site of infection, the kind of organisms involvedand their susceptibility patterns, time to diagnosis and institutionof fluid resuscitation and the types of antibiotics selected forempiric and definitive treatment.

Our local data shows that among enterobacteriaceae (E.coli,klebsiella, enterobacter) resistance patterns are as follows:3rd generation cephalosporins currently ranges from 50-80%,ciprofloxacin 30-70%, piperacillin-tazobactam 20-30% andcarbapenems between 2-15% .16

Of special consideration is the fact that carbapenem resistanceenterobacteriaceae (CRE) is associated with a high sepsis-related mortality of up to 80%.17 The numbers of deaths are 2-fold higher among patients with bacteremia caused by CRE ascompared with carbapenem-sensitive enterobacteriaceae (CSE).18

In our context, mortality secondary to bacteremia caused bycarbapenem- resistant gram negatives (includingCRE,acinetobacter and pseudomonas) has been 54% vs 36%due to sensitive organisms. With additional resistance to 3rd

generation cephalosporins and ciprofloxacin the mortalityapproaches 90% .19

Please see Appendix 5 for details.

Intravenous Fluids and Vasopressors:

Intravenous Access:The following is suggested:o Initial IV access for sepsis should be an upper extremity

peripheral lineo Initial IV access for severe sepsis and septic shock should

be either single or two upper extremity peripheral lines of18 gauge or larger when possible.

Pedal peripheral IV access (foot IVs) should be avoided becauseof the increase in transit time from the foot to the centralcirculation that can result from vasoconstriction from hypotension,shock, and/ or the administration of vasopressors.20, 21

Type of fluid for resuscitation:o For sepsis there is no recommendation for routine fluid

administration.o For severe sepsis and septic shock crystalloids (Normal

saline or Ringers Lactate) are recommended over colloids.9, 22

Dose and method of fluid administration for severe sepsisand septic shock.10, 23, 24

o In the 3 Hour bundle fluid is administered to a total doseof 30cc/kg.

o Crystalloid is administered as a fluid bolus250 cc over 10 minutes500 cc over 15 minutes1000 cc over 30 minutes

o Administer as follows:10 ml/kg over 15-30 minutesIf a systolic blood pressure of 90mmHg or greater or aMean Arterial Pressure of 65 mmHg or greater is notreached after the above bolus then repeat 5ml/kg over 15minutes and repeat every 15 minutes until the above bloodpressure goal is reached or a total of 30 cc/kg administered.

Vasopressorso If vasopressors are used to support the blood pressure while

the fluid boluses are given or after the 30ml/kg total doseis given and the patient is still in shock the following isrecommended.9, 25

Norepinephrine is the first line vasopressor for use in septicshockDopamine may be used in place of Norepinephrine ifNorepinephrine is not availableIf the maximum dose of Norepinephrine or Dopamine isreached, and the patient is still in shock, then Epinephrinecan be added.Vasopressin in doses of 0.03-0.04 units/min can be addedto norepinephrine to help attain the above blood pressure


targets or to reduce the dose of norepinephrine being given.

Recommendations for implementation and monitoring ofthe 3 Hour Bundle

Order sets provide a method of funneling the decisionmatrix of the bedside provider along best practicerecommendations.26 An example of an order set modifiedand developed at the Aga Khan University Hospital (AKUH)Karachi Pakistan is presented in the appendix section ofthis document (Appendix 4). The addition of a carbon backto the order set allows easy storage and later abstractionof data for monitoring of compliance with the bundleelements.When following compliance of the 3 hour bundle in anemergency room, time zero is when the patient presents totriage.An algorithm for the 3 hour bundle, modified and developedat the AKUH Karachi Pakistan is presented in Appendix 1.Following an institution’s lactate test volume and volumeof norepinephrine units used can help an organizationfollow the penetration of the sepsis bundle in thatinstitution.12

Glossary of Terms:

1. Bundle:A set of evidence based diagnostic and therapeutic interventions(usually 3-5 elements) that together can help improve patientoutcomes.27

2. Systemic Inflammatory response (SIRS)28 2 or more ofthe following:a. Temp>38 or <36b. Heart rate >90 beats/min

c. Resp rate >20 breaths/mind. WBC >12,000 or <4,000 or 10% bands

3. Sepsis28

SIRS + Infection (known or suspected source) = Sepsis

4. Severe Sepsis28

Sepsis criteria + evidence of organ dysfunction or Sepsiscriteria + Lactate >/ 4mmol/L

Cardiovascular: Systolic BP ≤90 mmHg, MAP ≤70 mmHg for at least 1 hour despite volume resuscitation, or theuse of vasopressors.Renal: Urine output < 0.5 ml/kg body weight/hr for 1 hourdespite volume resuscitation. Creatinine > 2.0 mg/dLPulmonary: PaO2/FiO2 ≤250 if other organ dysfunctionpresent or ≤ 200 if the lung is the only dysfunctional organ.Hematologic: Platelet count ≤100K or decreased by 50%in 3 daysMetabolic: pH ≤ 7.3 and plasma lactate > 4Altered Mental StatusBilirubin > 2mg/dL

5. Septic Shock10

Persistent arterial hypotension despite adequate volumeresuscitation

6. Hypotension10

Systolic blood pressure of <90 mmHg, or a mean arterialpressure (MAP) < 65 mmHg, or a decrease of >/ 40mmHg inthe systolic blood pressure from baseline

7. Shock10

Life threatening, generalized form of circulatory failureassociated with inadequate oxygen utilization by the cellsresulting in cellular dysoxia

( See appendex 1 to 5 from next page.)


Appendix 1

Start Antibiotics (Refer to Hospital Antibiogram)

Recognize Severe Sepsis. FILL OUT SEPSIS ORDER SET

ADVANCE RESUSCITATION : CALL EXPERT CONSULTATION

SIRS criteria• Temp>38°C or <36°C• Heart rate >90 beats/min• Resp rate >20 breaths/min• WBC >12,000 or <4,000 or 10% bands

SIRS + Infection = SEPSIS

Sepsis criteria + evidence of organ dysfunction or Sepsis criteria + Lactate 4mmol/L

• CV: Systolic BP ≤90 mmHg, MAP ≤70 mm Hg for at least 1 hour despite volume resuscitation,or the use of vasopressors.

• Renal: Urine output < 0.5 ml/kg body weight/hr for 1 hour despite volume resuscitation• Pulmonary: PaO2/FiO2 ≤250 if other organ dysfunction present or ≤200 if the lung is the only dysfunctional organ.• Hematologic: Platelet count ≤100K or decreased by 50% in 3days• Metabolic: pH ≤7.3 and plasma lactate > 4 mmol/L• Altered Mental Status

SEVERE SEPSIS

Cultures + Lactate, CBC, Basic Metabolic panel PT/PTT INR, LFTs, Urine DR

Radiology: CxR and others

Early Surgical Consultation is recommended if the above work up suggests a surgical amenable focus of infection

If MAP <65 mmHg and/or Lactate > 4mmol/L and/or signs/labs of organ dysfunction START IVF Crystalloid(10 ml/kg) over 15-30 minutes THEN If BP goal not attained give 5ml/kg every 15 minutes up to 30 ml/kg

PATIENT REMAINS WITH MAP < 65 mmHg AFTER I L of FLUID STARTNOREPINEPHRINE AT 0.01 µg/kg/min and titrate to MAP 65 mmHg. CONTINUE FLUID RESUSCITATIONUNTIL TOTAL 30cc/kg/ given

Insert Central Venous Access at 3 hours if patient still in shock and titrate Norepinephrine to obtain a MAP >/ 65 mmHg.IF PATIENT IN SHOCK AFTER 3 HOURS OR REPEAT LACTATE 4 mmol/L at 3 HOURS THEN:


Evaluation for Severe Sepsis Screening Tool

Appendix 2

Instructions: Use this optional tool to screen patients for severe sepsis in the emergency department, on themedical/surgical floors, or in the ICU.

1. Is the patient’s history suggestive of a new infection?

Pneumonia, empyema Bone/joint infection Implantable device infectionUrinary tract infection Wound infection Other infectionAcute abdominal infection Blood stream catheter infectionMeningitis EndocarditisSkin/soft tissue infection Yes No

2. Are any two of following signs & symptoms of infection both present and new to the patient? Note: laboratoryvalues may have been obtained for inpatients but may not be available for outpatients.

Hyperthermia > 38.3 °C (101.0 °F) Tachypnea > 20 bpm Hyperglycemia (plasma glucoseHypothermia < 36 °C (96.8°F) Leukocytosis (WBC count >12,000 µL–1) >140 mg/dL) or 7.7 mmol/L inAltered mental status Leukopenia (WBC count < 4000 µL–1) the absence of diabetesTachycardia > 90 bpm

If the answer is yes, to both questions 1 and 2, suspicion of infection is present:

Obtain: lactic acid, blood cultures, CBC with differential, basic chemistry labs, bilirubin.At the physician’s discretion obtain: UA, chest x-ray, amylase, lipase, ABG, CT scan.

Yes No

3. Are any of the following organ dysfunction criteria present at a site remote from the site of the infection that areNOT considered to be chronic conditions? Note: in the case of bilateral pulmonary infiltrates the remote site stipulationis waived.

SBP < 90 mmHg or MAP <65 mmHgSBP decrease > 40 mm Hg from baselineCreatinine > 2.0 mg/dl (176.8 mmol/L) or urine output < 0.5 ml/kg/hour for 2 hours Bilirubin > 2 mg/dl (34.2 mmol/L)Platelet count < 100,000 µL Lactate > 2 mmol/L (18.0 mg/dl)Coagulopathy (INR >1.5 or aPTT >60 secs)Acute lung injury with PaO2/FiO2 <250 in the absence of pneumonia as infection sourceAcute lung injury with PaO2/FiO2 <200 in the presence of pneumonia as infection source

Yes No

If suspicion of infection is present AND organ dysfunction is present, the patient meets the criteria for SEVERESEPSIS and should be entered into the severe sepsis protocol.

Date: _/ / (circle: dd/mm/yy or mm/dd/yy) Time: _: (24 hr. clock)Version 7.2.13


Appendix 3

Technique for Proper Blood Culture Sampling

1. Gather necessary equipment: 20 ml syringe, 2 aerobicand 2 anaerobic bottles for 2 sets of blood cultures, skinprep swabs, tourniquet, sharps disposal box, gloves.

2. Verify the patient's identification and explain procedure

3. Wash or sanitize hands before and after removing gloves.Follow Standard Precautions for all patients. Wear cleangloves.

Masks with face shields may be worn for drawing bloodcultures depending on the clinical situation.

4. Assemble necessary equipment before preparation of thepatient's skin.1. Remove dust caps from culture bottles.2. Clean surface with alcohol wipe.3. Leave the alcohol wipe on the bottle top during skin

preparation.

5. Remove alcohol wipe just prior to inoculating the bottles- do not use iodine.

6. Apply tourniquet to the extremity and identify thephlebotomy site.

7. Preparation of the phlebotomy site: clean site withchloraprep or 70% isopropyl alcohol for 30 secondsprior to chlorhexidene or povidone iodine (if chorhexideneis not available,inferior)

1. Using chloraprep, use a firm scrubbing motion for

30 seconds over a 5 cm area of the skin using circularmotion starting at the site and working outward.

2. Allow 30 seconds for drying before venipuncture.3. Using sequential chlorhexidene or povidone iodine,

cleanse a 5 cm area using circular motion startingat the site and working outward.

4. Allow to dry for a at least 30 seconds to allowantiseptic effect

5. Clean patient's skin with alcohol to remove excessiodine (to prevent iodine burns).

If unable to use either of the abovea) Use alcohol to cleanse the patient's skin, using a

circular motion starting at the site and movingoutward.

b) Repeat times two.c) Allow to dry.

6. Do not touch the venipuncture site after skinpreparation. If palpation is absolutely necessary,sterile gloves must be applied immediately prior topalpation.

7. Insert needle into vein and withdraw 20 ml of blood.Do not collect blood through IV cannula even it isfreshly inserted.

8. Inject 10 ml of blood into each culture bottle. Needlesshould not be changed before inoculating culture medium.

9. If an inadequate amount of blood was obtained (lessthan 5 ml), and repeat phlebotomy cannot be performed;all blood should be preferentially inoculated in theaerobic culture bottle.

10. Label culture bottles with patient's name and MR number.

11. Fill out Microbiology lab slip.1. Indicate site from which blood was collected using

comment section. If using aVascular Access Device(VAD) to draw culture, you must indicate type andsite of VAD in the comments section (i.e., leftsubclavian triple lumen).

2. Indicate suspected diagnosis, if necessary (requiredfor R/O endocarditis).

3. Include date and time of collection.4. Document that cultures were obtained on appropriate

form

12. Send specimens to the laboratory as soon as possible.Do not refrigerate blood culture specimens.

Contd. on next page


13. Send second set of blood cultures using the sameprocedure as above. If a different peripheral site ispossible, the second set may be drawn immediately. Ifusing the same site, wait at least 10 minutes for thesecond set, and if possible (i.e. not waiting to giveantibiotics) draw second set 1-3 hours later.

Biosafety Considerations:Discard used syringes in the sharps bin; used gloves andswabs should be discarded as controlled medical waste.

14. In order to rule out diagnoses, more specific bloodculture procedures may be necessary.1. Suspected catheter sepsis

1. Draw two culture sets, using a fresh syringefor every venipuncture/draw

2. One set is obtained from a suspected site.3. Second set must be from a separate peripheral site.4. If catheter is removed, send tip (3 cm) using

sterile procedure for cultures. Do not sendcatheter tipwithout sending concomitantblood cultures.

2. Acute endocarditis1. Draw two culture sets from two separate sites

during the first 1-2 hours of evaluation.2. Begin therapy.

3. Subacute endocarditis1. Draw 2-3 blood culture sets on day 1.2. If all are negative additional sets can be drawn

on days 2 and 3 (no more than 4 sets in a 24hour period).

3. Immediate antibiotics are less important thanestablishing a specific microbial diagnosis.

4. Endocarditis patients on anti-microbial therapy1. Draw resin blood culture sets on each of three

successive days.2. Indicate "R/O endocarditis" in special

instructions on lab requisition.


Appendix 4 Patient Identification & Location

PHYSICIANS’ ORDERS

ADULT SEPSIS ORDER SET PAGE 1 of 3

Note: Check Box Where Appropriate

PLEASE WRITE ALL ‘MEDICATION AND IV FLUID’ ORDERS ON THE SEPARATE SHEET,DESIGNED FOR THE PURPOSE

RN SIGNATURE - TIMEDATE AND TIME ORDER, SIGNATURE AND TITLE

IV Saline Lock with flush of Normal Saline 3ml every 12 hours

Allergies:Attending Physician:Primary Diagnosis:

Sepsis Severe Sepsis Septic Shock

Secondary Diagnosis: Pneumonia Urinary Tract InfectionAbd Infection Other

Condition: Critical Stable

Admit to:

Oxygen:Vital Signs every 15 minutes X 1 hour, then per unit policyStrict Intake and OutputActivity: Bed Rest OtherDiet: NPO Other

LABS:Sepsis Panel (IF CHECKED SEND ENTIRE LABS BELOW): CBC with Differential and Platelet Count: PT/PTT: BUN/Creatinine: Serum Electrolytes (Na, K, Cl, HCO3, BUN, Creatinine): Lactic Acid and Repeat every 6 hours for next 24 Hours: Urine DR: Procalcitonin: Blood Cultures X2: Urine Cultures: Sputum Cultures: Cultures Other

Additional Labs:

Chest X-RayOther Imaging Studies

EKG 12 Lead


Patient Identification & LocationPHYSICIANS’ ORDERSADULT SEPSIS ORDER SET PAGE 2 of 3



RN SIGNATURE - TIMEDATE AND TIME ORDER, SIGNATURE AND TITLEDVT Prophylaxsis:

ANTIBIOTICS: ADJUST FOR RENAL and/or HEPATIC DYSFUNCTION

SEPSIS SOURCE UNKNOWN:Piperacillin-Tazobactum 3.375gm Q6 Hours IVORImipenem Cilastatin 500mg Q8 Hours IVORCefoperazone Sulbactam 400mg Q12 Hours IVAnd Clindamycin 600mg Q8 Hours IV

IF MRSA SUSPECTED THEN ADDVancomycin:Load 20mg/kg IV

Maintenance 15mg/kg Q 12 Hours IVSUSPECTED

Piperacillin-Tazobactum 3.375gm Q6 Hours IVAnd Levofloxacin 500mgQ24 Hours IV

Ceftriaxone 2gm Q24 Hours IVAnd Levofloxacin 500mg Q24 Hours IV

OR

SUSPECTED SKIN & SOFT TISSUE INFECTION:Cefazolin 2gm Q8 Hours IV

IF Penicillin AllergicClindamycin 600mg Q8 Hours IVAndCiprofloxacin 400mg Q12 Hours IV

IF MRSA SUSPECTED THEN ADDVancomycin: Load 20mg/kg IV

Maintenance 15mg/kg Q 12 Hours IV

SUSPECTED ABDOMINAL INFECTION:Piperacillin-Tazobactum 3.375gm Q6 Hours IVORImipenem Cilastatin 1 gm Q8 Hours IVORCeftriaxone 2gm Q24 Hours IVAnd Amikacin 15mg/kg Q24 Hours IVAnd Metronidazole 500mg Q8 Hours IV


IF AFTER RECEIVING FLUID BOLUS 30ml/kg PATIENT STILLIN SHOCK AND/OR REPEAT LACTATE 4 OR GREATER OBTAINURGENT EXPERT CONSULTATION

Patient Identification & LocationPHYSICIANS’ ORDERSADULT SEPSIS ORDER SET PAGE 3 of 3



RN SIGNATURE - TIMEDATE AND TIME ORDER, SIGNATURE AND TITLESUSPECTED UROSEPSIS

Piperacillin-Tazobactum 3.375gm Q6 Hours IVORImipenem Cilastatin 500mg Q8 Hours IV

IF RISK OF ENTEROCCOCUSVancomycin: Load 20mg/kg IV

Maintenance 15mg/kg Q 12 Hours IVSUSPECTED CNS INFECTION

Ceftriaxone 2gm Q24 Hours IVANDVancomycin: Load 20mg/kg IV

Maintenance 15mg/kg Q 12 Hours IVORMeropenem 2gm Q8 Hours IV

SUSPECTED HOSPITAL ACQUIRED INFECTIONPiperacillin-Tazobactum 3.375gm Q6 Hours IVAND Vancomycin: Load 20mg/kg IV

Maintenance 15mg/kg Q 12 Hours IVOR

Imipenem Cilastatin 500mg Q8 Hours IVAND Vancomycin: Load 20mg/kg IV

Maintenance 15mg/kg Q 12 Hours IVAND Colistin 9MU stat and 3MU Q 8hours IV

See appendix 5 for detailsFOR PATIENTS WITH SYSTOLIC BLOOD PRESSURE LESS THAN 90 mmHgInitial intravenous fluids per kg estimated ideal body weightTarget BP greater than 90 mmHg systolic and Mean Arterial Pressure(MAP) greater than 65 mmHg

Normal saline (10 ml/kg) ml over 15-30 minutes THENIf BP goal not attained 5ml/kg every 15 minutes up to 30 ml/kgRingers Lactate (10 ml/kg) ml over 13-30 minutes THENIf BP goal not attained 5ml/kg every 15 minutes up to 30 ml/kgVASOPRESSOR (Titrate to systolic BP greater than 90 mmHg andMAP greater than 65 mmHgNorepinephrine mg in ml D5W at micrograms/minContinuous Infusion


Before Selecting Empirical therapy

Appendix 5 Antibiotics in Sepsis

• Selection of antibiotic must be based on clinical assessment of site of infection• Viremia, severe malaria or fungemia must be considered as possible causes of sepsis• Antibiotics must be administered as soon as possible, within 2 hours of admission to ER or ICU• Two sets of blood cultures, urine analysis and urine culture must be drawn prior to institution of antibiotic• Obtain history of previous use of antibiotics in past 3 months. Avoid same antibiotic if possible• Dose must be prescribed on weight basis• Dose must be adjusted for renal or hepatic insufficiency, diabetes• Hematologic malignancy or febrile neutropenia must be considered• Combination therapy and use of colistin may be considered for suspected highly resistant pathogens, e.g sepsis in patients with pro-longed hospital or ICU stay, transfer from another healthcare facility, immunocompromised status.• Only intravenous antibiotic should be used until there is clinical improvement• Empiric antifungal therapy may be considered initially in specific circumstances, e.g. perforated viscus or prolonged

antibacterial therapy in immunocompromised patients

During antibiotic therapy

• Once culture and sensitivity reports are available, de-escalate to a narrower spectrum antibiotic• Once patient shows clinical improvement and is stable, de-escalate to oral preparation, if an equally effective oral

preparation is available• Antibiotic should be given for no longer than 7-10 days• Source control is essential, i.e. drainage of abscess, repair or resection of perforated viscus, removal of cannula, catheter,

devices, debridement of infected tissue.

Table 1

Best empirical antibioticSource of infection Likely pathogen

Urinary tract E. coli Carbapenem or Piperacillin –tazobactam orCefaperazone-sulbactam

Genital tract E. coli, Enterococcus, S hemolyticus, Carbapenem or Piperacillin –tazobactam orAnaerobes Cefaperazone-sulbactam + vancomycin

Respiratory tract (CAP) S. pneumoniae, atypical Ceftriaxone +levofloxacin or clarithromycinRespiratory tract (HAP) GPC, GNR, atypical Carbapenem or Piperacillin –tazobactam or

Cefaperazone-sulbactam + levofloxacin or clarithromycin

Respiratory tract (VAP) GNR, MRSA Carbapenem or Piperacillin –tazobactam+ Colistin (colistimethate sodium)9MU stat and3MU Q 8hours IV orCefaperazone-sulbactam + vancomycin+ Colistin9MU stat and 3MU Q 8hours IV

Intra-abdominal Gram negatives, anerobes Carbapenem or Piperacillin –tazobactam orCefaperazone-sulbactam + vancomycin

SSTI (necrotizing fasciitis) S. aureus, Streptococci anerobes Amoxicilin/clavulanate or clindamycin+vancomycin

Burn sepsis S. aureus, Streptococci, Pseudomonas, Carbapenem or Piperacillin –tazobactam orCandida Cefaperazone-sulbactam + vancomycin

Line sepsis S. aureus, (MSSA, MRSA), Pseudomonas Ceftazidime or amikacin+ vancomycinInfected device S. aureus, (MSSA, MRSA), Pseudomonas Ceftazidime or amikacin+ vancomycinBacterial meningitis S pneumoniae, Meningococcus Ceftriaxone + vancomycin + steroid


References1. www.pjcm.net/Data/Year2013/pdf_v19_n2_a5.pdf Last accessed Feb 23

20152. Kissoon N, et al. World Federation of Pediatric Intensive Care and Critical

Care Societies: Global Sepsis Initiative. Pediatr Crit Care Med. 2011;12(5):494-503.

3. Siddiqui S, Jamil B, Nasir N, Talat N, Khan FA, Frossard P, Hussain R.Characteristics and outcome of sepsis - A perspective from a tertiary carehospital in Pakistan. International Journal of Scientific & EngineeringResearch 2013; Volume 4(9):1013-1023

4. The ProCESS Investigators. A randomized trial of protocol-based carefor early septic shock. N Engl J Med 2014;370:1683-93

5. The ARISE Investigators and the ANZICS Clinical Trial Group. GoalDirected Resuscitation for Patients with Early Septic Shock. N Eng JMed 2014;371:1496-506

6. Lilly CM. The ProCESS Trial-A New Era of Sepsis Management. N EnglJ Med 2014;370;1750-1751

7. Simon L, Gauvin F, Amre DK, Saint-Louis P, Lacroix J. Serum procalcitoninand C-reactive protein levels as markers of bacterial infection: a systematicreview and meta-analysis. Clinical infectious diseases. 2004;39(2):206-17

8. Riedel S. Procalcitonin and the role of biomarkers in the diagnosis andmanagement of sepsis Diagnostic Microbiology and Infectious Disease

2012; 73: 221-2279. Dellinger RP, Levy MM, Rhodes A, Annane D, Gerlach H, Opal SM,

et al. Surviving Sepsis Campaign: international guidelines for managementof severe sepsis and septic shock, 2012. Intensive care medicine.2013;39(2):165-228.

10. Cecconi M, De Backer D, Antonelli M, Beale R, Bakker J, HoferC, et al. Consensus on circulatory shock and hemodynamic monitoring.Task force of the European Society of Intensive Care Medicine. Intensivecare medicine. 2014;40(12):1795-815.

11. Nguyen HB, Kuan WS, Batech M, Shrikhande P, Mahadevan M, Li CH, et al. Outcome effectiveness of the severe sepsis resuscitation bundlewith addition of lactate clearance as a bundle item: a multi-nationalevaluation. Crit Care. 2011;15(5):R229.

12. Casserly B, Phillips GS, Schorr C, Dellinger RP, Townsend SR, OsbornTM, et al. Lactate measurements in sepsis-induced tissue hypoperfusion:results from the surviving sepsis campaign database*. Critical caremedicine. 2015;43(3):567-73.

13. Englehart MS, Schreiber MA. Measurement of acid-base resuscitationendpoints: lactate, base deficit, bicarbonate, or what? Curr Opin Crit Care2006; 12:569-574

14. Mutschler M, Nienaber U, Brockamp T, Wafaisade A, Fabian T, PaffrathT, et al. Renaissance of base deficit for the initial assessment of traumapatients: a base deficit-based classification for hypovolemic shockdeveloped on data from 16,305 patients derived from the TraumaRegisterDGUÂ®. Crit Care. 2013;17(2):R42.

15. Kumar A, Roberts D, Wood KE, Light B, Parrillo JE, Sharma S, et al.Duration of hypotension before initiation of effective antimicrobial therapyis the critical determinant of survival in human septic shock*. Criticalcare medicine. 2006;34(6):1589-96.

16. http://www.parn.org.pk/index_files/MULTI.RESISTANT.html Lastaccessed Feb 23, 2015

17. Falagas ME, Lourida P, Poulikakos P, Rafailidis PI, Tansarli GS. Antibiotic

Table 2

Class Spectrum Available preparations Route of Effective against Not effective administration against

Carbapenem Broad Meroponem/imepenem Intravenous GPC, GNB, MRSA, VRE./ ertapenem anaerobes Ertapenem ineffective

against pseudomonasB lactamase Broad Piperacillin – tazobactam Intravenous GPC, GNB, MRSA, VREinhibitor anaerobes.3rd genCephalosporin Broad Cefaperazone- sulbactam Intravenous GPC, GNB, MRSA, VRE

anaerobes.1st gencephalosporin Narrow Cefazolin, Cephradine Intravenous Strept MRSA,VRE,anaerobes3rd genCephalosporin Broad Ceftriaxone Intravenous Strept, GNR MRSA,VRE,anaerobes3rd genCephalospor Broad Ceftazidime Intravenous GNR, esp pseudom MRSA,VRE,anaerobesGlycopeptide Narrow Vancomycin Intravenous MRSA,enterococcus GNR, anaerobesAminoglycoside Narrow Amikacin, Tobramycin, Intravenous GNRStrept and Entero,, anaerobes

GentamicinB lactamaseinhibitor Broad Amoxicillin- clavulanate Intravenous GPC, some GNR, E coli, enterobacteriacae

and oral anaerobesFluoroquinolonesBroad Levofloxacin Intravenous GPC,atypical resp Anaerobes

and oral pathogensMacrolides Narrow Azithromycin, Oral Atypical resp GNR, anaerobes

clarithromycin pathogensLincosamide Narrow Clindamycin Intravenous Strep, Staph GNR

and oral (MSSA)

GPC= gram positive cocci, GNR= gram negative rodsMSSA= methicillin sensitive Staph aureusMRSA= methicillin resistant Staph aureus

Submitted by : Dr. Naseem SalahuddinEndorsed by : Members of Medical Microbiologists and

Infectious Disease Society of Pakistan(MMIDSP)


treatment of infections due to carbapenem-resistant Enterobacteriaceae:systematic evaluation of the available evidence. Antimicrob Agents.Chemother. 2014;58(2):654-63

18. Falagas ME, Tansarli GS, Karageorgopoulos DE, Vardakas KZ. Deathsattributable to carbapenem-resistant Enterobacteriaceae infections. EmergInfect Dis. 2014 Jul;20(7):1170-5.

19. Kalam K, Qamar F,Kumar S,Ali S,Baqi S. Risk factors for carbapenemresistant bacteraemia and mortality due to gram negative bacteraemia ina developing country. J Pak Med Assoc. Vol. 64, No. 5, May 2014. 530-536

20. Doan LA Peripheral versus central venous delivery of medications duringCPR Ann Emerg Med 1984 Sep; 13(9 Pt 2): 784-6

21. Emerman CL, Pinchak AC, Hancock D, Hagen JF. Effect of injection sitein circulation times during cardiac arrest. Crit Care Med 1988 Nov;16(11):1138-1141

22. Raghunathan K, Murray PT, Beattie WS, Lobo DN, Myburgh J, SladenR, et al. Choice of fluid in acute illness: what should be given? Aninternational consensus. British journal of anaesthesia. 2014;113(5):772-83.

23. Hoste EA, Maitland K, Brudney CS, et al for the ADQI XII InvestigatorsGroup. Four phases of intravenous fluid therapy: a conceptual model.British Journal of Anaesthesia 2014; 113(5): 740-747

24. Vincent JL, Weil MH. Fluid challenge revisited. Crit Care Med 2006; 34:1333-1337

25. De Backer D, Biston P, Devriendt J, Madl C, Chochrad D, Aldecoa C,et al. Comparison of dopamine and norepinephrine in the treatment ofshock. New England Journal of Medicine. 2010;362(9):779-89.

26. Thiel SW, Asghar MF, Micek ST, Reichley RM, Doherty JA, Kollef MH.Hospital-wide impact of a standardized order set for the management ofbacteremic severe sepsis*. Critical care medicine. 2009;37(3):819-24.

27. Bundle Definition: http://www.ihi.org/topics/bundles/Pages/default.aspxLast accessed Feb 19, 2015

28. ACCP-SCCM Consensus Conference; definition of sepsis and multipleorgan failure and guidelines for the use of innovative therapies in sepsisCrit Care Med 1992; 20:864-874

Corresponding Author: Ejaz A. Khan,Department of Pediatrics, Shifa International Hospital Ltd,Sector H-8/4, Islamabad.Email: [email protected]

Volume 24 Issue 01

CASE REPORT

Autoimmune hemolytic anemia in a child with AIDS

Seema Sakina*, Ayesha Junaid**, Ejaz Ahmed Khan*

*Department of Pediatrics and **Hematology, Shifa International Hospital Ltd, Islamabad, Pakistan

Abstract

Anemia in Human Immunodeficiency Virus (HIV) disease isa common problem, and associated with multiple etiologies.Autoimmune hemolytic anemia in HIV infected people is arare disease and it is not frequently reported in medical literature.We report a seven year old child with HIV-AcquiredImmunodeficiency syndrome (AIDS) who presented withautoimmune hemolytic anemia and will review literature ofHIV-associated autoimmune hemolytic anemia.

Key wordsHIV, autoimmune hemolytic anemia, antiretroviral therapy

IntroductionPediatric mortality due to Human Immunodeficiency Virus (HIV)has declined remarkably from 40% in 2009 to 31% in 2013 dueto the availability of improved antiretroviral therapy (ART).1 HIVassociated anemia is a major burden on morbidity and mortalityof patients.2 These children have poor over all prognosis,3 survival4

and poorer short-term virologic response to antiretroviral therapy.5

Many factors may be contributory for anemia in HIV infectedchildren such as HIV itself, micronutrient deficiency, opportunisticinfections, anemia of chronic disease and antiretroviral drugs.Autoimmune hemolytic anemia (AIHA) has rarely been describedin HIV-infected children, which may be fatal.3 We are reportinga case of a seven year old child with Acquired-Immune DeficiencySyndrome (AIDS) who presented with severe anemia and positivedirect and indirect coombs test. A review of HIV-associated AIHAwith clinic-pathological features is presented here.

Case reportA one year old boy presented in in our clinic with cough andfever since 20 days, and positive maternal HIV infection. Hewas born by cesarean section at term and was breast-fed forone month. There was no history of blood products and heremained well prior to presentation. He achieved hisdevelopmental milestones on time. His father died a month agodue to AIDS. Initial evaluation in the child showed positiveHIV antibody, CD4 count 374 cells/mm3 and HIV-I RNA1.32x105 copies/mm3. Rest of his workup was unremarkable.The child was started on ART containing lamivudine, zidoviduneand nelfinavir.

During his treatment the patient had regular follow up, was onco-trimoxazole (Septran) prophylaxis, had recommendedvaccinations, supplemental iron and multivitamins. He showedprogressive clinical (weight gain) and immunologic (peak CD4count 1046 cells/mm3 at 2 years) improvement over next fewyears. During course of next 3 years the patient developed someopportunistic infections including oral candidiasis and repeateddiarrheal episodes. His nelfinavir was switched to nevirapineat age 3.5 years. At age 5.5 years the child presented withpersistent fever and lymphadenopathy for 6 months, whichsettled after starting anti-tuberculous therapy that was givenfor nine months.

At 7 years he presented with weakness, fatigability and poorweight gain. On examination he had pallor, oral thrush,hepatomegaly with drop in CD4 count to 10 cells/mm3 andHIV viral load of 3x104 copies/mm3. Further evaluation forHIV drug-resistance showed protease inhibitor mutations (L90M,L10F), reverse transcriptase mutations (M41L, A62V, M184V,D67N, L210W, T215Y and K219N) and non-nucleoside reversetranscriptase inhibitor mutations (K101Q, Y181I). This translatesto high level resistant to nelfinavir, lamivudine, emtricitabine,delaviridine and nevirapine. Patient was switched to secondline available ART regimen including efavirinz, lamivudineand lopinavir/ritonavir.

After a few months the child again presented with anorexia,pallor and lethargy. On examination he had severe pallor,jaundice and hepatosplenomegaly. Investigations showedhemoglobin of 4.2 g/dl, LDH 762 IU/L and positive Coombstest (direct and indirect). The peripheral blood picture showedvery high reticulocyte of 75% (Figure 1a, 1b).

Jan - Mar 2015. 797

Fig 1a & 1b. Reticulocytes shown on supra vital stain after370C incubation of blood.

Fig 1a Fig 1b


On basis of the examination and laboratory findings a diagnosisof HIV-associated AIHA was made. He was managed withpacked red blood cell transfusions and prednisone 2 mg/kg/dosewith ART discontinued for 7 days.

Child showed significant improvement with steroids withhemoglobin of 8.4 gm/dl and reticulocytes decreased to50% in 2 weeks and improved to normal values after 4weeks. The steroids were given for 2 weeks and thentapered over next 2 weeks. After no further evidence ofhemolysis child was restarted on second line ART regimen.He had no further episodes of hemolysis. However hecontinued to remain unwell off and on over next few yearswith fevers, diarrheas, growth failure and falling CD4 to10 cells/mm3. He eventually expired at age of 10 yearsprobably due to drug-resistant-HIV and opportunisticinfections (tuberculosis, pneumonia, recurrent GE andfevers).

DiscussionThe prevalence and etiologies of anemia in HIV-infected childrenmay be diverse as shown in many studies. Anemia occurs in20-70% of HIV-infected children especially those with AIDS.6 It is an independent risk factor for disease progression anddeath.3,4 A systemic review of anemia in children with HIV/AIDSin both western and tropical countries showed anemia incidenceranging from 0.41 to 0.44 per person-year.3 Mild (hemoglobin<11 gm/dl) and moderate (hemoglobin <9 gm/dl) anemia weremore prevalent with HIV infection. There was limited data onmore severe anemia (hemoglobin <7 or <5 g/dl). The reviewconcluded that anemia is a very common complication ofpediatric HIV infection and is associated with poor prognosis.3

A retrospective study of 248 HIV children from India showedan overall prevalence of anemia to be 66% with 8% havingsevere anemia (Hb<7 g/dL).7 It highlighted some independentrisk factors such as age younger than 6 years, advance HIVdisease stage, stunting, rural residence and co-infection withpulmonary tuberculosis. A recent adult series and literaturereview has shown that cases of AIHA occurred in HIV patientswho are severely immunosuppressed.8

The etiology of anemia in children with HIV infection is notentirely clear and may be multifactorial in origin. These factorscan suppress the bone marrow, which then fails to produce anadequate number of red blood cells (RBCs). Patients with HIVinfection often have lower than normal levels of vitamin B12,folate and iron. Possible causes include decreased red cellproduction, nutritional deficiency of iron, folic acid and vitaminB12. Opportunistic infections, HIV-associated nephropathycausing decrease production of endogenous erythropoietin,myelosuppression by drugs such as zidovidune orproinflammatory cytokines (IL-1 and TNF) or increased redcell destruction due to autoantibodies causing autoimmunehemolysis have been implicated as other causes.2 The HIV virus

may infect the progenitorstem cell in bone marrow9 or causesit to function abnormally.10 Autoimmune diseases andautoantibodies may also develop at different stages of HIVinfection. 11

Autoimmune antibodies in HIV infected children have rarelybeen reported. Literature shows very limited cases reported inchildren with HIV-associated AIHA. Literature shows one casereport of severe AIHA and was associated with disseminatedintravascular coagulation in a 5 months old infant.12 Anotherhospital based screening study for AIHA from Nigeria of 98adult patients found a frequency of 3.06%.13 It suggested thatall patients with HIV infection and anemia should be screenedfor AIHA before giving any blood transfusions to avoid fataltransfusion reactions. Autoantibodies may be a significant causeof anemia but reticulocytopenia may lead to the under diagnosisof AIHA in HIV-infected patients.14

The clinical presentation of AIHA in HIV infected patients ispallor, jaundice, dark urine, fever, splenomegaly, severehemolytic anemia and positive direct and indirect coombs test.Differential diagnosis should include infections, RBC membraneor enzyme defects, malignancy, immunodeficiency, drugs andautoimmune diseases. Evaluation should include CBC,reticulocyte count, direct coombs test and peripheral smear tolook for microspherosytosis.

In AIHA, including those with HIV infection, treatment isdirected to reduce production and efficiency of antibodies withremoval of possible offending cause(s) and treatment ofunderlying disease such as HIV infection. Corticosteroids andimmunoglobulins are considered the first line treatment inAIHA with remission rates >60%.15 For refractory casessplenectomy or immunosuppressive drugs may be an option.16

Transfusion with washed RBCs or matched with donor’s cellsmay be used in severe cases.15 Prevention of anemia in HIV isrecommended to decrease mortality. Use of transfusions, ironsupplementation, epoetin alfa and other strategies are beingadvocated as well.2,6,7 However safety and efficacy data areneeded for such interventions in HIV infected children.3

ConclusionAIHA may occur in a child with HIV infection. Physiciansshould do appropriate workup for underlying etiology andinstitute early therapy including steroids.

References1. WHO. Global health sector strategy on HIV/AIDS 2011—

2015. Geneva: World Health Organization 2011. Availablehttp://www.who.int/hiv/pub/hiv_strategy/en/.

2. Volberding PA, Levine AM, Dieterich D, Mildvan D, Mitsuyasu R, SaagM. Anemia in HIV Working Group; Anemia in HIV infection: Clinicalimpact and evidence-based management strategies. Clin Infect Dis 2004May 15;38(10):1454–63. Epub 2004 Apr 27.

3. Calis JC, van Hensbroek MB, de Haan RJ, Moons P, Brabin BJ, Bates I.HIV-associated anemia in children: a systematic review from a global

Volume 24 Issue 01

perspective. AIDS Jun 2008:22(10);1099-112. doi:10.1097/QAD.0b013e3282fa759f.

4. Ellaurie M, Burns ER, Rubinstein A. Hematologic manifestations inpediatric HIV infection: Severe anemia as a prognostic factor. Am J PediatrHematol Oncol 1990;12:449-453.

5. Nyesigire Ruhinda E, Bajunirwe F, Kiwanuka J.Anemia in HIV-infectedchildren: severity, types and effect on response to HAART. BMC Pediatr2012 Oct 31;12:170.doi: 10.1186/1471-2431-12-170.

6. Volberding P. Consensus statement: Anemia in HIV infection—currenttrends, treatment options, and practice strategies Anemia in HIV WorkingGroup. Clin Ther 2000 Sep;22(9):1004-1020; discussion 1003.

7. Shet A, Mehta S, Rajagopalan N, Dinakar C, Ramesh E, NM Samuel,et al. Anemia and growth failure among HIV-infected children in India:a retrospective analysis. BMC Pediatr 2009; 9: 37. Published online 2009June 16. doi: 10.1186/1471-2431-9-37.

8. Iordache L, Launay O, Bouchaud O, Jeantils V, Goujard C,Boue F.et al.Autoimmune diseases in HIV-infected patients: 52 cases and literaturereview. Autoimmun Rev 2014 Aug;13(8):850-7. doi:10.1016/j.autrev.2014.04.005. Epub 2014 Apr 18.

9. Moses A, Nelson J, Bagby GC Jr. The influence of human immunodeficiencyvirus-1 on hematopoesis. Blood 1998;91:1479-1495.

10. Swartz BN, Kessler SW, Rothwell SW, Burrell LM, Reid TJ, Meltzer MS,et al. Inhibitory effects of HIV-1 infected stromal cell layers on theproduction of myeloid progenitor cells in human long term bone marrowcul tures . Experimental Hematology 1994;22:1288-1296.

11. Zandman-Goddard G, Shoenfeld Y. HIV and autoimmunity. AutoimmunRev 2002 Dec;1(6):329-37.

12. Rheingold SR, Burnham JM, Rutstein R, Manno CS.HIV InfectionPresenting as Severe Autoimmune Hemolytic Anemia With DisseminatedIntravascular Coagulation in an Infant. J Pediatr Hematol Oncol 2004Jan;26(1):9-12.

13. Olayemi E, Awodu OA, Bazuaye GN. Autoimmune hemolytic anemia inHIV-infected patients: a hospital based study. Ann Afr Med 2008 Jun;7(2):72-

6.14. Telen MJ, Roberts KB, Bartlett JA. HIV-associated autoimmune hemolytic

anemia: report of a case and review of the literature. J Acquir ImmuneDefic Syndr 1990;3(10):933-7.

15. King KE, Ness PM. Treatment of autoimmune hemolytic anemia. SeminHematol 2005 Jul;42(3):131-6.

16. Oliveira LA, Oliveira BM, Murao M, Vieira ZM, Gresta LT, Viana MB.Clinical course of autoimmune hemolytic anemia: an observational study.J Pediatr 2006;82:58-62.

Jan - Mar 2015. 799


NEWS & VIEWS

International Forum on Infectious Diseases (IFID) 2015

An Interactive session of 5th IFID in cooperation with MMIDSP and College of Family Medicine, Pakistan was conducted byGETZ Pharma with the theme “Regions United against Infectious Diseases: Multidimensional Perspective”. It provided aunique experience relating to different aspects of Infectious diseases from different countries in Africa and Asia. It was held inBangkok Thailand, Malaysia on April 27, 2015. Dr Ejaz A. Khan (ex-president MMIDSP represented the society and welcomedthe guests. Topics of interest varied and included:

“Antibiotic Stewardship – a need for national, regional and international cooperation”(Key note Speaker Dr Ejaz A Khan- Pakistan),“MDR TB: Pakistan Perspective” (Dr. Saqib Saeed- Pakistan),“Infectious Diseases Threats in the Present Century” (Dr. Jennifer Chua- Philippines),“Mycoplasma Burden in Developing Countries” (Dr. Sri Lal De Silva- Sri Lanka),“Dengue Haemmorhagic Fever-Myanmar Perspectives”(Dr. Mar MarKyi- Myanmar) and“The Role of Commensals in ENT Infections”(Dr. Feroze Din- Kenya).

The list of topics itself was a very exciting one even for an average participant. The speakers were well known figures in theirfield and elaborated more about these threatening infections in each of our regions to more than 150 participants from 7 countries.The platform itself has made its name over last few years and will continue to contribute in a very positive way in future as well.Medical Microbiology and Infectious Disease Society of Pakistan (MMIDSP) was pleased to endorse efforts from GETZ Pharmain bringing together experts to give views on different topics.

ASP within Rawalpindi/Islamabad

Further ASP activities by a multidisciplinary team mademore advocacy and awareness sessions within the twin cities of Rawalpindiand Islamabad. Some other major private and public hospitals where these useful sessions were held included Railways Hospital,PIMS, Polyclinic, Military Hospital and Ali Medical center.The following team members with topics were discussed:

Antimicrobial Stewardship (ASP) in Public Hospitals: Dr. Ejaz A. KhanRole of Microbiology-Knowing your bug: Dr Usman JanjuaRole of Pharmacy in ASP- Knowing your drug Ms Komal FizzaRole of Infection Control in Hospital settings Ms Ruth Samuel

The team is planning to cover all major hospitals in the coming months and will printed educational material prepared for thispurpose with large main messages on Standees.

Antibiotics Stewardship Activities

INSTRUCTIONS FOR AUTHORS

AbstractAbstract should not exceed 250 words and must be structuredin to separate sections headed Background, Methods, Resultsand Conclusions.

Please do not use abbreviations or cite references in the abstract.A short list of four to five key words should be provided tofacilitate.

BackgroundThe section must clearly state the background to the researchand its aims. Controversies in the field should be mentioned.The key aspects of the literature should be reviewed focusingon why the study was necessary and what additional contributionwill it make to the already existing knowledge in that field ofstudy. The section should end with a very brief statement ofthe aims of the article.

Materials and MethodsPlease provide details of subject selection (patients orexperimental animals). Details must be sufficient to allow otherworkers to reproduce the results. The design of study and detailsof interventions used must be clearly described. Identify preciselyall drugs and chemicals used, including generic name(s) androute(s) of administration All research carried out on humansmust be in compliance with the Helsinki Declaration, andanimal studies must follow internationally recognized guidelines.The authors are expected to include a statement to this effectin the Methods section of the manuscript. A description of thesample size calculation and statistical analysis used should beprovided.

ResultsPresent results in logical sequences in the text, tables andillustrations. Articles can have a maximum of 5 illustrations(in a combination of figures and tables) per article. The resultsshould be in past tense and repetition of results presented inthe tables should be avoided. Exact P-values should be reportedalong with reporting of OR and RR with their ConfidenceIntervals where applicable.

DiscussionEmphasize the new and important aspects of the study andconclusions that follow from them. Do not repeat the detailsfrom the results section. Discuss the implications of the findingsand the strengths and limitations of the study. Link theconclusions with the goals of the study but avoid unqualifiedstatements and conclusion not completely supported by yourdata.

AcknowledgmentsAcknowledge any sources of support, in the form of grants,equipment or technical assistance. The source of funding (ifany) for the study should be stated in this section. Please seebelow for format of References, Figures and Tables.

Instructions to Authors

ScopeThe Infectious Diseases Society of Pakistan sponsors theInfectious Disease Journal of Pakistan (IDJ). The Journal acceptsOriginal Articles, Review Articles, Brief Reports, Case Reports,Short Communications, Letter to the Editor and Notes andNews in the fields of microbiology, infectious diseases, publichealth; with laboratory, clinical, or epidemiological aspects.

Criteria for publicationAll articles are peer reviewed by the IDSP panel of reviewers.After that the article is submitted to the Editorial Board. Authorsmay submit names and contact information of 2 persons whopotentially could serve as unbiased and expert reviewers fortheir manuscript, but IDSP reserves the right of final selection.

Submission of the ManuscriptManuscripts must be formatted according to submissionguidelines given below, which are in accordance with the“Uniform Requirements for Manuscripts Submitted toBiomedical Journals” (originally published in N Engl J Med1997;336:309-15). The complete document appears atwww.icmje.org. Please submit one complete copy of themanuscript and all enclosures to The Managing Editors,Infectious Diseases Journal of Pakistan, Department ofPediatrics & Child Health, The Aga Khan University,Stadium Road, P.O. Box 3500, Karachi 74800, Pakistan.An electronic copy of the manuscript must also be sent [email protected]. All manuscripts submitted to IDJP mustbe accompanied by an Authorship Declaration stating that ‘Theauthors confirm that the manuscript, the title of which is given,is original and has not been submitted elsewhere.Each authoracknowledges that he/she has contributed in a substantial wayto the work described in the manuscript and its preparation’.Upon submission a manuscript number will be assigned whichshould be used for all correspondence.

Manuscript Categories

I. Original ArticlesArticles should report original work in the fields of microbiology,infectious disease or public health.The word limit for originalarticles is 2000.

Title pageThis should list the (i) title of the article, (ii) the full names ofeach author with highest academic degree(s), institutionaladdresses and email addresses of all authors. (iii) Thecorresponding author should also be indicated with his/her name,address, telephone, fax number and e-mail address. (iv) A shortrunning title of not more than 40 characters (count letters andspaces) placed at the foot end of the title page.(v) a conflict ofinterest statement should also be included in this section.

Oct - Dec 2014. 801Volume 24 Issue 01

II. Review ArticlesAuthoritative and state of the art review articles on topicalissues are also published, with a word limit of 2000. It shouldconsist of critical overview of existing literature along withreference to new developments in that field. These should becomprehensive and fully referenced. Articles should containan Abstract; Main Text divided into sections, Conclusions andReferences.

III. Brief ReportsShort clinical and laboratory observations are included as BriefReports. The text should contain no more than 1000 words,two illustrations or tables and up to 10 references.

IV. Case ReportsInstructive cases with a message are published as case reports.Routine syndromes or rare entities without unusual or newfeatures are invariably rejected. The text should contain nomore than 1000 words, two illustrations or tables and up to 10references. The authorship should not exceed 3-4 persons.

V. Letter to the EditorThese may relate to material published in the IDJP, topic ofinterest pertaining to infectious diseases, and/or unusual clinicalobservations. A letter should not be more than 300 words, onefigure and 3-5 references.

VI. News and ViewsInformative, breaking news updates in infectious diseases fromaround the world (approx. 200 words).

VII. NoticesAnnouncements of conferences, symposia or meetings may besent for publication at least 12 weeks in advance of the meetingdate. Details of programs should not be included.

ReferencesNumber references consecutively in the order in which theyare first mentioned in the text. Identify references in text, tablesand legends by Arabic numerals (in superscript). Referencescited only in tables or in legends to figures should be numberedin accordance with a sequence established by the firstidentification of the particular table or illustration. Bibliographyshould be given in order. Authors, complete title, journal name(Abbr), year, vol, issue, page numbers. According to “Uniform

Requirements of Manuscripts submitted to Biomedical Journals”,as cited in N Engl J Med 1997; 336:309-15.

Tables and FiguresData reported either in a table or in a figure should be illustrativeof information reported in the text, but should not be redundantwith the text. Each table must be presented on a separate sheetof paper and numbered in order of appearance in the text. Tableshould be numbered consecutively in Arabic numerals. Tablesand Figures legends should be self-explanatory with adequateheadings and footnotes.Results which can be described as shortstatements within the text should not be presented as figuresor tables.

IllustrationsIllustrations should be numbered, given suitable legends andmarked lightly on the back with the author’s name and the topedge indicated. Original drawings may be submitted althoughhigh quality glossy photographs are preferable. They shouldbe kept separate from the text. If possible, figures should besubmitted in electronic format as either a TIFF (tagged imagefile format) or JPEG format. Minimum resolution for scannedartwork is:

v Black& white line illustration (e.g. graphs): 600 dpiv Black & white halftone illustrations (e.g. photographs):

300 dpiv Color illustrations: 400 dpi (note that color images should

be split CMYK not RGB)

PlagiarismAuthors should refrain from plagiarism and should doublecheck their work before submitting it for publication. Adequatereferences should be provided for text from other sources.

Authorship criteriaThose who have contributed sufficiently to the conceptualization,design, collection and analysis of data and writing of themanuscript should be granted authorship. Ideally all authorsshould be from the same department except for studies that aremulti center or multispecialty.

Instructions updated - April 2012.Editor IDJ


infectious issn 1027-0299 recognised and … 1027-0299 recognised and registered with the ... case...

Documents