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dr. Mustofa Aidid

dr. Siti Fatonah Sp.PK(K)

NAAT (Nucleic Acid Amplification Test) 2

Yu CY, Chan KG, Yean CY, Ang GY. Nucleic Acid-Based Diagnostic Tests for the Detection SARS-CoV-2: An Update. Diagnostics. 2021; 11(1):53. https://doi.org/10.3390/diagnostics11010053
NAAT (Nucleic Acid Amplification Test) 3

Classic PCR Steps

https://journals.asm.org/doi/epub/10.1128/CMR.00228-20
Treponemal Detection Tests 4

https://www.frontiersin.org/articles/10.3389/fcimb.2020.574806/full https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4999316/ https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2095002/ https://www.cdc.gov/nchs/data/nhanes/nhanes_01_02/l36_b_met_syphillis_rapid_plasma_reagin.pdf

Treponemal Detection Tests 5

Syphilis Staging
 Primary syphilis
 This stage is characterized by a painless genital, anal, or less commonly oral ulcer or
chancre. This lesion occurs at the site of inoculation. The ulcer is typically indurated
and is usually without exudate. There may be regional lymphadenopathy.
 Secondary syphilis
 This stage typically develops weeks or a few months after acquisition in a portion of
untreated patients. The most common manifestation is a diffuse maculopapular skin
rash involving the trunk, extremities, palms, and soles. This is a systemic illness and
the rash is often accompanied by fever, malaise, myalgias, sore throat, headaches,
and weight loss. Hepatitis, patchy alopecia, and renal insufficiency may also be
seen. Ocular manifestations can include uveitis, retinitis, and optic neuritis. Invasion
of the CNS is also seen in the early stages of untreated disease and can be associated
with aseptic meningitis, cranial neuropathies, or meningovascular manifestations.
https://journals.asm.org/doi/epub/10.1128/CMR.00228-20 https://journals.asm.org/doi/pdf/10.1128/CMR.19.1.29-49.2006
Treponemal Detection Tests 6

Syphilis Staging

 Latent syphilis
 This stage is characterized by the absence of any signs or symptoms of infection,
but associated with positive serologic tests. Early latent syphilis has been
defined as infection of 1 year or less. Other asymptomatic states are classified as
late latent syphilis or latent syphilis of unknown duration.

 Late syphilis (tertiary)

 A progressive dementing illness (general paresis) and tabes dorsalis are
considered the classic late neurologic manifestation of the disease. Aortitis and
gummatous syphilis (nodular lesions more commonly present in the skin and
bones) are other clinical manifestations in this stage.

https://journals.asm.org/doi/epub/10.1128/CMR.00228-20 https://journals.asm.org/doi/pdf/10.1128/CMR.19.1.29-49.2006
Treponemal Detection Tests 7

Syphilis Staging

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4999316/
Treponemal Detection Tests 8

Interpretation

https://www.researchgate.net/figure/Syphilis-symptoms-stages-and-serologic-titers-timeline_fig1_334751332
Treponemal Detection Tests 9

Testing
 Serologic diagnosis of syphilis always requires detection of two types of antibodies. [1]
 Drect diagnostic methods include the detection of T pallidum by microscopic
examination of fluid or smears from lesions, histological examination of tissues or
nucleic acid amplification methods such as polymerase chain reaction (PCR). [2]
 Indirect diagnosis is based on serological tests for the detection of antibodies.
Serological tests fall into two categories: nontreponemal tests for screening, and
treponemal tests for confirmation [2]
 The traditional approach to the diagnosis of syphilis begins with a nontreponemal
assay, either the venereal disease research laboratory (VDRL) test or, more commonly,
the RPR test that detect anticardiolipin antibodies (Fig. 1). Since these antibodies are
not specific for syphilis, reactive nontreponemal assay results must be confirmed with
an assay that detects antibodies produced against T. pallidum. [3]

[1] https://www.medlabmag.com/article/1236 [2] https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2095002/ [3] https://journals.asm.org/doi/full/10.1128/JCM.06347-11

Treponemal Detection Tests 10

Screening Algorithm
 (a) In the traditional screening
algorithm, serum samples are first
tested by a nontreponemal test, such
as the rapid plasma reagin (RPR).
 Patients testing nonreactive by RPR
are considered negative for syphilis.
Samples testing reactive by RPR are
reflexed to a treponemal test, such as
fluorescent treponemal antibody
absorption (FTA) for confirmation.
 Patients testingnonreactive by FTA
are considered negative for syphilis.
Patients testing reactive by RPR and
FTA are considered positive for
untreated or recently treated syphilis.

Binnicker, M. J. (2012). Which algorithm should be used to screen for syphilis? Current Opinion in Infectious Diseases, 25(1), 79–85. https://sci-hub.se/10.1097/QCO.0b013e32834e9a3c
Treponemal Detection Tests 11

Screening Algorithm
 (b) In the reverse screening algorithm,
samples are initially tested by an
automatedtreponemal test, such as enzyme
immunoassay (EIA).
 Patients testing nonreactive by the screening
EIA are considered negative for syphilis.
 Samples testing reactive by EIA are reflexed
to a semi-quantitative, nontreponemal test,
such as RPR. If the RPR is reactive, the
patient is considered positive for untreated or
recently treated syphilis.
 If the RPR is nonreactive, the sample is
reflexed to a second treponemal test, such as
Treponema pallidum particle agglutination
(TP-PA).
 A nonreactive TP-PA is typically interpreted
as negative for syphilis, whereas a reactive
TP-PA is suggestive of past, successfully
treated syphilis; late/ latent syphilis; or early
syphilis.

Binnicker, M. J. (2012). Which algorithm should be used to screen for syphilis? Current Opinion in Infectious Diseases, 25(1), 79–85. https://sci-hub.se/10.1097/QCO.0b013e32834e9a3c
Treponemal Detection Tests 12

Interpretation

https://journals.asm.org/doi/epub/10.1128/CMR.00228-20
CLIA vs ECLIA 13

Interpretation

https://journals.asm.org/doi/epub/10.1128/CMR.00228-20
Terima
Kasih

14
Treponemal Detection Tests - RPR 15

RPR Test - Overview

 Rapid plasma reagin (RPR) is macroscopic, non treponemal, flocculation card test used to
screen for syphilis caused by Treponema pallidum.
 RPR is simple test can be done within few minutes.
 Autoantibodies are produced in 2-3 weeks of treponemal infection due to tissue damage.
These auto antibodies are often referred to as cardiolipin antibodies because they can be
detected in serological test using cardiolipin antigen.
 This test doesn’t look for antibodies against actual bacterium but rather for antibodies
against substances released by cells they are damaged by Treponema pallidum.
 The anti-lipodial antibodies are antibodies that are not produce only in syphilis infection
but also in other non treponemal disease of an acute and chronic nature in which tissues
are damaged.
 RPR measures IgM and IgG antibodies to lipodial materials released from damaged host
cells as well as lipoprotein like material and possibly cardiolipin released from treponems.

https://microbiologyinfo.com/rapid-plasma-reagin-rpr-test-for-the-diagnosis-of-syphilis/
Treponemal Detection Tests 16

RPR Test - Overview

 Antigen used in RPR test contain cardiolipin lecithin, cholesterol, 10% choline
chloride, EDTA, charcoal in buffer.
 This test cannot be performed on CSF .Serum or plasma can be used for testing,
serum not heated.
 This test tends to give negative results during late syphilis.

https://microbiologyinfo.com/rapid-plasma-reagin-rpr-test-for-the-diagnosis-of-syphilis/ https://www.cdc.gov/nchs/data/nhanes/nhanes_01_02/l36_b_met_syphillis_rapid_plasma_reagin.pdf
Treponemal Detection Tests 17

RPR Test - Principles

 RPR is 18 mm circle card test is a macroscopic flocculation test for syphilis.

 The antigen is prepared from modified VDRL (Venereal Disease Research
Laboratory), antigen suspension containing
 choline chloride and EDTA (ethylenediamine tetraacetic acid) to enhance stability of
suspension,
 finely divided charcoal particles as visualizing agents.
 In this test antigen is mixed with unheated serum on plastic –coated card.
 This test measures IgM & IgG antibodies to lipodial material released from
damaged host cells as well as possibly cardiolipin released from treponems.
 If antibodies are present, they combines with lipid particles of the antigen, causing
them to agglutinate .The charcoal particles co-agglutinate with antibodies and
shows black clumps on white cards. If antibodies are not present, the test mixture is
uniformly gray.
https://microbiologyinfo.com/rapid-plasma-reagin-rpr-test-for-the-diagnosis-of-syphilis/ https://www.cdc.gov/nchs/data/nhanes/nhanes_01_02/l36_b_met_syphillis_rapid_plasma_reagin.pdf
Treponemal Detection Tests 18

RPR Test - Qualitative

 Place 50µl of serum or plasma on 18mm circle of RPR test using a disposable
dispenstirs or a safety pipetting device.
 Spread serum or plasma to fill the entire circle. Don’t spread the specimen
beyond the confines of the circle.
 Gently shake the antigen dispensing bottles to re-suspend the particle.
 Dispense several drop[s of antigen (17µl of ag) suspension to each circle
containing serum or plasma.
 Mix the suspension well in one direction.
 Rotate card for 4-8 mins and observed for flocculation.

https://microbiologyinfo.com/rapid-plasma-reagin-rpr-test-for-the-diagnosis-of-syphilis/ https://www.cdc.gov/nchs/data/nhanes/nhanes_01_02/l36_b_met_syphillis_rapid_plasma_reagin.pdf
Treponemal Detection Tests 19

RPR Test - Quatitative

 Dilute the endpoint titre all serum specimen with rough non-reactive results in qualitative test. Test
each specimen undiluted (1:1) and in 1:2, 1:4, 1:8, 1:16 dilution.
 Place 50µl of 0.9% saline in circles. Don’t spread saline.
 Using safety pipette device, place 50µl of serum in circle labeled 1 and 50µl of serum in circle 2. Mix
the saline and serum in circles.
 Transfer 50µl from circle 2 (1:2) to circle3, & mix
 Transfer 50µl from circle 3 (1:4) to circle 4 &Mix
 Same way transfer 50µl from circle (1:8) to circle (1:16), mix and discard the last 50µl.
 Spread the serum dilution using clean dispenstirs to fill entire circle.
 Gently shake the dispensing bottles to re-suspend the antigen particles.
 Add (17µl of ag) antigen suspension in each circle.
 Place the card in rotator for 8 min at 100v 2rpm under humidifying cover.
 Remove card from rotator and tilt the card by hand (three or four to and fro motions) to aid in
differentiating non-reactive from minimally reactive results.
https://microbiologyinfo.com/rapid-plasma-reagin-rpr-test-for-the-diagnosis-of-syphilis/ https://www.cdc.gov/nchs/data/nhanes/nhanes_01_02/l36_b_met_syphillis_rapid_plasma_reagin.pdf
Treponemal Detection Tests 20

RPR Test - Quatitative

Image from: https://microbiologyinfo.com/rapid-plasma-reagin-rpr-test-for-the-diagnosis-of-syphilis/

https://microbiologyinfo.com/rapid-plasma-reagin-rpr-test-for-the-diagnosis-of-syphilis/
Treponemal Detection Tests 21

RPR Test - Interpretation

 Positive Result (Reactive):

 Clumping (Characteristic clumping ranging from marked and intense (reactive)
to Reactive (R) slight but definite (minimally to moderately) reactive).

 Negative Result (Non-Reactive):

 No Clumping or slight roughness.

 Note: A negative RPR Test result does not mean the patient is free of syphilis,
particularly if the exposure was recent. It can take up to several weeks for
antibodies to reach detection levels in the blood. As a result, repeat RPR testing
may be required at a later date.

https://microbiologyinfo.com/rapid-plasma-reagin-rpr-test-for-the-diagnosis-of-syphilis/
Urine Dipstick parameters 22

pH
The pH test relies on a combination of three indicators: methyl red,
bromthymol blue and phenolphthalein. A pH range of 5–9 is eflected in a
color gradation from orange to yellow-green and finally blue.

Normal: Day Time (4.8–7.4) Morning(5–6)

High pH Interferences Low pH Interferences
Run over effect Run over effect
Neglected Sample Metabolic Acidosis

Compendium of urinalysis, Urine test strips and microscop. Roche, 2010. https://www.sciencedirect.com/science/article/pii/S2352551718301082