tes lab penyakit infeksi dan tropis (april 2011)

Bagian Patologi KlinikFK UNHAS

Pengamatan Pada :- Eritrosit, Lekosit, Trombosit- Manifestasi : Anemia, lekositosis /lekopeni dan DIC

Lekositosis- Umumnya Netrofil , bentuk muda - Netrofilia lanjut infeksi kronik- Netrofilia menghebat + sel muda reaksi leukemoid

* Non-Ganas > 25-30 x 106/µl* Inflamasi, Stress, Trauma.

Lekopeni* Netropenia, mis Demam Tifoid, Brucellosis* Infeksi hebat netropenia hebat prognosis buruk

Perubahan morfologik pada sepsis* Dohle Bodies* Granulasi Toksik* Vakuolisasi

Eosinofilia :* Non-bakterial, biasanya alergi/infeksi parasit

Anemia* Bisa timbul sekalipun cadangan besi cukup* Anemia akut : perdarahan / destruksi eritrosit

(misalnya : cold aglutinin sehubungan dengan Mycoplasma pneumoniae)* Anemia kronik, dengan :

- Cadangan besi yang normal atau meninggi

di sistem retikuloendotelial- Penurunan besi dalam plasma- Penurunan TIBC

Infeksi serius + bakteremia* Gram negatif DIC (gram positif kurang)

- Trombus - PT memanjang- FDP - Fibrinogen

Trombositopenia bisa juga menjadi tanda sepsis bakterial dan bisa bermanfaat dalam mengobservasi respon pasien terhadap terapi.

(A) Direct Detection

1. Electron Microscopy - direct demonstration of viruses

- standard morphology of virus particles

- “catch-all” method- helpful for virus that fails to

grow in cell culture - virus may be recovered

direct from clinical sample and needs to be purified or concentrated

106 virus particles per ml required for visualization

X 50,000-60,000 magnification normally used

Viruses may be detected in the following specimens:

Faeces Rotavirus, AdenovirusNorwalk like virusesAstrovirus, Calicivirus

Vesicle Fluid HSV,VZV

Skin scrapings papillomavirus, orfmolluscum contagiosum

Direct Detection:Direct Detection: Electron Microscopy

Adenovirus Rotavirus

(courtesy of Linda Stannard, University of Cape Town, S.A.)

Direct Detection:Direct Detection: Electron Microscopy

Enterovirus

2.Virus Isolation

Methods of isolation:

(A)Cell Culture

(B)Chick Embryo/ Eggs

(C)Animals (eg: Suckling mouse brain inoculation)

Specimens for viral isolation:

- Collect during acute phase

- Intact cells important

- Prompt delivery

- Refrigerate if stored less 24 h

- Seal well if stored on dry ice (pH change)

(A) Cell Cultures

- ‘Catch-all’ cell culture-based

- Labour and resource intensive

- CPE and haemadsorption based

- Roller tubes, flasks or multi-well plates

(B) Chick Embryo/ Eggs

- now rarely used

- demonstration of poxviruses and propagation of orthomyxoviruses and

paramyxoviruses

Cytopathic effect of enterovirus 71 and HSV in cell culture: note the ballooning of cells. (Virology Laboratory, Yale-New Haven Hospital, Linda Stannard, University of Cape Town)

Virus Isolation:Virus Isolation: Cell culture Cell culture

Viruses readily isolated by cell

culture

Less frequently isolated viruses

Herpes SimplexCytomegalovirusAdenovirusPoliovirusCoxsackie B virusesEchovirusesInfluenza virusesParainfluenza virusesMumpsRespiratory Syncytial virus

Varicella-ZosterMeaslesRubellaRhinovirusesCoxsackie A viruses

Problems with cell culture:- Long period (up to 4 weeks) required for

result- Often very poor sensitivity (depends on a

large extent on the condition of the specimen)

- Susceptible to bacterial contamination- Susceptible to toxic substances which may

be present in the specimen.- Many viruses will not grow in cell culture

e.g. Hepatitis B, Diarrhoeal viruses, parvovirus, papillomavirus

Virus Isolation:Virus Isolation: Cell culture Cell culture

Direct DetectionDirect Detection

3. Detection of Viral AntigensDetection of Viral Antigens

Tests include: - Immunofluorescence (IFA)- Enzyme Immuno Assays (EIA)- Agglutination

(B)Other assays:Enzyme immunoassay (EIA/ELISA)Radio Immunoassay (RIA)Latex agglutination Reverse passive haemagglutination

(RPHA) Eg: HbsAg (serum)

p24 HIV Ag (serum)

2 methods:(A) Ab-staining technique

(i) Immunofluorescence technique - label used is fluorescein/rodamine

dye– immunofluorescence microscope

(ii) Immunoperoxidase technique - label used is horseradish

peroxidase – light microscope

Positive immunofluorescence test for rabies virus antigen. (Source: CDC)

(Virology Laboratory, Yale-New Haven Hospital)

Direct Detection: Direct Detection: Antigen detectionAntigen detection (IF)(IF)

Direct Detection: Direct Detection: Antigen detectionAntigen detection

4. Nucleic Acid (NA) Detection

- Methods based on the detection of viral genome molecular methods. - PCR and related techniques

Direct Detection Direct Detection

- Molecular techniques: 2 types: (a) No amplification involved

(e.g hybridisation with nucleic acid probes)(b) Amplification (PCR, LCR, NASBA etc.)

Advantages of PCR:Extremely high sensitivity, may detect down to

one viral genome per sample volumeFast turnaround time (5 to 10 hours) Clinical samples – small in size/volumeDetect viruses that are unculturableDoes not rely on viability of virus or intact

genomeOnly target NA amplified – non infectious

Direct Detection: Direct Detection: NA detectionNA detection

Disadvantages of PCR

•Reagents and equipment – expensive•High degree of operator skill required •Extremely liable to contamination (surfaces, reagents, equipment)•A positive result may be difficult to interpret, especially with latent viruses such as CMV, where any seropositive person will have virus present in their blood irrespective whether they have disease or not.



Polymerase chain reaction

RT PCR Conventional PCR


Post PCR Analysis:

1.Agarose gel with ethidium bromide staining2.Southern/dot blot3.Restriction fragment length polymorphism

(RFLP)4.NA sequencing



Gel Electrophoresis

Southern Blot


(B)(B) Indirect Detection: Indirect Detection: SerologySerology

- Serology forms the mainstay of viral diagnosis.

- Viruses are good antigens and infection

elicits a prompt antibody response and can remain at a high level for many years after infection.

- Following exposure, the first antibody to appear is IgM (7 to 10 days), which is followed by a much higher titre of IgG.

Note that during reinfection, IgM may be absent or present at a low level transiently

Indirect Detection: Indirect Detection: Serology Serology


Criteria for Diagnosing Primary Infection

(a) Presence of IgM- the earliest Ab to appear- problems with IgM assays:

interference by rheumatoid factor (ab IgM class)

(false positive) re-infection (absent or low level) unexplained persistence of IgM years after

primary infection

(b) Rise in titre- 4 fold or more increase in titre of IgG or

total antibody between acute and convalescent sera

- 2 serum samples [acute (before 7days of illness) and convalescence (1-2 weeks later)

(c) Seroconversion- changing from a previously antibody

negative state to a positive state e.g. seroconversion against HIV following a needle-stick injury, or

against rubella following contact with a known case.

(d) A single high titre of IgG/total ab (High stationary titre)

- considerably higher than the general population this is a very unreliable means of serological diagnosis since the cut-off is very difficult to define.


Criteria for Diagnosing Re-infection/Re-activation

-Difficult to differentiate re-infection/re-activation from a primary infection.

-Under most circumstances, it is not important to differentiate between a primary infection and re-infection.

-Important under certain situations, such as rubella infection in the first trimester of pregnancy: primary infection is associated with a high risk of fetal damage whereas re-infection is not

-IgM is usually low or absent in cases of re-infection/ re-activation

-A sharp large rise in antibody titres is found in re-infection



Serological Tests

(i) Neutralization test

- measures Abs that neutralize virus infectivity

- Ab prevents virus infection of cells

- Ab can be detected by neutralization of virus CPE in tissue culture

- variation, plaque reduction test

- highly specific, can type virus strains

- time-consuming and laborious

Serology:Serology: Serology Tests Serology Tests

(ii) Complement fixation test (CFT) - not all viruses will agglutinate RBCs.

- CFT can get around this problem by using complement to detect the presence of antibody. - not to react with an antigen or an antibody alone but to enter into combination with antigen-antibody

complexes (mixture of ab, ag and complement)

- indicator system used in CFT is sheep red blood cells (RBCs)

- use for herpesviruses and respiratory viruses

(can be used to identify either viral antigens or antibodies)

Positive or reactive test

The complement is bound to an antigen-antibody complex, and is not free to interact with sensitized RBCs so they remain unlysed and settle to the bottom of the well to form a button.

Negative or nonreactive test

The complement remains free since there is no antigen-antibody complex for it to bind to, and it interacts with the sensitized RBCs causing them to lyse

Serology Test:Serology Test: CFT CFT

Complement fixation test (CFT)

Ag + Serum (Ab pos)+ complemen Complemen fixed + RBC (sheep) RBC unlysed POSITIF TEST

Ag + Serum (Ab neg)+ complemen Complemen unfixed + RBC (sheep) RBC lysed NEGATIF TEST

Serology Test:Serology Test: CFT CFT

- Reliable only when test carefully standardized.

- All reagents involved must be used at optimal reactivity.

- All reagents to be carefully prepared and standardized to insure a completely balanced system titrations of sheep RBCs, hemolysin, complement and antigens.

- Rather difficult and time consuming (2 days to complete)

- Many of the test antigens and antisera are not available commercially.

Serology Test:Serology Test: CFT CFT Complement fixation test (CFT)

(iii) Haemagglutination-inhibition (HAI) test

- many viruses have the ability to agglutinate erythrocyte of various species (e.g. influenza, parainfluenza, adenoviruses, rubella, alphaviruses, flaviviruses)

- this agglutination can be inhibited by specific Ab

- if virus Ab present can see button- mostly type specific except in

flaviviruses (cross-react)- IgG, IgM and IgA- main use today, influenza and

parainfluenza

Microplate ELISA for HIV antibody: coloured wells indicate reactivity

Indirect Detection: Indirect Detection: Serology Serology (iv) Enzyme-linked Immunoassay

- The most commonly used sensitivity (ability to detect small amounts of Ag or Ab),

specificity (ability to discriminate between closely related but antigenically different molecules)- Ease of automation (large numbers of tests).

- Generally considered safer than radioisotopes used in RIA (radioimmunoassay).

- False positives can result from impure reagents

To detect antibody (indirect ELISA)


No Abs

Abs present

ELISA Test Kit ELISA Machine

LABORATORY DIAGNOSIS (ANTIBODY TEST: ELISA)

Tests that can be performed in less than 30 minutes, easy to perform.

Disadvantages:• subjective interpretation• more expensive• sensitivity may not match the ELISA

Rapid Test


long length of time required for diagnosis for paired acute and convalescent sera

mild local infections such as HSV genitalis may not produce a detectable humoral immune response

Extensive antigenic cross-reactivity between related viruses may lead to false positive results

immunocompromised patients often give a reduced or absent humoral immune response

Patients with infectious mononucleosis and those with connective tissue diseases such as SLE may react non-specifically giving a false positive result

Patients given blood or blood products may give a false positive result due to the transfer of antibody.


LABORATORY DIAGNOSIS OF DENGUE

Methods

Virus Isolation

Serological Test

Genome Detection

Method

(1) Cell culture: Mosquito cell linesPrimitive cell lines

(2) Mosquitoes: LarvaeAdult

(3) Animals: Suckling mice

Virus Isolation- C6/36 Cells

Cell culture

Cytopathic effect

Larvae Inoculation: Toxorhynchites splenden

Suckling mice

Serological Tests

1. Rising Ab titreDetection of specific IgG AbFour-fold or greater rise in Ab titre against a virus

2. Detection of specific IgM antibody

3. High stationary titre

Ab

Level

1o Infection 2o Infection

Onset of Sx Onset of Sx

IgG

Virus VirusIgMIgM

IgG Cut Off

IgM Cut Off

Serological Tests

1. Traditional TechniquesHaemagglutination Inhibition Test

(HI)Neutralization Test (NT)Complement Fixation Test (CFT)

2. Newer TechniquesELISA-IgMDengue Blot assayPan Bio Dengue Rapid Test

Serological Profile Of Dengue Virus Infection

20 60 3

Infection

Days Weeks Months Years

Titre

Viraemia

Neutralising Ab

Complement Fixing Ab

Haemagglutination Inhibition Ab

IgM Ab

TRADITIONAL TECHNIQUES

Detection of specific IgG antibody-2 serum sample-acute and convalescence

(1) Haemagglutination Inhibition (HI)-Test very pH dependent-Takes 3-5 days to complete-Able to differentiate between

primary and secondary-Problem of “original antigenic sin”

(2) Neutralization Test (NT)-The most specific and sensitive-Useful for research purposes-Laborious-At least 5 days to complete

(3) Complement Fixation Test (CFT)-Less sensitive than HI or NT-CFT can be used to show rises in Ab

titer later in infection than other serological tests

NEWER TECHNIQUES

ELISA 1. Anti-dengue IgM

- Infeksi Primer, akut 7-10 hari2. IgG (post/kronik)

- Infeksi sekunder, sesudahnya

BLOT ASSAY(i) IgG(ii) IgM

Dengue Blot Assay

Positive Control

Negative Control

Invalid

Panbio Dengue Rapid Test

M

G

C

M

G

C

M

G

C

M

G

C

Primary Dengue Secondary Dengue

Secondary Dengue Suspected Negative

HASILIgG IgM

INTERPRETASI

+ + D Sekunder

- + D Primer

+ - Duga D sekunder

- - Non-D

Primer sangat dini

(1) Polymerase Chain Reaction•Universal primers for Dengue virus followed by Specific primers for 4 serotypes•Multiplex PCR•1 set of primers which amplify different sites and sizes of each serotypes•Serotyping/Surveillance

-Specimen within 5 days of symptoms

-Serum/Plasma/PBMC/Biopsy samples

-Expensive

Genome Detection

(2) DNA Sequencing

- Genotyping/Molecular epidemiology study

RT-PCR using consensus primers

RT-PCR for detection of dengue serotypes

Laboratory findings

* Hematology

- Leukopenia

- Trombocytopenia

- serum aminotransferase (AST, ALT) elevations

* The diagnosis is made by lab test seroloimmunology

1. Hemaglutination tests

2. Complement Fixation test

3. Neutralization Test

- IgM ELISA or

- paired serology during recovery or

- by antigen-detection ELISA or

- RT-PCR during the acute phase

* Virus is readily isolated from blood in the acute phase if mosquito

inoculation or mosquito cell culture

Diagnosis Demam Dengue/Demam Berdarah Dengue

Suspicion of dengue infection

Delay after onset of fever

Unknown or ≤ 5 days >5 hari

NS1 Antigen Antisipated Diagnosis IgM/IgG Serologis

Improve patient Management+ - + -

Confirmed early IgM serology Late acute orDengue is infection past-infection unlikely

Acute infection

+ -

Presumably early early acute infectionAcute infection is unlikely A second sample isrequested for confirmation

EARLY DIAGNOSIS LATE DIAGNOSIS

In denguePresent by the 2nd day of feverBy the 4th or 5th day, the wbc count 2000 to 4000/ml,

20 to 40 % granulocytesModerate albuminuria and a few casts may be found

- Dengue may be confused with Colorado tick fever, typhus, yellow fever, or other hemorhagic fevers.

Serologic diagnosis may be made by - Hemagglutination inhibiting and complement fixation

test using paired sera - but is complicated by cross-reactions with other

flavivirus antibodies

In dengue hemorrhagic fever• Hct >50 %: ipresent during shock• WBC count in 1/3 of patients• Coagulaive abnormalities

- Trombocytopenia (<100.000/ml)- positive tourniquet test- prolonged PT- Minimal proteinuria may be present.- AST levels may be moderately- Serologic test usually show high complement fixation antibodies titers againts flavivirus, suggestive of a secondary immune response

WHO clinical criteria for diagnosis of denguehemorrhagic fever :• Acute onset of high, continous fever lasts for 2

to 7 days• Hemorrhagic manifestations, including at least a

positive tourniquet tes and petechiae, purpura, ecchymoses, bleeding gums, hematemesis or melena

• Hepatomegaly• Trombocytopenia (<100.000/ml);or

hemoconcentration (Hct increased by >20%)• Those with dengue shock syndrome also have a

rapid weak pulse with narrowing of the pulse pressure (<20 mmHg) or hypotension with cold, clammy skin and restlessness.

Laboratory tests are generally not necessary (viral cultures and Tzanck smear will confirm diagnosis in patient with atypical presentation)

Antibody to appropriate serotype :- Seroconversion- Increase- Direct immunofluorescent antibodies slide test (rapid diagnosis)

Tzanck preparation :- Base of lesion- Multinucleate giant cells

Laboratory test are generally not necessary (viral cultures and Tzanck smear will confirm diagnosis in patient with atypical presentation)

The Tzanck preparation shows multinucleate giant cells for both varicella-zoster virus and HSV

Darah - lekopeni- serum amilase dalam 10 hari- Serologi :

* cold agglutinin* IgM , max 2 minggu, menetap 6-9

bulan; kadar serum konvalesens 4x dr pd serum

akut* tes fixasi komplement thd atb positif

minggu pertama

Biakan- Virus dari ludah 1-5 hari

Komplikasi :- Inflamasi testis / ovarium : lekositosis, LED- Pankreatitis : lekositosis, amilase , hiperglikemia- Meningitis : sel LCS < 500/µl, mononuklear; glukosa normal, protein agak (20-125 mg/dl)

Temuan Laboratorium- Darah :

* Lekosit terutama limfosit dan segmen* Serologi : EIA

- IgM : Fase akut ( ± 1-2 hari)- IgG : >10 hari

- Sekret :* Apusan + pulasan imunofluorosen* Pulasan Tzanck : Multinucleated Giant

Cells - Biakan :

* Bahan : sekret resp dan urin* Identifikasi : jaringan

Tes Lab yang bisa dilakukan:- Sediaan Apus :

* Bahan : Kerokan dasar vesikel* Pulasan : Tzanck Multinucleated Giant

Cells* Sensitivitas ± 60 %

- Darah :* Serologi :

- Titer atb serum konvalesent 4x dpd serum akut.- Hemaglutinasi- ELISA- FAMA

* PCR : deteksi DNA Virus

Giemsa – stained

* Show inclusion bodies within many large cells or extracellularly

DNA typing : Circular – doubel – stranded , 8000 bp* Cross–hybridization :

- > 50% : type seperation- < 50% : subtype seperation

Generally not necessary Gram stain and C&S to confirm the

diagnosis when the clinical presentation is unclear

Sedimentation rate parallel to activity of the disease

Anti-DNAse B and anty hyaluronidase Urinalysis : hematuria with erytrocyte casts

and proteinuria in patients with acute nephritis

(A) Screening Test

Enzyme-linked Immunosorbent Assay =ELISA Simultaneously detection of antibodies to HIV-1 and

HIV-2 in human serum or plasma.

Detect antibodies directed against major group of HIV-1 and HIV-2 proteins:

HIV-1: envelope protein of gp41core structural protein of p24

HIV-2: envelope protein of gp36

Methods: indirect, competitive, antigen sandwich

ELISA for HIV antibody

Microplate ELISA for HIV antibody: coloured wells indicate reactivity

Agglutination Test

Requires greater than 30 minutes but has procedures that can be performed easily without instrumentation.

Within this class of tests are agglutination assays in which antigen-coated particles (red blood cells [RBC], latex particles or gelatin particles) are allow to react with serum antibodies to form visible clumping (agglutination).

If RBC are used, the technique is termed passive haemagglutination (PHA); latex particles is known as latex

agglutination (LA); gelatin particle is known as particle agglutination (PA).

Good sensitivity, low cost and ease of performance.

It incorporates a quality system to detect nonspecific antibodies directed toward the gelatin particles themselves;sample need to be treated by adsorption and retested.

False-negative reaction due to prozone (inhibition of agglutination when excess of antibody present, events

optimal combination) - perform dilution.

Results can be obtained within 2 hours.

Agglutination Test

Particle Agglutination Test

(B) Supplementary/Confirmation Tests 1. Western Blot (WB)

- “gold standard” for validation of HIV results.

- Based on electrophoretic technique to separate HIV antigens derived from viral lysate grown in culture and the separate antigens are transferred (blotted) onto nitrocellulose membrane.

- Commercial WB are supplied with individual strips of nitrocellulose containing the blotted, separated HIV antigens. Application of ELISA technique in detection of specific antibodies to each viral antigens.

- Modified WB has the ability to identify and differentiate infections by HIV-1 and HIV-2.

Western Blot

Line Immunoassay (LIA)

Another alternative to the classic Western blot.

Recombinant or synthetic peptide antigens are applied onto nylon strip rather than electrophoresed as in the Western blot.

Use of “artificial” antigens decreases the presence of contaminating substances derived from cell culture

that can cause interference and sometimes false reactions.

A number of reports have verified that the accuracy is equivalent to the Western blot.

LABORATORY DIAGNOSIS (ANTIBODY TEST: SUPPLEMENTARY/CONFIRMATORY)

Line Immunoassay

Interpretation criteria for Immunoblot tests

Organization Minimum band requirements for “reactive” pattern

American Red Cross At least one band from each gene product group: gag AND pol

AND env

Centres for Disease Any two of p24, gp41 or gp120/160Control (CDC)

World Health Organization Two env bands (gp160, gp120 or gp41)(WHO) +/- pol bands, +/- gag bands

Du Pont p24 AND p31 AND gp41 or gp120/160

(C) Other Antibody Test: Rapid Test

less than 30 minutes, easy to perform.

Qualitative, intended for use as point-of-care

Technical errors are common, must be performed carefully by experienced personnel.

Application includes: emergency rooms, physician's offices, autopsy rooms. Not recommended for use in a blood transfusion centre or home-use.

Example: (i) "dot blot" or "immunoblot” (incorporate a built-in control that indicates that the test was performed correctly)(ii) “dipsticks” (antigen is attached on the "teeth" of comb- like devices.

Several of these rapid tests have the ability to differentiate HIV-1 and HIV-2.

Disadvantages:• subjective interpretation• more expensive• sensitivity may not match the ELISA

Result obtained, must always be confirmed

Types of samples: blood, serum, plasma, saliva and urine

HIV RAPID TEST KIT

HIV Testing Strategies

WHO recommends three testing strategies to:

-Maximize accuracy

-Minimize cost

Choice of strategy depends upon:

-objective of test

-prevalence of HIV in population

Objective of testing Prevalence of

Infection

Testing Strategy

Transfusion/donationsafety

All I

Surveillance>10% I

<10% II

Diagnosis

Clinical signs/symptoms of HIV infection/AIDS

all II

Asymptomatic>10% II

<10% III

WHO Testing Strategies

STRATEGY I:

All samples are tested with one ELISA or rapid/simple assay.

Samples that is reactive is considered HIV Ab positive.

STRATEGY II:

All samples are first tested with one test.

Any reactive samples are subjected to second test based on different principle and/or a different antigen preparation.

STRATEGY III:

All samples are first tested with one test.

Any reactive samples are subjected to second test based on different principle and/or a different antigen preparation. Requires a third test if samples are found reactive on the second test.

POSITIVEHigh Risk Group (WHO 2-tests strategy)

ELISA - ReactivePA - Detected

Low Risk Group (WHO 3-tests strategy)ELISA - Reactive

PA - DetectedImmunoblot - Positive

NEGATIVEBoth screening tests: EIA - non-reactive and

PA - not-detected

FALSE POSITIVE

The ELISA test may produce false positive results on some blood from uninfected individuals.

Employs at least 2 different tests.

May due to nonspecific reaction, autoimmune diseases (Systemic Lupus Erythematosus), etc

FALSE NEGATIVE

Test concludes HIV is not present, when in fact the person is infected

This occurs when the blood is taken during the window period.

If there has been exposure to risk activities, it is advisable to repeat the test 3-6 months later.

EQUIVOCAL

Results that neither clearly positive nor clearly negative (one of the screening tests is positive)

To test with third test (LIA or Wblot)

Follow-up sample after a minimum of 2-weeks

If produces an equivocal result, person is considered to be HIV Ab Negative

Units of donated blood must be discarded

Once a diagnosis of HIV infection had been made, it is important to monitor the patient at regularly for signs of disease progression and response to antiviral chemotherapy.

Tests use:HIV antigen tests (p24)

Detect the presence of HIV p24 antigenNot as good as serial CD4 counts (does not

check presence of HIV, monitor immune function)CD4 T-cells – marker of progression of HIV infection

HIV viral loadHIV viral load in serum may be measured by assays

which detect HIV-RNA e.g. RT-PCR, NASBA, or bDNA. HIV viral load has now been established as having good prognostic value, and in monitoring response to antiviral chemotherapy.

Sediaan darah malariaKegunaan sediaan darah malaria :

Menentukan ada tdknya parasit malariaMenentukan spesies & stadium plasmodium Dapat melacak 10 – 100 parasit/μL darahMenentukan kepadatan parasit

104

Sediaan darah tebalCara terbaik menemukan parasit malariaMudah dibuatDiperiksa paling sedikit 100 lapangan pandang

Sediaan darah tipisDigunakan utk identifikasi jenis Plasmodium bila dgn sediaan darah tebal sulit ditentukanDiperiksa paling sedikit 200 lapangan pandang

105

Kelebihan sediaan darah tebalLebih banyak sel darah yg diperiksaParasit lebih mudah ditemukan

Kekurangan sediaan darah tebalTdk dpt membandingkan ukuran Plasmodium dgn ukuran eritrositSpesies Plasmodium sukar ditentukan

106

Kelebihan sediaan darah tipisMorfologi eritrosit jelasSpesies Plasmodium bisa ditentukanPerbandingan ukuran Plasmodium terhdp ukuran eritrosit bisa dilihat% parasitemia bisa dihitung

Kekurangan sediaan darah tipisJumlah parasit dlm lap. pandang sangat sedikit.

107

Bentuk TropozoitCincin kecil (⅓ ө eritrosit normal)Sitoplasma biru Kromatin inti merah

Bentuk SkizonJarang ada dlm sirkulasi darah tepiJk ditemukan dlm darah tepi tanda malaria berat

108

Bentuk GametositSgt khas yaitu elips (crescent)Berpigmen warna hitamSitoplasma kuning

109

Teknik Quantitative Buffy Coat (QBC)Prinsip : Tes FluoresensiEritrosit yg terinfeksi Plasmodium akan terlihat berfluoresensi di bwh mikroskop fluoresensi

Cepat namun peralatannya mahalTdk dpt membedakan spesies Plasmodium & tdk dpt digunakan utk hitung parasit.

110

Mendeteksi Ab/Ag spesifik terhadap parasit malaria atau eritrosit yg terinfeksi PlasmodiumTes imunoserologis yg melacak Ab tdk dipakai utk keperluan diagnosisTes imunoserologis malaria :

Radioimmunoassay (RIA)Enzyme Linked Immunoassay (ELISA)Immunochromatographi (ICT)Indirect Fluorescent Antibody Test (IFAT)

111

112

Radioisotop sebagai labelKadar Ag atau Ab pada sampel dpt ditentukan scr kuantitatifLow detection limit 50 parasit/μL darahSensitifKurang praktis & berbahaya

113

Lebih praktis dibanding RIAEnzyme direaksikan dgn substrat kromogen intensitas warna sebanding dgn kadar bahanMendeteksi Ag & Ab spesifik terhadap Plasmodium

114

Rapid Diagnostic TestMelacak Ag parasit malaria melalui pengikatan Ag oleh Ab monoklonalAda 3 jenis Ag utama yg sering dijadikan target ICT utk mendiagnosis malaria yaitu :

Histidine-rich protein (HRP) Parasite specific lactate dehydrogenase (pLDH) Plasmodium Aldolase

115

Keunggulan tes ICTPraktisTdk membutuhkan alat pembantu lainTdk memerlukan tenaga terampil

Kelemahan tes ICTHanya dpt melacak parasit > 100 parasit/μL darahMembutuhkan biaya pemeriksaan yg relatif sdgTdk dpt memberi informasi derajat parasitemia

116

Mendeteksi Ab spesifik terhdp PlasmodiumKeadaan dimana parasit sangat minimalTdk utk menentukan infeksi baruMendeteksi keempat spesies PlasmodiumManfaat utk penelitian epidemiologi

Mendeteksi DNA spesifik terhadap parasit Plasmodium dlm darah penderita malariaTeknik Polymerase chain reaction (PCR)Dpt melacak sampai 5 parasit/μL drhDpt mengidentifikasi spesies PlasmodiumWaktu pemeriksaan cepatSensitivitas & spesifisitasnya tinggiMahal

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Jenis tes laboratorium untuk tuberkulosis terdiri dari: Tes Mikrobiologi, terdiri dari:

Tes seluler: Tes BTA Sputum Tes Biakan dan Identifikasi M.tuberculosis Tes Kepekaan Antibiotika

Tes molekuler: PCR Tes Serologis, terdiri dari:

Semirapid: TB-Dot, ELISA, Tb-kompleks Rapid: Mycodot, ICT-TB

Anamnesis Pemeriksaan Fisis Pemeriksaan Radiologis Tes Laboratorium

Tes MikrobiologiTes seluler: Tes Apusan BTA

Tes Biakan & Identifikasi Tes Kepekaan Antibiotika

Tes molekuler: PCRTes Serologis

Semirapid: TB-Dot, ELISA, Tb-kompleksRapid: Mycodot, ICT-TB seperti Mycotec TB

Mikroskopik Ziehl Neelsen Dekontaminasi

Kultur (pembiakan) medium Lowenstein-Jensen

PCR Isolasi DNA: Metode Boom Proses : denaturasi, annealing,

elongasi IS6110

Tes Imunoserologi Deteksi Ab terhadap Ag mikobakterial

spesifik atau gabungan beberapa antigen Ag60: ELISA Ag16 : 16 kDa,

spesies-spesific epitop imunodominan stadium awal infeksi M.tbc & TB primer

38kDa spesies-spesific epitop imunodominan

ESAT-6 kontak baru terjadi, konversi & meningkatnya

risiko penyakit

Pemeriksaan lab. meliputi :

1. Pemeriksaan bakteriologik

- menegakkan diagnosis dan memantau pengobatan

- kerokan kulit/mukosa hidung (pewarnaan Ziehl Neelsen)

- negatif : bukan berarti tdk mengandung M. leprae

- indeks bakteriologi (IB) dan indeks morfologi (IM)

- IB : banyaknya kuman M. leprae tiap satuan lap. tertentu

- IM : prosentase basil utuh dlm semua basil yg dihitung

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Cara menghitung IB dan IM :

IB : total kepadatanjumlah tempat pengambilan

IM : jumlah basil utuh jumlah basil yang diperiksa

x 100%

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Penilaian skala algoritme Ridley :- negatif (-) : tdk ditemukan BTA pd 100 lap. penglihatan (LP)- 1 (+) : 1 – 10 basil/100 LP- 2 (+) : 1 – 10 basil / 10 LP- 3 (+) : 1 – 10 basil/1LP- 4 (+) : 10 - 100 basil/1 LP- 5 (+) : 101 – 1000 basil/1 LP- 6 (+) : > 1000 basil/LP

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2. Pemeriksaan histopatologik- menegakkan diagnosis (manifestasi klinik tdk jelas)- biopsi kulit & imunohistokimia

3. Pemeriksaan imunologik- tdk utk diagnosis menentukan klasifikasi &

perjalanan peny. kusta

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Tes lepromin- kemampuan individu bereaksi scr seluler thd M. leprae

- lepromin : suspensi steril dr jaringan yg dihancurkan & sbg tes kulit secara intradermal

a. lepromin Mitsuda : lepromin dr suspensi jaringan, mengandung kuman M. leprae yg sdh disterilkan dlm autoklaf (manusia / binatang)

b. Lepromin Dharmendra : dr ekstraksi fraksi protein dgn kloroform eter (tipe lepromatous)

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Reaksi kulit terhadap lepromin :1. reaksi dini (reaksi Fernandez)

- berbentuk infiltrat eritematosa (12 – 72 jam)- hipersensitivitas yg telah ada thd antigen- pembacaan : 48 jam sth penyuntikan

2. reaksi lambat (reaksi Mitzuda)- btk noduler, tampak pd hr ke-21 – 30 (paling

jelas)- respon thd imunitas seluler- pembacaan : sth hr ke-21

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3.2 Tes histamin- secara intradermal pd kulit normal dilatasi

kapiler - bintul berwarna merah (histamin flare)

- ukuran bintul merah derajat kerusakan saraf 3.3 Tes serologis

- ELISA mendeteksi antibodi phenolic glicolipid-1 (PGL-1)

reaksi antigen antibodi dgn enzim sbg label- imunokromatografi menggunakan antigen PGL-1

neoglycoconjugate, sensitivitas 91,7%, spesivisitas 78,1%

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3.4 Polymerase Chain Reaction (PCR)- mendeteksi adanya organisme dgn cepat dan

tepat- mendeteksi sejumlah kecil basil dr biopsi kulit- kolonisasi M. leprae pd mukosa/apusan

hidung penderita atau orang sehat- diagnosis pasti tipe tuberkuloid- follow-up hasil pengobatan- menggantikan pemeriksaan adanya BTA

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Tes lain:1. Tes pengeluaran keringat

- mengetahui integritas saraf kulit - tergantung pd saraf parasimpatik- respon kelenjar keringat thd obat kolinergik

berkurang2. Tes pilokarpin

- melihat perubahan warna pada kulit setelah ditaburi tepung amilum

- warna amilum tetap (ada kerusakan saraf)

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Diagnosis : pemeriksaan Klinik (bakteriologi, histopatologi, imunologi)Tanda-tanda kardinal :1. Anestesi2. Penebalan saraf di daerah yang terkena 3. Adanya lesi kulit berupa hipopigmentasi, eritema, infiltrat, nodul4. Ditemukannya kuman tahan asam (BTA positif)Diagnosis : 2 dari 3 tanda kardinal I, terlebih BTA positif

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Generally not necessary Gram stain and C&S to confirm the

diagnosis when the clinical presentation is unclear

Sedimentation rate parallel to activity of the disease

Anti-DNAse B and anti hyaluronidase Urinalysis : hematuria with erytrocyte

casts and proteinuria in patients with acute nephritis

Diagnosis definitif tergantung pada isolasi C.diphtheriae yang diambil dari bahan lesi-lesi lokal

Pihak laboratorium harus diberitahukan bahwa bahan disangka diphteri.

Gram stains of secretion :club-shaped organism, appear as “Chinese

letters”

CSF :* Aseptic meningitis* Elevated WBCs* Elevated protein* Normal glucose

Kultur :* Darah : positif dalam 10 hari pertama* Tinja & urin positif dalam minggu 3-5 * Sumsum tulang

Serologi :* Tes Widal : Serum sembuh 4x drpada sakit

Darah rutin : lekopeni

Felix-Widal test

- measures levels of agglutinating antibodies against O and H antigens

- usually appear after 5-8 days of infection - limited clinical utility in acutely ill patients because positive

results may represent previous infection in endemic areas - paired titration should be preformed - In the absence of recent immunization, a high titre of

antibody to O antigen > 1:640 is suggestive but not specific. - false positive (cross reactivity)

An ELISA for antibodies to the capsular polysaccharide Vi antigen is useful for detection of carriers but not for the diagnosis of acute illness

Typhidot test that detects presence of IgM and IgG in one hour (sensitivity>95%, Specificity 75%)

Typhidot-M, that detects IgM only (sensitivity 90% and specificity 93%)

Typhidot rapid (sensitivity 85% and Specificity 99%) is a rapid 15 minute immunochromatographic test to detect IgM.

IgM dipstick test

Isolasi vibrio cholerae dari bahan tinja identifikasi serogroup 01 atau 139

Serologi :* tes agglutinasi menggunakan antiserum spesifik

Mandell, Douglas, and Bennett's Principles and Practice of Infectious Diseases, 5th Edition, 2000

Lab studies: Confirm diagnosis Epidemiologic investigations

Direct microscopy: Dark field microscopy: at least 104 org/ml to be

able to see 1 spirochete per HPF. Silver staining DF using mouse monoclonal AB IP Insitu hybridisation using DNA probes Electron microscopy

Isolation: Possible using special media containing serum. may not be of value as diagnostic test. Generation time

of the bacteria is about 12-14 days. It takes 4-8 weeks to be positive

Sensitivity is very low . 1st week :

Blood (Blood in EDTA bottle) CSF

2nd week: urine

Immuno diagnosis:Antigen detection:

RIA, ELISA (Monoclonal antibody Based), Chemiluminiscent immunoassay Immunomagnetic antigen capture combined with

fluoroimmunoassay can detect as low as 102 lepto/ml of cattle urine.

For biopsy or post mortem tissues: immunohistochemistry

tes lab penyakit infeksi dan tropis (april 2011)

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