Autoimmune diseases serology -ґ

1- Rheumatoid Factor (RF)A rheumatoid factor (RF) blood test measures the amountof the RFantibody present in the blood. Normally,antibodies are produced by the immune system to helpdestroy and eliminate invading bacteria and viruses thatcan cause disease. But the RF antibody can attach tonormal body tissue, resulting in damage.

A high level of rheumatoid factor can be caused byseveralautoimmune diseases (including rheumatoidarthritis) and some infections. Occasionally an elevatedlevel of RF is present in healthy people.

Sensitivity 80% in patients with RA.

The amount of rheumatoid factor in blood can bemeasured in two ways:

Agglutination tests. One test method mixes bloodwith tiny rubber (latex) beads that are covered withhuman antibodies. If RF is present, the latex beads clumptogether (agglutinate). This method is best used as a first-time screening test for rheumatoid arthritis. Anotheragglutination test mixes the blood being tested with asheep's red blood cells that have been covered with rabbitantibodies. If RF is present, the red blood cells clumptogether. This method is often used to confirm thepresence of RF.

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Nephelometry test. This test mixes the blood beingtested with antibodies that cause the blood to clump if RFis present. A laser light is shined on the tube containingthe mixture and the amount of light blocked by the bloodsample is measured. As levels of RF increase, moreclumping occurs, causing a cloudier sample and less lightto pass through the tube.

Normal values vary from lab to lab. Results are usuallyavailable in a day or two.

Rheumatoid factor (RF)

Titers1:20 to 1:80

Units14 units/mL to 60 units/mL

2- C-REACTIVE PROTEIN

CRP is an acute phase protein (synthesized by the liver)found in the blood, the levels of which rise in response toinflammation. it was named CRP after the C-polysaccharide of Pneumococci, because it was discoveredfirst as substance reacting with this structure and rising inPneumococcus infection. Later turned out that it rises inother inflammations as well.

CRP usually appears in the sera of patients in the acutestages of a number of inflammatory conditions such asmost bacterial and some viral infections; acuterheumatoid fever with or without carditis; rheumatoidarthritis and most other collagen diseases; and in anumber of other conditions characterised by inflammation.CRP is considered to be a sensitive indicator ofinflammation. Changes in the serum level of CRP with timefrom the same patient can be used as an index ofrecovery. The use of the CRP test to measure the

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effectiveness of therapy is of great clinical significance incases such as acute rheumatoid fever.

Normal values: CRP: < 0.8 mg/dlCRP rises up to 50,000-fold in acute inflammation, such asinfection. It rises above normal limits within 6 hours, andpeaks at 48 hours. The CRP test is a general test to checkfor inflammation in the body but it cannot pinpoint theexact location.

A more sensitive CRP test, called a high-sensitivity C-reactive protein (hs-CRP) assay, is available to determinea person's risk for heart disease. Many consider a highCRP level to be a risk factor for heart disease.

*low risk of developing cardiovascular disease if hs-CRPlevel is lower than 1.0 mg/L * high risk for cardiovascular disease, if hs-CRP level ishigher than 3.0 mg/L

Principle: (general overview)

When latex particles complexed human anti-CRP are mixedwith a patient’s serum containing C -reactive proteins, anagglutination reaction will take place.

3- Systemic Lupus Erythematosus (SLE)

Systemic lupus erythematosus is a chronic, multisystem,inflammatory disorder of autoimmune etiology, occurringpredominantly in young women. Common manifestationsmay include arthralgias and arthritis; malar and other skinrashes; pericarditis; renal or CNS involvement; andhematologic cytopenias. Diagnosis requires clinical andserologic criteria.

Of all cases, 70 to 90% occur in women (usually of child-bearing age). SLE is more common among blacks andAsians than whites.

ANA : A group of antibodies that bind to various nuclear(and some cytoplasmic) antigens. ANA stands for

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Antinuclear Antibody. This literally means 'substanceagainst the cell nucleus'. The nucleus is the 'headquarters'of the living cell, therefore the ANA can damage or destroycells & tissues.

ANA test : Specific autoantibody tests

Fluorescent ANA: The fluorescent test for ANA is the bestscreen for SLE; positive ANA tests (usually in high titer: >1:80) occur in > 98%. However, positive ANA tests canalso occur in RA, other connective tissue diseases,cancers, and even in the general population. The false-positive rate varies from about 3% for ANA titers of 1:320to about 30% for ANA titers of 1:40 among healthycontrols. Drugs such as hydralazine

Sensitivity 95% in patients with SLE

The ANA test is very sensitive, but it is not specific for SLE,thus the need for other autoantibodies to establish thediagnosis. These include Ro [SSA], La [SSB], Smith [Sm],ribonucleoprotein [RNP], and double-stranded DNA. Ro ispredominantly cytoplasmic; anti-Ro antibodies areoccasionally present in ANA-negative SLE patientspresenting with chronic coetaneous lupus. Anti-Ro is thecausal antibody for neonatal lupus and congenital heartblock. Anti-Sm is highly specific for SLE but, like anti–double-stranded DNA, is not sensitive. Anti-RNP occurs inpatients with SLE, mixed connective tissue disease, andoccasionally other systemic autoimmune disorders andsystemic sclerosis

Fluorescent ANA

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Anti-Nuclear Antibody: ELISA

Anti-DNA

Normal Range = < 1:10 titer.

Anti-DNA is an immunoglobulin specific against native(double-stranded) DNA . It is increased in Systemic lupuserythematosus & is highly specific to SLE. Sixty to eightypercent of patients with active SLE have a positive anti-

DNA test .

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Anti-ds-DNA antibody is not found in drug-induced lupus .Titers of anti-ds-DNA correlate well with disease activity

and with occurrence of glomerulonephritis .

*Specific for SLE (60-70%)

*Single stranded DNA nonspecific

*Farr assay preferable to ELISA

П- Infectious Disease Serology

As a rule, acute and convalescent sera must be submittedfor serological testing. The acute serum should becollected as soon after the onset of illness as possible, theconvalescent serum should be collected 14 days from thetime the acute specimen was collected.

1-Typhoid Fever test Typhoid should be considered in any patient withprolonged unexplained fever in endemic areas and inthose with a history of recent travel to endemic area.

Widal test is a tube agglutination test employed in theserological diagnosis of enteric fever. Widal test measurestitres of serum agglutinins against somatic (O) andflagellar (H) antigens which usually begin to appear duringthe 2nd week. In the absence of recent immunization, ahigh titre of antibody to O antigen > 1:640 is suggestivebut not specific.

2- Rose Bengal test A rapid agglutination slide test for the detection ofBrucella specific agglutinin

A suspension of Brucella possessing active antigen willagglutinate when exposed to homologous Brucellaantibody. This agglutination forms clumps of bacteriawhich becomes macroscopically visible. The test is usedfor the early detection of Brucella agglutinins (BrucellaAbortus, Melitensis and Suis), Brucella antigens arebacterial suspensions for use in slide agglutination tests to

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detect the presence of bacterial agglutinins associatedwith bacterial infection or previous exposure to a relatedorganism.

3-Serology for Tularemia

This blood test looks for antibodies against Francisellatularensis, the bacteria that causes the disease tularemia,multisystem bacterial disease transmitted from rodents byticks, biting flies, and mosquitoes

Serology is currently carried out for diagnosis of tularemiain humans,serology may be employed, either on sera oron lung extracts , virulent F. tularensis palaearctica couldbe used as antigen in all serological tests.

a) Tube agglutinationThe most commonly used serological test is the tubeagglutination test. The antigen is a culture of F. tularensison Francis medium.The bacteria are stained by addingcrystal violet , and adjusted by adding normal saline toprovide an antigen that when tested on a slide givesreadily visible stained agglutination reactions against aclear fluid background.The test is performed in tubes containing a fixed amountof antigen (0.9 ml) and different dilutions of serumcommencing with 1/10, 1/20, 1/40, etc. The results areread after 20 minutes of shaking, or after 1 hour in awater bath at 37°C followed by overnight storage at roomtemperature. The agglutinated sediment is visible to thenaked eye or, preferably, by using a hand lens. b) Enzyme-linked immunosorbent assayAnother serological test, the enzyme-linkedimmunosorbent assay (ELISA), also allows an earlydiagnosis of tularemia . This method is now widely usedfor clinical purposes. Different antigens, whole bacteria aswell as subcellular components. For routine diagnosis,wholeheat-killed (65°C for 30 minutes) bacteria can be used asantigen. Bacteria can be coated to plastic plates,followedby serial dilutions of serum to be tested. Positive reactionscan be visualised by anti-antibodies labelled with enzyme.

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4- Weil-Felix testRickettsiae are gram-negative, obligate, intracellularorganisms. Diagnosis of infection is by serology sinceRickettsia is difficult to culture. The Weil-Felix test relies onthe agglutination of certain strains of the entericbacterium, Proteus vulgaris (Proteus OX-19 Abs; ProteusOX-2 Abs; Proteus OX-K Abs), which shares cross-reactiveantigens with certain Rickettsia. The results of the Weil-Felix test are nonspecificand insensitive, yet many hospitals use it despite theavailability of better methods. Patients with priorinfections due to Proteus sp. (i.e., urinary tract or woundinfections) will have false-positive results with thismethod.

*Antigen suspension of Proteus OX19 antigen suspensionreacts strongly with sera of patients with typhus grouprickettsiae and rocky mountain spotted fever. *ProteusOX2 reacts strongly with sera of patients with spottedfever infections,*while the Proteus OXK antigen suspensionreacts strongly with sera of patients infected with scrubtyphus.

Agglutination obtained within one minute is a positivereaction and indicates the presence of the correspondingantibody in the patient serum.No agglutination is a negative test result and indicates theabsence of the corresponding antibody in the patientserum.Slide Semi-quantitative MethodThe reactions obtained are roughly equivalent to thosewhich would occur in a tube agglutination test with serumdilutions of 1:20, 1:40, 1:80, 1:160, and 1:320respectively. If a positive reaction is observed it isadvisable to confirm the result and establish the titre by atube test. A tube test is indicated when results do notconform to clinical findings. False results may be obtainedif the reagents are not allowed to reach room temperature(22 to 300C) before use. False positive reactionsare also likely if the test is read beyond one minute aftermixing.

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The indirect fluorescent assay (IFA) specific for Rickettsiarickettsii (Rocky Mountain spotted fever) and Rickettsiatyphi (typhus fever) antibodies replace the nonspecific andinsensitive Weil-Felix test

5- Cold Agglutination testSerology testing for cold agglutinins are commonlyrequested in suspected cases of primary atypicalpneumonia, where this rapid screening test has provenuseful. Cold agglutinin antibodies are found in the serumof approximately 55% of the patients with primary atypicalpneumonia, a respiratory disease caused by Mycoplasmapneumoniae. These antibodies cause agglutination ofadult red blood cells at 4°C, but not at normal bodytemperature (37°C). Cold agglutinin antibody levels areoften detectable by the end of the first or second week ofthedisease, increasing to their maximum by the second tofourth week and decreased or absent by the sixth week.In M. pneumoniae, a positive correlation exists betweenthe level of cold agglutinin antibodies and the severity ofthe disease, the extent of pulmonary involvement andduration of illness. Extremely high titers are sometimesfound in cases of hemolytic anemia. A fourfold or greaterrise in titer from paired sera (where one sample is takenearly in the disease and another sample is drawn severaldays or a week later) is significant of acute disease.

Principle:When serial dilutions of serum containing a cold agglutininantibody with anti-I specificity are mixed with 1% group Oadult red cells and refrigerated, a positive reaction ofagglutination will occur in those tubes containing sufficientantibody. The end point is determined as the last tubedemonstrating the agglutination, and the reciprocal of thedilution is reported as the titer.

6- STREPTOCOCCAL SEROLOGY6- STREPTOCOCCAL SEROLOGY

The antistreptolysin O (ASO) test is the most widely usedserological test for the detection of Streptococcus Asequelae. Since streptolysin is only one of several

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Streptococcus A exoenzymes, the ASO test will not detectthe other antibodies to exoenzymes of Streptococcus A.In the course of streptococcal infections, numerous exo- numerous exo-antigens are produced and excreted as the cellantigens are produced and excreted as the cellmetabolizes (Streptolysin O, DNase, Hyaluronidase,metabolizes (Streptolysin O, DNase, Hyaluronidase,Nicotinamide, Adenine dinucleotidase (NADase),Nicotinamide, Adenine dinucleotidase (NADase),Streptokinase)Streptokinase), these extracellular products of the bacteriaact as antigens to which the body responds by producingspecific antibodies. Streptolysin O (SLO) is one of twohemolysins (the other being Streptolysin S) produced byvirtually all strains of Streptococcus pyogenes. The letter“O” indicates that this toxin is oxygen labile. The SLO toxinhas direct toxic effects on heart tissue. The toxic effects ofSLO can be demonstrated in-vitro, as when added to asuspension of redblood cells, hemolysis will occur inminutes. In the course of astreptococcal infection, SLOstimulates the production of specific antistreptolysinantibodies, which in-vitro, neutralize the hemolyticproperties of the antigen, SLO.A titer of antistreptolysin O (ASO) is useful in theinvestigation of the disease processes related tostreptococcal infections, such as acute poststreptococcalglomerulonephritis and rheumatic fever.

Principle:This is a neutralization procedure. A specified quantity ofstreptolysin O (bacteria produced antigen) is added toprogressively decreasing amounts of patient serum. In thetubes where patient antistreptolysin O antibody is presentin sufficient amount to neutralize the antigen, nohemolysis will occur when indicator red cells aresubsequently added. When the antigen exceeds theantibody, the excess streptolysin O will cause hemolysis ofthe indicator red blood cells. Put another way, in a testsystem, the patient’s serum containing antistreptolysin Oantibody is added to streptolysin O, an antigen-antibodyreaction takes place, which, depending on the antibodylevel, completely or partially neutralizes thehemolytic action of the streptolysin O.

The ASO Latex Test is a stabilised buffered suspension ofpolystyrene latex particles that have been coated withStreptolysin O. When the latex reagent is mixed with aserum containing ASO, agglutination occurs. The sensitivity

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of the latex reagent has been adjusted to yield agglutinationwhen the level of ASO is greater than 200 IU/ml, a leveldetermined to be indicative of disease by epidemiologicaland clinical studies. Sera having titers of between 200 IU/mland 3500 IU/ml will be reactive.

Normal value

Adults: less than 200 units/ml

Children: less than 300 unit/ml

*Anti-DNase B Testing

= may appear earlier than ASO

= increased sensitivity for detection of glomerulonephritis

= macro- and micro-titer, ELISA, and neutralization techniques are available

*Anti-Hyaluronidase Testing

= test patient serum for antibodies which inhibit actionof Hyaluronidase

= after performance of the test, a clot will form into thetubes where enzyme activity of Hyaluronidase has beenneutralized by patient antibody

= Hyaluronidase produced by patients with throat or skininfections, ASO produced in response to throat infectionsonly

*Streptozyme Testing

= hemagglutination procedure to detect antibodies tonumerous Streptococcal antigens

= sheep RBC’s are coated with Streptolysin, Streptokinase,Hyaluronidase, DNase, and NADase

= patient serum diluted 1 : 100, mixed with sheep RBC’sand observed for agglutination

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= rapid and simple to perform, more false positive andnegative results occur

7- Infectious mononucleosis (IM)

Infectious mononucleosis (IM) is an acute or subacuteinfectious lymphoproliferative disease that is associatedwith the Epstein-Barr virus (EBV). The disease most oftenaffects children and young adults and is characterized byan increase in atypical lymphocytes, enlargement of thelymph nodes and spleen, fever and production ofcharacteristic heterophile antibodies. Serological testing for mononucleosis became available asa result of the research of Paul and Bunnell on heterophileantibodies.

*Heterophile antigens are a group of similar antigensfound in unrelated animals

*Heterophile antibodies produced against heterophileantigens of one species will cross react with others

*Forssman antigen is an example of a heterophile antigenand is found on the RBC’s of many species

*Forssman antibodies formed against Forssman antigenswill agglutinate sheep RBC’s

Paul-Bunnell testHeterophile tests measure IgM antibody directed towardbovine, sheep or horse erythrocytes..not differentiatebetween heterophile antibodies types

Davidsohn testDavidsohn modified the original Paul-Bunnell test byadding steps to absorb out cross reacting Forssman andserum sickness antibodies leaving behind the IMheterophile antibody to react with sheep red cells.

Absorbtion steps include : (a) Guinea-pig kidney

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absorption: not more than a three tube reduction in titre ofunabsorbed serum , after absorption with guinea-pigkidney .(b) Ox cell absorption: at least a four tube decrease in titreof unabsorbed serum after absorption with ox cells .

Recent researchers have developed tests for infectiousmononucleosis heterophile antibodies using treated horseor bovine red cells, or extracted antigens from bovineRBCs that have been attached to latex particles or coatedin tubes for enzyme-linked immunoassay testing (EIA).

Infectious Mononucleosis slide tests

The original heterophile test (Paul-Bunnell) has beenmodified several times to improve performancecharacteristics (MonoCard, MonoSpot, MonoTest).

Horse RBC’s possess antigens which react with theantibody associated with IMPatient serum after absorbtionmixed with horse RBC’s, agglutination is positive

Specific EBV serology tests measure specific antibodyagainst various antigens associated with EBV infection.IgM and IgG antibody against viral capsid antigen (VCA),antibody against early antigen (EA) and IgG antibodyagainst Epstein-Barr nuclear antigen (EBNA) are frequentlyreported. The pattern of antibody response successfullyidentifies the type of EBV infection in 96% of patients,ELISA and immunofluorescence techniques mostcommonly used

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II -Laboratory Diagnosis of Sexually TransmittedInfections (STDs)

The laboratory approach to the diagnosis of STDs isrelated to the sex of the patient , although some infectionsare common to both sexes like gonorrhea, syphilis andchlamydial infection but there are usually difference in thepresenting symptoms , the sites and methods ofspecimens collection in these infections.

Genital infections and STDs in women

These include :

1- Acute vaginitis:Is caused by Trichomonas vaginalis ( protozoan) andCandida albicans or other yeast.

- Trichomoniasis is primarily an infection of theurogenital tract; the most common site of infection is theurethra and the vagina in women, is caused by the single-celled protozoan parasite Trichomonas vaginalis

Symptoms include inflammation of the cervix (cervicitis),urethra (urethritis), and vagina (vaginitis) which producean itching or burning sensation. There may also be ayellow-green, itchy, frothy, foul-smelling vaginaldischarge. In rare cases, lower abdominal pain can occur.Symptoms usually appear in women within 5 to 28 days ofexposure

Trichomoniasis is diagnosed by visually observing thetrichomonads via a microscope

-Vulvovaginal candidiasis is caused by Candidaalbicans , squamous epithelial cell of vaginal wail isinvaded and inflamed causing vaginal discharges andpain.

2-Bacterial vaginosis (BV) or very uncommonlyvaginal bacteriosis

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Is caused by Gardnerella vaginalis, and anaerobic cocci ,anaerobic vibrios and Mycoplasma hominis which ispresent in 50% of the ore sever cases of BV.

Clinical manifestations include discomfort and pungentodor, a gray, thin, homogenous discharge, but rarelydysuria. Characteristically, signs of inflammation are notpresent in the vaginal walls, although a few leukocytes

may be present.

-What causes a BV? Oftentimes, it derives from changes inthe local microflora and overgrowth of 1 or more bacterialtypes. This may be due in part to a reduction or loss inLactobacillus that normally keeps the vagina slightlyacidic, and/or a reduction or loss of peroxide-producing

bacterial strains, which protect against BV.

3 -Cervisitis with or without Urethritis :

Is caused by gonococci or Chlamidia trachomatis .

4 -Uterine sepsis :

Is caused by Streptococcus pyogenes, Staphylococcusaureus, coliform bacilli, Bacteroides , Clostridium and

Mycoplasma hominis.

5-Toxic shock syndrome : is caused by Staphylococcusaureus.

6 -Genital ulceration

Is caused by T. pallidum , Haemophilus ducreyi andChlamydia group A.

7 -Tuberculosis of uterus: is caused by Mycobacteriumtuberculosis.

8-Viruses : Cytomegalo virus . Herpes , human papillomacan cause STDs.

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Specimens collection

Vaginal and urethral discharges are collected by swabsmade of cotton or ragon that has been treated withcharcoal to adsorb toxic material to gonococci ,

Mycoplasma and Chlamydia.

-For vaginitis or uterine sepsis the specimen is highvaginal swabs

-For Trichomonas special specimens should be collectedfrom vagina , urethra and cervix , the swab placed in clearTrichomonas transport medium or saline for microscopy

and culture.

-For gonorrhea the vaginal swab is unsuitable becausegonococci tend to die in the acid vaginal secretion and ifremaining viable , are likely to over grown by vaginalcommensal bacteria, so that endocervical swab must becollected and urethral and pharyngeal swabs should be

taken.

Swabs for culture should be placed in tubes of Amiestransport medium or modified Stuart's media andtransport to the lab.(held at room temperature until

inoculating)

-For cervisitis endocervical swab helps to avoidcontamination with vaginal flora and its useful for isolation

of Herpes , Mycoplasma and Chlamydia.

- Since Chlamydia is intracellular pathogens , it isimportant to remove epithelial cells with swabs from theurethral mucosa. Swabs for isolaton of Mycoplasma andChlamydia are transport in sucrose buffer with antibiotic(A useful medium has 0.2 mol/L sucrose in 0.02 Mphosphate buffer, pH 7.0– 7.2, with 5% fetal calf serum.Other transport media may be equally suitable. Thetransport medium should contain antibiotics to suppressbacteria other than chlamydia species. Gentamicin, 10

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μg/mL, vancomycin, 100 μg/mL, and amphotericin B, 4μg/mL, can be used in combination since they do notinhibit Chlamydia ) . If specimens cannot be processedrapidly, they can be refrigerated for 24 hours; otherwise,they should be frozen at −60 °C or colder until processed.

Microscopic examination

Both a wet film and gram stained film should beexamined, the wet film is examined for the presence of

Trichomonas ( motile , rounded shaped)

-flourescein conjugate monoclonal Ab reagent are quitesensitive for Trichomonas.

-Examine under dark field microscope for T.pallidum.

-Fluorescein-conjugated monoclonal antibodies for Chlamydia can be used for direct examination of specimens from the genital tract and ocular specimens butare not as sensitive as chlamydial culture or molecular diagnostic tests.

-Gram stained film should be examined for candidiasisand bacterial vaginitis .

Candida G+ve yeast form and G+ve hyphe( pseudomycelium).

The presence of G-ve diplococcic intracellularly withlimited proportion of PMNs almost diagnosis as gonorrhea.

Bacterial vaginitis G – ve bacilli (Gardnerella vaginalis ) ,squamous epithelial cells covered with many such bacilli

( clue cells).

To make a diagnosis of bacterial vaginitis the swabs shouldbe tested for:

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1- Thin homogenous vaginal discharge vary from white togrey in color.

2-A characteristic "fishy" odor on wet mount. This test,called the whiff test, is performed by adding a smallamount of 10% potassium hydroxide (KOH) solution to amicroscopic slide containing the vaginal discharge. Acharacteristic fishy odor is considered a positive whiff testand is suggestive of bacterial vaginosis.

3-Loss of acidity (pH of vaginal fluid >4.5) . To controlbacterial growth, the vagina is normally slightly acidic witha pH of 3.8–4.2. A swab of the discharge is put onto litmuspaper to check its acidity. A pH greater than 4.5 isconsidered alkaline and is suggestive of bacterialvaginosis.

4-Gram stain demonstrates a shift in vaginal flora, with adecrease in large Gram-positive rods (lactobacilli) and anincrease in small Gram-variable (consisting Gardnerellavaginalis, , anaerobic vibrios such as Bacteroides andMycoplasma hominis).

5-The presence of clue cells on wet mount. Similar to thewhiff test, the test for clue cells is performed by placing adrop of sodium chloride solution on a slide containingvaginal discharge. If present, clue cells can be visualizedunder a microscope. They are so-named because theygive a clue to the reason behind the discharge. These areepithelial cells that are coated with bacteria.

At least three of the four criteria should be present for aconfirmed diagnosis so that bacterial vaginitis does notdepend on the isolation of the bacteria.

The diagnosis of acute vaginitis as followings:

1-Trichomonas vaginalis

-Copious , yellow-green or discolored discharge.

- Motile trichomondas

-Vaginal pH is >4.5, and the whiff test is usually negative

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-Because of the low sensitivity of direct microscopy,culture may be used, where available, to isolate theparasite from urethral swabs, urine sediments, prostatefluid and vaginal specimens.

2- Candida albicans

- Thick ,white ( cottage cheese ) discharge

-The vaginal pH is normal (<4.5), and the whiff test isnegative.

- Wet-mount preparation with 10% KOH shows buddingyeast and/or branching pseudohyphae.

Culture:

The specimen should be inoculated on two plates of a richBlood agar , one incubated at 35-37c˚ in 5% CO2 withmoisture & the other in an aerobic atmosphere with CO2.

Candida albicans can be recognized on the aerobic Bloodagar and grow well on Colombia agar base + 5% Sheepblood agar + Naldixic acid ( CAN) , Sabouraud agar or Maltextract agar , placing 50U Nastatin disc & 20 µgamphotericin disc will assist in the recognition of Candidaalbicans ( sensitive ) from resistant Staphylococcal andother bacterial colonies .

N. gonorrhoeae grow well on Thayer Martin ( TM )medium wich contains the antibiotics ( Vancomycin ,Colistin , Nastatin ) , although Thayer Martin mediumwidely used but 3 - 10% of gonococcal strains are inhibitedby Vancomycin so that a modified TM medium which isModified New York City medium (MNYC medium ) ispreferred because it gives better growth and the use ofLincomycin as a selective agent avoids the problem ofVancomycin sensitivity , incubated in air plus CO2 ; but ifthe clinical features or the appearances in the gram smearsuggest that there is gonococcal infection the specimen

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should be inoculated additionally on Chocolate agar andincubated at 35-37 c˚ in air plus 5-10 % CO2 .

For further diagnosis of N. gonorrhoeae CF test andIndirect fluorescent Abs can be used.

Gardnerella and Sterptococci grow on CAN.

Mycoplasma and Ureplasma urrelyticum grow on Diphasicmedium and SP-4 medium.

For Trichomonas vaginalis the Cysteine peptone liverinfusion maltose ( CPLM) medium is used under anaerobiccondition.

Cell culture techniques are recommended for the isolationof Chlamydia species. Usually involves inoculation of theclinical specimens onto cycloheximide-treated McCoycells. One technique uses a confluent growth of McCoycells on 13- mm coverslips in small disposable vials. Theinoculums is placed in duplicate vials and centrifuged ontothe monolayers at approximately 3000 × g followed byincubation at 35 °C for 48–72 hours and stained. To detectChlamydia immunofluorescence, Giemsa’s stain, or iodinestain is used to search for intracytoplasmic inclusions.Immunofluorescent techniques are the most sensitive ofthe three stains but require special IF reagents andmicroscopy. Giemsa is more sensitive than iodine, but themicroscopy is more difficult.

Enzyme immunoassays (EIAs) are used for detectingchlamydial antigens in genital tract specimens frompatients and PCR( 16S RNA sequence) .

Genital infections in men

The infections in men are mostly caused by the sameorganisms as in women , include :-

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1-Urethritis : is classified as gonococcal or nongonococcal (NGU) depending on whether or not gonococciare found in gram film or culture of discharge . Most casesof NGU are caused by Chlamydia trachomatis andUreaplasma in 10% of cases .

2-Prostatitis : is usually caused by gonococci orChlamydia. Sub acute or chronic prostatitis found in oldermen is usually associated with the presence of coliformbacilli or enterococci.

3-Ulceration : caused by Herpes simplex virus ( usuallytype 2) , T. pallidum , Haemophilus ducreyi andChlamydia.

Collection of specimens and laboratoryexamination

Urethral discharge may be expressed directly on ti slidefor gram stain and be inoculated immediately onChocolate agar and selective medium for the culture ofgonococci . If specimens have to be transported to thelaboratory the exudates from ulcers should be collected ona swab and put into a tube of Amie ҆ s transport medium.

For isolation of Haemophilus ducreyi a special agar( Mueller Hinton Chocolate Horse Blood agar) enrichedwith 1% Iso vitale X and Vancomycin ( 3µg/ ml) incubatedin 5-7% CO2 at 37 c ˚.

The gram stained films may show small pleomorphic G –verods or coccobacilli arranged in chains and groups.

Herpes simplex virus examined by immunofluorescent Absor by ELISA.

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SYPHILIS

Syphilis is a contagious venereal disease caused by the spirochete Treponemapallidum. The organism enters the body through a break in mucosa or epitheliallayer. After a 10-60 day incubation, a painless inflammatory reaction producing acharacteristic ulcerated lesion called a chancre usually appears at the site of entry.

Syphilis is usually cured by penicillin, if treated early. If untreated, a generalizedskin rash and other abnormalities will begin appearing six weeks to six monthsfollowing the disappearance of the chancer (secondary stage syphilis). Again, the

clinical symptoms may disappear (latent stage syphilis).

The latent syphilis may continue throughout life, it may terminate withspontaneous cure, or it may advance to tertiary syphilis. Pregnant women withactive syphilis (even primary stage) can transmit the organism to the unborn child

(congenital syphilis).

Tertiary Stage occurs anywhere from months to years aftersecondary stage, typically between 10 to 30 years (gummatoussyphilis ,cardiovascular syphilis ,neurosyphilis ).

- Congenital Syphilis Transmitted from mother to fetus Fetus affected during the second or third trimester 40% result in syphilitic stillbirth Live-born infants show no signs during first few weeks 60-90% develop clear or hemorrhagic rhinitis rash especially around mouth, palms of hands and soles of

feet general lymphadenopathy, hepatosplenomegaly, jaundice,

anemia, painful limbs & bone abnormality

SYPHILIS - - DIAGNOSIS

Evaluation based on 3 factors

-Clinical findings

-Demonstration of spirochetes in clinical specimen

-Present of antibodies in blood or CSF

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= more than one test should be performed

A- CULTURE

No cultural methods are available.

B- DIRECT SMEARS

1-Darkfield microscopy

Dark field microscopy is used to demonstrate Treponemapallidum in material from lesions or lymph nodes. The presence ofT. pallidum constitutes a definitive diagnosis of syphilis. Since T.pallidum is identified by characteristic morphology and motility,the preparation must be fresh and the organisms actively motile.Considerable expertise is required not only for the correctidentification of the spirochetes but also for proper use of a darkfield microscope. For these reasons, the test should only beperformed in a setting where it is routinely done.

2- Direct fluorescent antibody (DFA-TP)

As an alternative to dark field microscopy, fixed smears fromlesions, serous fluid, or lymph node aspirates may be sent toreference laboratories for staining with fluorescent-conjugatedantibody to T. pallidum. The results, however, are usually notavailable for days to weeks and thus, may not be helpful inguiding patient management.

C- SEROLOGICAL TESTS1- Nontreponemal or reagin tests

This group of common nontreponemal tests measure antibody toa nonspecific cardiolipin lecithin antigen. The tests are

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moderately specific for syphilis (false-positives occur), but highlysensitive.

Non-specific or non-treponemal serological test to detect reagin

= Reagin is an antibody formed against cardiolipin

= Found in sera of patients with syphilis as well as other diseases

= Non-treponemal tests become positive 1-4 weeks afterappearance of primary chancre, in secondary stage may havefalse positive , in tertiary 25% are negative, after successfultreatment will become non-reactive after 1 to 2 years.

Because they are easily performed, the nontreponemal tests areuseful screening tools. The tests can be quantitative to obtain atiter and, thus, are useful in monitoring patient response totherapy.

*Venereal Disease Research Laboratory=VDRL

The (VDRL) is a nontreponemal serological screening for syphilisthat is also used to assess response to therapy, to detect CNSinvolvement, and as an aid in the diagnosis of congenital syphilis.The basis of the test is that an antibody produced by a patientwith syphilis reacts with an extract of ox heart (diphosphatidylglycerol). It therefore detects anti-cardiolipin antibodies (IgG, IgMor IgA).

= Flocculation test, antigen consists of very fine particles that precipitate out in the presence of reagin

= Utilizes antigen consists of cardiolipin, cholesterol and lecithin

= serum must be heated to 56 C for 30 minnutes to remove anti-complimentary activity which may cause false positive

= reported as Non-reactive, weakly reactive and reactive

= used primarily to screen CSF

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Many other medical conditions can produce false positive results,including some viruses (mononucleosis, hepatitis), drugs,pregnancy, rheumatic fever, rheumatoid arthritis, lupus, andleprosy.

*The RPR (Rapid Plasma Reagin) test

uses the same antigen as the VDRL, but in that test it has beenbound to several other molecules including a carbon particle toallow visualization of the flocculation reaction without the need ofa microscope. The RPR test utilizes the VDRL cardiolipin antigen ismodified with choline chloride to make it more stable and isattached to charcoal particles to allow macroscopic reading.

= general screening test

= cannot be performed on CSF

= serum or plasma may be used for testing, serum is not heated

= results are read macroscopically

= appears to be more sensitive than the VDRL

= easy and cheap, used for screening

= reported as a titer

= used to follow treatment ,sensitive except in late syphilis

* Other tests which use modified VDRL Ag

A. USR – unheated serum reagin test

= modified VDRL Ag, uses choline chloride/EDTA

= microscopic flocculation test

B. RST – reagin screen test

= modified VDRL Ag with Sudan Black

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= Sudan Black makes flocculation reaction macroscopicallyvisible

*The Wassermann test

Is based on complement-fixation. A sample of blood orcerebrospinal fluid is taken and introduced to the antigen -cardiolipin extracted from bovine muscle or heart, theWassermann reaction of antiphospholipid antibodies (APAs),Syphilis non-specific antibodies (coreagin) which react with lipidin presence of complement then indicator system ( Sheep RBCscoated with anti-Sheep RBCs) , no hemolysis consider as positiveresult .

The reaction is not specific to syphilis and will produce a positivereaction to other diseases, including malaria, tuberculosis, andnumerous other diseases. It is possible for an infected individualto produce no reaction and for a successfully treated individual tocontinue to produce a reaction (known as being "Wassermannfast" or "fixed").The Wassermann test it is rarely used today.Replacement tests such as the VDRL test and the RPR test.

2- Treponemal Tests

These tests measure antibody specific for T. pallidum. They arehighly specific and highly sensitive. Treponemal tests are notcurrently used for general screening because they are expensiveand time consuming to perform. Their use is limited toconfirmation of positive reagin tests (to identify false-positivediagnoses) and in the diagnosis of late syphilis when reagin testsmay be nonreactive.

●-Treponema Pallidum Immobilization Test (TPI)

- measures ability of (patient produced) antibody and complementto immobilize live (reagent) treponemes. Live T. pallidum becomeimmobilized by antibody in serum of infected persons , expensivetest.

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●-Hemagglutination Tests

(includes Treponema pallidum Hemagglutination – TPHA andadapted to microtechniques (MHA-TP) ) utilize tanned sheep RBC’sare coated with T. pallidum antigen from Nichol’s strain. Serum ispre-treated with non-pathologic Reiter Strain treponemes to limit

non-specific reactions. Agglutination indicated is positive. Moresensitive and more specific, even in late syphilis . Reported aspositive or negative

● Fluorescent treponemal antibody absorption(FTAABS) Test

- an indirect fluorescent antibody test requiring diluted heat-inactivated patient serum. The serum is mixed with non-pathologic Reiter Strain treponemes to remove nonspecific cross-reactive antibodies.

The ‘absorbed’ serum is then tested with the Nichols Strain of T.pallidum, washed, stained with an antibody conjugate (antimmunoglobulin with a fluorescein isothiocyanate label) andexamined under a fluorescent microscope.

= one of the most used confirmatory test

= requires experienced personnel to read

= highly sensitive and specific

●ELISA

= tubes coated with T. pallidum antigen, antibody in serumattaches to antigen

= following washing, add an anti-antibody tagged with enzymealkaline phosphatase , detectable color changes occur.

Interpretation:

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1- If the test is negative, but the physician still suspects theinfection is present, the more specific treponemal tests should beordered.

2- Reagin tests (VDRL & RPR) are considered screening tests. Ifpositive results are obtained, the more specific treponemal testing(FTA-ABS, MHA-TP, etc.) should be performed. Specimens givingany degree of clumping should be subjected to further serologicalstudy.

3 -However it must be noted that all treponemal specific tests willremain positive for life once a person has been infected withsyphilis, even if syphilis has been adequately treated. Therefore,these types of tests cannot be used to monitor the treatment ofsyphilis. automated RPR test(ART)is available for large scale tests.

Time course of antibody development duringsyphilis

4-Biologic False Positives (BFP)

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A. Collagen diseases such as arthritis, LE, etc., sometimesresult in increased amount of reagin

B. Certain infections : IM, malaria, leprosyC. Other treponemal infections

5- False negativesA. Very early in disease or latent, inactive stageB. Immunosuppressed patients

Leptospirosis

Leptospirosis is a typical zoonosis that can be transmitted fromanimals to humans and occurs worldwide. Leptospirosis is abacterial disease that affects humans and animals. It is causedLeptospira interrogans, more than 200 serovars are known.

L. interrogans can enter the body through scratches or breaks inthe skin. The bacteria use blood as a means of travel, travelingthroughout the body and infecting different organs. It is believedthat the bacteria can attach to several different receptors on thehost cells which is why it can effect so many different hosts andattack different organs. The kidney is where L. interrogans surviveand multiply the best, causing kidney infections. Havingleptospirosis is usually not fatal to humans but has been known tocause death in a human at least once. There are two phases ananimal goes through when being infected by L. interrogans, theleptosipremic acute phase and the immune leptospirosis phase.The symptoms during the first phase include fever, nausea,headaches, and muscle pain. An organism experiencing thesecond phase also will have fever and may develop meningitis.Organ failure and renal failure may occur in severe cases. It isduring this phase that the bacteria leave the body through theurine .

Leptospirosis is confirmed by laboratory testing of a blood orurine sample.

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-Microscopic agglutination test (MAT) :is the standard test todetect serovar specific antibodies. However, this method cannotbe performed in general laboratories because it requires culturesof reference leptospire strains of many serovars as a series ofantigens for testing. The method is also complicated, timeconsuming and needs killed for operation

-Lepto Latex Test( Latex Agglutination) for Leptospirosistest(LA): is a simple serodiagnostic method for leptospirosis byusing the latex agglutination test to detect antibodies toleptospires.

Leptospira interrogans serovar pyrogenes was cultured inneopeptone medium. Leptospiral cells was inactivated (killed)with 0.1% formalin for 2 hours. The antigen-coated latex wasstable for at least 12 month when kept at 4-10 C.

The Lepto Latex Test kit is used for detection of anti-Leptospiresin the human serum or plasma which capable to react withantigen of Leptospires sp. that coated on Latex bead. The reactionof antigen and antibodies will form agglutination clearly within 5minutes by normal eye vision. When no specific antibodies arepresent, the latex suspension will remain homogeneous (negativeresult).

The diagnosis of Leptospirosis by latex agglutination isconvenient, simple and rapid. This method is suitable for use as alaboratory screening test. LA is highly sensitive at 94.7% and

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specific 93.3% (of the patients confirmed as cases of leptospirosisby Microscopic agglutination test MAT)

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Skin ,Wound and Soft tissue InfectionsMany minor and superficial skin , wound & Soft tissueinfections are diagnosed by the doctor based on a physicalexamination, signs and symptoms . A clinical evaluationcannot, however, definitively tell the doctor whichmicroorganism is causing a wound infection or whattreatment is likely to be effective. For that, laboratorytesting is required.

Skin, Wound and Soft tissue Infections include :-

1- Folliculitis is a minor infection of the hair follicles.Itcaused by Pseudomonas aeruginosa , Staphylococcusaureus & Candida albicans

2- Acne : also involves inflammation of hair follicles and associatedsebaceous glands. It caused by Propionibacterium acnes

3- Furuncles is a small abscess that develops in the region of a hairfollicle. It caused by Staphylococcus aureus

4- Impetigo : Pyoderma, also termed impetigo, is acommon, sometimes epidemic, skin lesion. The initialesion is often a small vesicle that develops at the site ofinvasion and ruptures with superficial spreadcharacterized by skin erosion and a serous exudate, whichdries to produce a honey-colored crust. It caused by GroupA β-hemolytic streptococci, Staphylococcus aureus , rarely,Corynebacterium diphtheriae

5- Erysipelas is a rapidly spreading infection of the deeper layers of thedermis. It is associated with edema of the skin, marked erythema, pain. Itcaused by Group A β-hemolytic streptococci.

6- Nosocomial wound infection : caused by S. aureus ,Bacteroides fragilis, Clostridium perfringens , S.pyogenes , and coliform bacilli.

7-- Soft tissue infections (gangrene, necrotizing fasciitis,cellulitis): caused by a variety of aerobic and anaerobic

1

species of bacteria may be present either singly or incombination (synergistic infections) .

8- Burns : S. aureus and Pseudomonas aeruginosa .

9- Bone infections ( osyeomyelitis) :

- In infants caused by S. aureus , Streptococcus group B andcoliform bacilli.

- In children caused by Streptococcus group A and H. influenza ,skeletal tuberculosis may be occur.

- In adults caused by G-ve bacilli and various cocci , vertebraltuberculosis is seen.

Laboratory tests

Laboratory testing is primarily used to diagnose bacterialwound infections, to identify the microorganismresponsible, and to determine its likely susceptibility tospecific antimicrobial drugs. Sometimes testing is alsoperformed to detect and identify fungal infections.

Samples from Open Wounds and Sores

Sample collection may involve swabbing the surface of awound to collect cells or pus, aspiration of fluid or pus witha needle and syringe, and/or the collection of a tissuebiopsy. For fungal evaluation, scrapings of the skin may be

collected.

If the patient is febrile or in shock or the infectionaccompanied by bacteremia , a sample of blood should be

taken for culture.

Testing may include:

Naked eye examination:

The appearance of pus or exudates ( color , consistencyand odor ) should be noted.

- Staphylococcal lesion -------- the pus is typicallycreamy and thick in consistency with pus cellsevident on microscopy.

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- Streptococcus pyogenes infection -------- the pus isstraw colored and watery , with lysis of pus cellsseen on microscopy.

- Proteus infection ------- the pus with fishy smell.- Pseudomonas infection -------- the pus with a sweet-

musty odor and often a blue pigmentation.- Anaerobic organisms infection ----- the pus has an

offensive putrid smell.- Actinomycosis( caused by Actinomyces ) ---- the pus

contain small micro colonies that appear as sulphurgranules ( the pus yellowish granular ).

- Fungal infections such as yeast ( mycetoma) ------black or brown granules may be present .

Microscopic examination :

- Gram stain – This test is usually performed in conjunctionwith the wound culture. It is a special staining procedurethat allows bacteria to be evaluated under themicroscope. The results of this test are usually availablethe same day the sample is received in the laboratory andcan give the doctor preliminary information about themicroorganisms that may be causing the infection.

- Ziehl-Neelsen stain for Mycobacterium and Nocardia

- Immunofluorescent staining with specific antisera forsome pathogenic clostiridia.

- Bacterial culture – This is the primary test used todiagnose a bacterial infection. Part of this evaluationinvolves the identification of methicillin-resistantStaphylcoccus aureus (MRSA) when it is present. Resultsof bacterial wound cultures are usually available within 24-48 hours from the time the specimen is received in thelaboratory. Results of special cultures for slow-growingorganisms, such as fungi or mycobacteria, may requireseveral weeks.

– A follow-up test to the wound culture. When a pathogenis identified using the wound culture, this test is used todetermine the bacteria's likely susceptibility to certain

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SpecimenBloo

d agar

aerobically in 5-10% CO2

Anaerobically in N2 OR H2 + 5-10% CO2

Cooke meat

broth or Thioglycolate broth

Macconkey agar

Sabouraud

dextrose agar

Lowenstien

Jensen media

drug treatments. This information helps guide the doctorin selecting appropriate antibiotics for treatment. Theseresults are typically available about 24 hours afterisolation of the microorganism that is causing theinfection.

Other tests that may be ordered include:

KOH prep – A rapid test performed to detect fungi in asample. The sample is treated with a special solution,placed on a slide, and examined under a microscope.

Fungal culture – Ordered when a fungal infection issuspected. Many fungi are slow-growing and maytake several weeks to identify.

Blood culture – Ordered when infection from a woundmay have spread and septicemia is suspected.

Molecular testing to detect genetic material of aspecific organism.

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AcidfaststainG – ve

rodsBloodagar

yeast

Biochemical tests

Coagulase test+ Mannitolsalt agar Mycobacteriu

mAnaerobically in N2 orH2 +5-10%

CO2

Closteridia

Staphylococci

Bacteremia

Bacteremia is the presence of viable bacteria in the bloodstream. Bacteremia is different from sepsis (so-calledblood poisoning or toxemia), which is a condition wherebacteremia is associated with an inflammatory responsefrom the body (causing systemic inflammatory responsesyndrome, characterised by rapid breathing, low bloodpressure, fever, etc.). Bacteremia is the presence of viable bacteriain the blood. The term septicemia, the presence of microorganisms or

their toxins in the blood, is no longer used by the consensus committee.

Causes

In the hospital, indwelling catheters are a frequent causeof bacteremia and subsequent nosocomial infections,because they provide a means by which bacteria normallyfound on the skin can enter the bloodstream. Other causesof bacteremia include dental procedures, urinary tractinfections, peritonitis, Clostridium difficile colitis,intravenous drug use, and colorectal cancer. Bacteremiamay also be seen in oropharyngeal, gastrointestinal orgenitourinary surgery or exploration. Salmonella infection,despite mainly only resulting in gastroenteritis in thedeveloped world, is a common cause of bacteremia inAfrica. It principally affects children who lack antibodies to

Salmonella and HIV+ patients of all ages.

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Fungemia is the presence of fungi or yeasts in the blood. It is mostcommonly caused by Candida species (also known as Candidemia,Candedemia, and Invasive Candidiasis), but can be caused by otherfungi as well, including Saccharomyces, Aspergillus and Cryptococcus. Itis most commonly seen in immunosuppressed or immunocompromisedpatients with severe neutropenia, oncology patients, or in patients with

intravenous catheters .

Diagnosis

Bacteremia is most commonly diagnosed by blood culture,in which a sample of blood is allowed to incubate with amedium that promotes bacterial growth. Since blood isnormally sterile, this process does not normally lead to theisolation of bacteria. If, however, bacteria are present inthe bloodstream at the time the sample is obtained, thebacteria will multiply and can thereby be detected.

Blood culture is a microbiological culture of blood. It isemployed to detect infections that are spreading throughthe bloodstream (such as bacteremia, septicemia amongstothers). This is possible because the bloodstream is

usually a sterile environment.

A minimum of 10 ml of blood is taken through veinpuncture and injected into two or more "bloodbottles" with specific media for aerobic and anaerobicorganisms. A common media used for aerobies isTryptic soy broth and for anaerobes is thioglycollatebroth.

Collect enough blood

1-2ml in neonate

2-3ml in infants

3-5ml in children

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10-20ml in adolescent

-The blood is collected using aseptic technique. Thisrequires that both the tops of the culture bottles and thevein puncture site of the patient are cleaned prior tocollection with swabs 70% isopropyl alcohol.

- 2-3 blood cultures should be taken separated by 1 hr.intervals or less if treatment can not be delayed .Ordering multiple sets of cultures increases the probabilityof discovering a pathogenic organism in the blood andreduces the probability of having a positive culture due toskin contaminants , so that the chance of missing atransient bacteremia ( caused by S.epidermidis ) isreduced and the pathogenic role of S.epidermidis isconfirmed if they are recovered from multiple veinpunctures ( bacteremia in users of intravenous drugs) .

The blood should be mixed with 10 times its volume ofbroth( 5 ml blood in 50 ml broth) to dilute any antibioticpresent and to reduce the bactericidal effect of serum .After inoculating the culture bottles incubated at 37 c for 7days . A sterile culture shows a layer of sediment RBCscovered by a peal yellow transparent broth , microbialgrowth is evidenced by : a floccules deposit on top of theblood layer , turbidity , hemolysis , coagulation , gasproduction and white grains on the surface or deep in theblood layer.

If a culture bottle is positive, a microbiologist will performa Gram Stain on the blood for a rapid identification of thebacteria. The blood is also subcultured onto agar plates toisolate the pathogenic organism for culture andsuceptibility testing, which takes up to 3 days. This culture& sensitivity process identifies the species of bacteria.Antibiotic sensitivities are then assessed on the bacterialisolate to inform clinicians on appropriate antibiotics fortreatment.

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- It is necessary to determine the significance of a positive blood culture.The following criteria may be helpful in differentiating “true positives”from contaminated specimens:(1) Growth of the same organism in repeated cultures obtained atdifferent times from separate anatomic sites strongly suggests truebacteremia.

(2) Growth of different organisms in different culture bottles suggestscontamination.

(3) Growth of normal skin flora, eg, Staphylococcus epidermidis,diphtheroids (corynebacteria and propionibacteria), or anaerobic gram-positive cocci, in only one of several cultures suggests contamination.

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Blood culturebottles

Gram stain andsubculture

Blood agar withOptochin , Tellurite

and Bacitracindiscs

Mannitol saltagar

Bloodagar

StreptococciStaphyloco

cci

Macconkeyagar

G –verods

G +vecocci

Kligler iron agar ,motility , indol,urease mediumand citrate test

CAMP test ,Bile esculin

agar

Growth of such organisms in more than one culture enhances thelikelihood that clinically significant bacteremia exists.

(4) Organisms such as viridans streptococci or enterococci are likely togrow in blood cultures from patients suspected to have endocarditis, andgram-negative rods such as E coli in blood cultures from patients withclinical gram-negative sepsis. Therefore, when such “expected”organisms are found, they are more apt to be etiologically significant.

Meningitis - In infants ( to 2 months ) ------------ E. coli , Salmonella spp. ,

Citrobacter spp. , group B Streptococci and Listeriamonocytogenes .

- In all other age groups 1- purulent meningitis ( CSF is turbid , 100-3000 PMNs / mm3 -----

H. influenzae , N. meningitides , S. pneumoniae and Listeriamonocytogenes.

2- A septic meningitis ( CSF is clear or slightly turbid 10-500leukocyte / mm3 mostly lymphocytes ) ------------ Cryptococcusneoformans , Candida albicans , Leptospira , amoebae ( Naegleriaor Hartemanella ) and virus ( Polio , Echo , Coxsackie , Arboviruses) and M. tuberculosis .

Specimens

- CSF 3-5 ml is collected in two sterile tubes , one forchemical examination ( glucose and protein ) and thesecond one for microbiological examination andleukocytes count. The CSF is sterile , clear andcolorless fluid , contains 0-5 leukocyte/ mm3 and noRBCs.

- Blood culture should be collected because meningitisis often associated with bacteremia .

3- The appearance of CSF should be noted : clear ,turbid , purulent , yellow ( previous hemorrhage ) ,contamination with blood , fibrin web as in M.tuberculosis .

Microscopic examination :

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- Direct examination for leukocytes ( PMNs orlymphocytes ) , RBCs , Bacteria , yeasts , motileamoebae.

- India ink stain for Crtptococcus ( budding ) .- Gram stain --- very important because culture

depend on its result .- Acid fast stain for tuberculoses meningitis.

Table : Typical Cerebrospinal Fluid Findings inVarious Central Nervous System Diseases

Diagnosis Cells(per μL)

Glucose(mg/dL)

Protein(mg/dL)

OpeningPressure

Normal1 0–5lymphocytes

45–85 15–45 70–180mm H2O

Purulentmeningitis(bacterial)2

200–20,000PMNs

Low (<45)

High (>50)

++++

Granulomatous meningitis(mycobacterial, fungal)2,3

100–1000,mostlylymphocytes

Low (<45)

High (>50)

+++

Aseptic meningitis, viral orMeningoencephalitis 3,4

100–1000,mostlylymphocytes

Normal Moderatelyhigh (> 50)

Normalto +

Spirochetal meningitis (syphilis,leptospirosis)3

25–2000,mostlylymphocytes

Normalor low

High (>50)

+

1: CSF glucose level must be considered in relation to bloodglucose level. Normally, CSF glucose level is 20–30 mg/dL lowerthan blood glucose level, or 50–70% of blood glucose normalvalue.2:Organisms in smear or culture of CSF.3:PMNs may predominate early.

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4::Virus isolation from CSF early; antibody titer rise in pairedspecimens of serum.

Culture :

CSF

Blood agar chocolate agar blood agar withLowenstein Jensen

a streak of S. aureusmedia

N. meningitides H.influenzae M.tuberculosis

( satellite colonies )

Oxidase test , Slide aggl.

slide aggl.

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G + ve cocci G + ve rodG- ve rod

CAMP test Catalase test + ve,Macconkey agar ,

& optochin sensitivity Motility +ve ,biochemical tests for

for Streptococci bile esculin agar forEnterobacteriaceae

Listeria monocytogenes .

- Serology test------FTA-ABS for neurosyphilis.- CSF glucose decreased in meningitis .- CSF protein elevated in meningitis .

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