tes lab penyakit infeksi dan tropis (april 2011)
DESCRIPTION
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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.
Direct Detection: Direct Detection: NA detectionNA detection
Direct Detection: Direct Detection: NA detectionNA detection
Polymerase chain reaction
RT PCR Conventional PCR
Direct Detection: Direct Detection: NA detectionNA detection
Post PCR Analysis:
1.Agarose gel with ethidium bromide staining2.Southern/dot blot3.Restriction fragment length polymorphism
(RFLP)4.NA sequencing
Direct Detection: Direct Detection: NA detectionNA detection
Direct Detection: Direct Detection: NA detectionNA detection
Gel Electrophoresis
Southern Blot
Direct Detection: Direct Detection: NA detectionNA detection
(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
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.
Indirect Detection: Indirect Detection: Serology Serology
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
Indirect Detection: Indirect Detection: Serology Serology
Indirect Detection: Indirect Detection: Serology Serology
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)
Indirect Detection: Indirect Detection: Serology Serology
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
Indirect Detection: Indirect Detection: Serology Serology
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.
Indirect Detection: Indirect Detection: Serology Serology
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
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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