LABORATORY Flashcards
refer to bacteriological diagnosis to confirm TB; requires collection of the necessary specimens for testing, performing the test, and making the
diagnosis based on the results.
Primary diagnostic tools
Once a presumptive TB case is identified by symptom-based screening or by chest Xray, diagnosis through [?] must be conducted.
bacteriologic confirmation
shall be the primary diagnostic test for PTB and EPTB in adults and children.
Xpert MTB/RIF
who are at high risk for Multidrug-resisant TB (MDRTB) shall be referred for Xpert MTB/ RIF testing.
All presumptive TB patients
shall be the alternative diagnostic test if Xpert is not accessible. Unavailability of Xpert MTB/RIF test shall not be a deterrent to diagnose TB disease bacteriologically.
Smear microscopy or loop mediated TB LAMP
may be utilized to process large sample loads especially in ACF activities, but not for children, PLHIV and MDR-TB risk groups.
TB LAMP
[?], patients shall be evaluated by the health facility physician who shall decide on clinical diagnosis
based on best clinical judgment.
If bacteriologic testing is negative or not available/accessible
[?] practices and procedures, containment equipment and facilities are required for non-aerosol-producing manipulations of clinical specimens such as preparation of acid-fast smears.
Biosafety Level 2
must be conducted in a Class I or II biological safety cabinet.
All aerosol-generating activities
[?] practices, containment equipment and facilities are required for laboratory activities in the propagation and manipulation of cultures of M. tuberculosis and M. bovis.
Biosafety Level 3
for screening are rapid, sensitive molecular tests for detecting TB.
Molecular WHO-recommended rapid diagnostics (mWRD)
Currently, mWRD available in the country and will be utilized by the National Tuberculosis Control Program( NTP) are:
- Xpert MTB/RIF (Cepheid, USA)
- TB LAMP (Eiken Chemical, Japan)
- Line Probe Assay (LPA)
B. Conventional tests
- Direct Sputum Smear Microscopy (DSSM)
- Cultural method
- Biochemical tests
an automated molecular a s s a y b a s e d o n t h e
extraction and amplification of genetic material in clinical
specimens.
Xpert MTB/RIF (Cepheid, USA)
used for the rapid and direct detection of MTBC; and simultaneously detects genes that encode rifampin
resistance.
Xpert MTB/RIF (Cepheid, USA)
Specimens for Xpert MTB/RIF test and corresponding volume
Sputum
Respiratory specimen other than sputum
Non-respiratory
Spot (at time of consultation) sputum collected by expectoration
Sputum
Other [?] and [?] can only be submitted to specifically designated laboratories equipped with certified biosafety cabinets such as in TB culture laboratories.
fluid aspirates and biopsy specimens
[?] are currently not accepted specimens for Xpert MTB/RIF testing.
Blood, urine and stools
a manual molecular assay to detect MTBC based on LAMP (loop-mediated isothermal amplification) techniques, a unique temperature-independent technique for amplifying DNA. It requires less than 1 hour to perform and can be read with the naked eye under ultraviolet light.
TB LAMP (Eiken Chemical, Japan)
can replace smear microscopy, especially in remote areas.
TB LAMP (Eiken Chemical, Japan)
it cannot detect rifampicin resistance and there is limited evidence of performance in comparison to Xpert MTB/RIF in children and people living with HIV (PLHIV) who have more smear negative pulmonary TB.
TB LAMP (Eiken Chemical, Japan)
It is family of DNA stripbased tests that determine the drug resistance profile of a MTBC through the pattern
of binding of amplicons ( D N A a m p l i f i c a t i o n
p r o d u c t s ) t o p r o b e s targeting the most common r e s i s t a n c e a s s o c i a t e d mutations to first- and second-line agents.
Line Probe Assay (LPA)
It is used for rapid detection o f d r u g r e s i s t a n c e t o rifampicin and isoniazid.
Line Probe Assay (LPA)
• It is recommended for direct testing of smear positive specimens (direct testing) or a cultured isolate of MTBC (indirect testing).
Line Probe Assay (LPA)
refers to the staining of AFB in direct smears of unconcentrated sputum specimen
Direct Sputum Smear Microscopy (DSSM)
• It can be performed either by brightfield microscopy or fluorescence microscopy.
Direct Sputum Smear Microscopy (DSSM)
• It serves as a basis for the diagnosis of TB cases. This is also used:
a. to monitor progress of patients with TB while they are on anti-TB treatment; and,
b. confirm cure at the end of treatment in drug sensitive TB cases.
Direct Sputum Smear Microscopy (DSSM)
Two (2) expectorated sputum specimens (3-5 ml) collected by expectoration (within 3 days, at most)
- First specimen (spot) at the time of consultation and second specimen after at least hour (spotspot one hour apart), or
- Spot early-morning specimens
- Follow-up within three days if patient fails to submit a second specimen unless the first specimen already tests positive for acid-fast bacillus (AFB) in which case the second specimen will not be necessary.
Optimum number of sputum for the test and corresponding volume required for DSSM
• For DSSM, examine the specimen to see that it is not just saliva. Mucus from the nose and throat, and saliva from the mouth are not good specimens.
Quality of sputum for DSSM
• Sputum is purulent, mucoid, or may be blood stained.
Quality of sputum for DSSM
• Microscopically, it will show greater than 25 WBC/LPO or 5 WBC/OIO, and presence of alveolar macrophages (dust cells).
Quality of sputum for DSSM
• For diagnosis of EPTB, facilities with the necessary capability can collect body fluid samples or tissue
biopsy sample from the suspicious site. Refer if necessary.
Extrapulmonary specimens
• It has been an adopted a policy that all EPTB cases should undergo a DSSM if feasible before
treatment so as not to miss out the potentially more contagious pulmonary forms of the disease. This
was also borne out of the realization that majority of extrapulmonary TB cases emanated from the a
pulmonary focus as well.
Extrapulmonary specimens
i. Label the slide.
ii. Make a smear with a loop or applicator stick.
iii. Spread the specimen by repeated coil type over an area of 3 cm long by 2 cm wide .
iv. Air-dry.
v. Heat-fix the smear.
Smear preparation
Two (2) types of staining procedures:
i. Carbol fuchsin-based staining
- Ziehl-Neelsen stain (hot method) - MOST COMMON
- Kinyoun stain (cold method)
ii. Fluorochrome staining
Staining and microscopic examination
A. Mycobacterium tuberculosis in clumps stained with [?] (1000x). Note the leukocytes in the background
B. Mycobacterium tuberculosis in single arrangement stained with [?] (1000x). The beaded appearance (due to non-acid-fast metachromatic Much’s granules) is notable.
C. Mycobacterium tuberculosis with [?] (400x)
A. Ziehl Neelsen acid fast stain
B. Kinyoun acid-fast stain
C. fluorochrome stain
Carbol fuchsin (hot stain)
3% acid-alcohol
0.3% Methylene blue
Ziehl-Neelsen Method
Carbol fuchsin (cold stain)
3% acid-alcohol
Malachite green
Kinyoun Method
Brightfield microscope,
1000x magnification
Ziehl-Neelsen Method
Kinyoun Method
Mycobacteria appear as red bacilli on blue background. Typical morphological features: slender, curved, beaded rods in various arrangements, often in clumps. Nonmycobacteria appear blue.
Ziehl-Neelsen Method
Mycobacteria appear as red bacilli on green background. Typical morphological features: slender, curved, beaded rods in various arrangements, often in clumps. Nonmycobacteria appear green.
Kinyoun Method
Read along two (2) imaginary horizontal lines from one end to the other end of the smear or three (3) vertical lines which correspond to 300 oil immersion fields to report as “0”.
Read at least one (1) imaginary horizontal line if the reading is positive.
Ziehl-Neelsen Method
Kinyoun Method
Auramine O with or without Rhodamine B
- 5% acid-alcohol
- 5% Postasium permanganate
Fluorochrome Staining
Fluorescence
microscope,
250-450x magnification
Fluorochrome Staining
Mycobacteria appear as slender rods that fluoresce bright yellow (Auramine O) or yellowred (Auramine ORhodamine B) against a dark background.
Non-acid-fast organisms will not fluoresce or may appear a pale yellow,
Fluorochrome Staining
Read one length of the smear (about 30 or 40
fields).
Fluorochrome Staining
Interpretation of results
i. Interpret the results of the two specimens. Write the reading (IUATLD/WHO Scale) and final laboratory diagnosis.
Direct Sputum Smear Microscopy (DSSM)
ii. Final laboratory diagnoses are reported as follows:
- [?] = at least one sputum smear is positive for AFB (+n, 1+, 2+, 3+)
- [?] = both sputum smears are negative for AFB.
Positive
Negative
Advantages of AFB Smear Microscopy:
- Simple convenient test.
- Requires minimal infrastructure and equipment.
- Highly accurate, inexpensive and fast.
- Accessible to the majority of patients.
- Prioritizes infectious cases
Limitations of AFB Smear Microscopy:
• Does not distinguish between viable and dead organisms.
• Follow-up specimens from patients on treatment may be smear positive yet culture negative.
• Limited sensitivity.
• High bacterial load >3000–5000 AFB /mL is required for detection.
• Limited specificity
– All mycobacteria are acid fast.
– Does not provide species identification.
• Cannot perform Drug Susceptibility Testing (DST).
Specimens are treated with agents to kill or reduce contaminating bacteria that can rapidly outgrow mycobacteria, and to liquefy mucus so that mycobacteria are released from mucin and/or cells. After digestion and decontamination, mycobacteria are concentrated, usually by high speed centrifugation (RCF of 3,000 g for 15 minutes is optimal for the recovery of mycobacteria) to enhance their detection by culture, and also for acid-fast staining.
Digestion and Decontamination of Specimens
Specimens requiring digestion and decontamination: Mucoidal specimens and/or specimens with normal flora: sputum, gastric lavages, urine, fece
Digestion and Decontamination of Specimens
Specimens NOT requiring digestion and decontamination: Tissues or body fluids collected aseptically, CSF, pleural fluid, joint fluid.
Digestion and Decontamination of Specimens
Traditional digestant and decontaminant. When the
reagent is diluted with an equal volume of
specimen, it provides a final concentration of 2%
NaOH, in which it is most effective as a mucolytic
agent. However, as a decontaminating agent, this
concentration is toxic to both contaminants and to
some mycobacteria. Time of exposure must be
carefully controlled to no more than 15 minutes.
4% NaOH
NALC is a mucolytic agent, to which an equal
volume of 4% NaOH is added. When the reagent is
diluted with an equal volume of specimen, it
provides effective digestion and decontamination
with a final concentration of 1% NaOH, which is less
toxic to mycobacteria. Limit exposure to NaOH to 15
minutes.
N-acetyl-L- cysteine (NALC) + 2% NaOH
Very effective mucolytic agent used with 2% NaOH.
Trade name of dithiothreitol is Sputolysin. Reagent
is more expensive than NALC. Limit exposure to
NaOH to 15 minutes.
Dithiothreitol + 2% NaOH
Preferred by laboratories that cannot carefully
control time of exposure to decontamination
solution. Zephiran should be neutralized by lecithin
and not inoculated to egg-based culture medium.
Trisodium phosphate, 13% + Benzalkonium chloride (ZephiranTM)
Can be used for decontamination of specimens
when exposure time cannot be completely
controlled. It is not as effective as TSP-Zephiran
mixture.
Trisodium phosphate, 13%
Most useful in processing specimens that contain
Pseudomonas aeruginosa as contaminant.
5% Oxalic acid
Effective as decontamination solution for sputum
specimens mailed from out-patient clinics. Tubercle
bacilli have survived 8-day transit without significant
loss.
Cetylpyridinium chloride, 1% + 2% NaCl
Contain whole eggs solidified by inspissation (i.e., heating to 85°C to 90°C for 30 to 45 minutes); glycerol is the preferred carbon source, thus enhances growth of mycobacteria; use of aniline
dye, (malachite green) helps to control contaminating bacteria which may produce proteolytic enzymes that will cause liquefaction of the medium.
EGG-BASED MEDIA
Growth occurs within 18-24 days
EGG-BASED MEDIA
Coagulated whole eggs,
salts, glycerol, potato flour,
malachite green (0.025 g/100
ml)
Lowenstein-Jensen (L-J)
Coagulated whole eggs, egg
yolks, whole milk, potato
flour, glycerol, malachite
green (0.052 g/100 ml)
Petragnani
Coagulated whole eggs,
potato flour, glycerol,
malachite green ( 0.02 g/100
ml).
American Thoracic Society (ATS) medium
L-J slant (see above) supplemented with penicillin (50 U/ml) and nalidixic acid (35 mg/ml), and RNA (5 mg/ 100 ml)
L-J Gruft
LJ slant (see above) with
cycloheximide (400 ug/ml), ,
lincomycin (2 ug/ml), and
nalidixic acid (35 ug/ml).
L-J Mycobactosel
Commonly used medium in many clinical diagnostic laboratories; good recovery of M. tuberculosis but poor recovery of many other species; M. genovense fails to grow
Lowenstein-Jensen (L-J)
C o n t a i n s t w i c e t h e concentration of malachite green than L-J medium; improves recovery from heavily contaminated specimens.
Petragnani
S e l e c t i v e m e d i u m containing antimicrobial agents used to suppress b a c t e r i a l a n d f u n g a l contamination, and use can i m p r o v e r e c o v e r y o f mycobacteria
L-J Gruft
Selective medium for
mycobacteria.
L-J Mycobactosel
Solidified by agar; Growth occurs within 10-12 days
AGAR-BASED MEDIA
Defined salts, vitamins, c o f a c t o r s , o l e i c a c i d , albumin, catalase, biotin, glycerol, malachite green (0.0025 g/100 ml),glucose
Middlebrook 7H10
Oleic acid and albumin, which protect Mycobacterium from toxic agents, helping for the growth of tubercle bacilli; biotin and catalase stimulate revival of damaged bacilli in clinical specimens; albumin binds toxic amounts of oleate and other compounds that might b e r e l e a s e d f r o m spontaneous hydrolysis of Tween 80 of Tween 80.
Middlebrook 7H10
Defined salts, vitamins, c o f a c t o r s , o l e i c a c i d , albumin, catalse, biotin, glycerol, malachite green ( 0 . 0 0 2 5 g / d l ) , c a s e i n hydrolysates (0.1%)
Middlebrook 7H11
Differs from Middlebrook 7H10 by addition of casein hydrolysate that improves the rate and amount of growth of mycobacteria resistant to isoniazid (INH).
Middlebrook 7H11
Middlebrook 7H10 with
cycloheximide (360 ug/ml),
lincomycin (2 ug/ml), and
nalidixic acid (20 ug/ml)
Middlebrook 7H10 Selective Agar
The addition of antimicrobial agents makes it selective and improves the recovery of m y c o b a c t e r i a f r o m specimens containing mixed flora
Middlebrook 7H10 Selective Agar
Middlebrook 7H11 with carbenicillin (50 ug/ml), amphotericin B (10 ug/ml), polymixin B (200 U/ml) and trimethoprim lactate 20 ug/ ml)
Selective Middlebrook 7H11
Mitchison’s medium
Selective medium
Selective Middlebrook 7H11
Mitchison’s medium
a. Middlebrook 7H9
b. Dubos Tween Albumin
c. BACTEC 12B medium
LIQUID MEDIA
For optimal recovery of [?], a combination of different culture media is required.
At least one solid medium and a liquid medium must be used. When growth is detected in a liquid medium, the material is subcultured to solid agar.
mycobacteria
Each Mycobacterium species has an optimal temperature for growth and a range of time for recovery in culture. The time to recovery varies
depending on the type of media used—the average time of recovery of mycobacteria on egg-based media is about [?], but ranges from as short as 3 to 5 days to as long as [?], depending on the species and the quantity of mycobacteria in the specimen.
21 days
60 days
Mycobacteria are [?] whose growth is stimulated by increased levels of CO2 by use of CO2 generator sachet, or other suitable system providing an aerobic atmosphere enriched with CO2. For reasons that are not well understood, mycobacteria do not grow well in candle extinction jars.
strict aerobes
From the skin or superficial lesions that are suspected to contain M. marinum or M. ulcerans, an additional set of solid media should be inoculated and incubated at [?]. In addition, a chocolate agar plate (or placement of an X-factor [hemin] disk on conventional media) and incubation at 25°C to 33°C is needed for recovery of M. haemophilum from these specimens.
25°C to 30°C
Preliminary identification of mycobacterial isolates depends on:
- Rate of growth
- Permissive incubation temperatures
- Colonial morphology
- Pigmentation
- Slow growth (12-25 days) at 37°C
- Small, friable, rough (dry, scaly, warty, or cauliflower-like)
- Nonpigmented (buff in color)
- Growth is described as “eugonic” because of its luxuriant nature in the presence of glycerol.
M. tuberculosis
Organisms growing on solid or liquid mycobacterial media are subjected to:
- Acid-fast staining, to confirm that the organisms are indeed mycobacteria.
- Biochemical Testing
Advantages of Mycobacterial Culture:
- More sensitive than smear microscopy, 10-100 AFB /mL is required to obtain positive result.
- Allows diagnostic confirmation of TB, if TB is suspected and sputum smears are negative.
- Allows for identification of mycobacterial species.
- Allows for drug susceptibility testing.
Limitations of Mycobacterial Culture:
- Cost.
- Technical complexity.
- May take weeks to get results.
- Requires ongoing quality assurance.
Therefore, more likely to be available in [?] only.
Avoid [?] appropriate TB treatment in suspicious cases while awaiting results.
major referral centers
delaying
Although the applications of molecular techniques are currently the standard for the identification of cultured mycobacteria, [?] remain useful and are discussed briefly here. This is not intended to provide a detailed presentation of the reagents, procedures, and interpretation of various laboratory techniques.
conventional biochemical test methods
Key biochemical characteristics of M. tuberculosis are as follows:
- Accumulation of niacin
- Reduction of nitrates to nitrites
- Ability to grow in the presence of Thiophene-2 carboxylic acid hydrazide (T2H)
- Lack of catalase (heat-stable) activity
- Principle:
All Mycobacterium species produce niacin ribonucleotide; however, virtually all strains of M. tuberculosis and M. simiae and occasional strains of M. africanum, M. bovis, M. marinum, and M. chelonae lack the enzymes to further convert niacin to nicotinamide adenine dinucleotide (NAD). Niacin accumulates in the culture medium, from which it can be extracted with sterile water or physiologic saline. The extract is placed in a small test tube to which a reagent-impregnated niacin filter strip is added.
Niacin Accumulation Test
Niacin Accumulation Test Results:
(+) - Development of [?] in the test
medium incubated with a reagent strip.
(-) - Liquid remains [?]
yellow color
milky white or clear
Principle:
Mycobacteria producing nitroreductase, most notably M. tuberculosis, are capable of catalyzing the reduction of nitrate to nitrite. The nitrite produced is detected by the addition of α-naphthylamine and sulfanilic acid, forming the red diazonium dye, p sulfobenzene-azo-αnaphthalamine. The nitrate reduction test is also a key test in the identification of
M. kansasii and M. szulgai.
Nitrate Reduction Test
Nitr ate Reduction Test Results:
(+) - Development of [?]
(-) - No red coloration
red-pink color
Principle:
Thiophene-2-carboxylic acid hydrazide has the property of inhibiting Mycobacterium bovis, but not other species of mycobacteria, a helpful feature differentiating M. bovis from M. tuberculosis.
Growth Inhibition by Thiophene-2-Carboxylic Acid Hydrazide (T2H)
Growth Inhibition by Thiophene-2-Carboxylic Acid
Hydrazide (T2H) Results:
(+) - [?] in T2H
(-) - No growth in T2H
Growth
Principle:
The heat-stable catalase test is based on the ability of the catalase enzyme to remain active after heating the culture at 68°C for 20 minutes. Catalase splits hydrogen peroxide into water and oxygen. The evolution of oxygen appears as effervescence (bubbles). The catalase is detected by using 30% hydrogen peroxide, Superoxol, (not 3% used in classic catalase test) in a strong detergent solution (10% Tween 80). The detergent helps disperse the hydrophobic tightly clumped mycobacteria from large aggregates to individual bacilli, maximizing the
detection of catalase. Most of the mycobacteria produce catalase; however, only some species are capable of producing a heat-stable catalase.Catalase activity is assessed semiquantitatively by measuring the height achieved by the column of bubbles produced when hydrogen peroxide is added to growing colonies in a tube culture.
Heat-stable Catalase Test
Heat-stable Catalase Test Results:
(+) -
(-) -
Effervescence (bubbles)
Lack of effervescence
Semiquantitative catalase test:
High catalase reaction
Low catalase reaction
- A column of effervescence > 45 mm
- A column of effervescence < 45 mm
High catalase reaction
Low catalase reaction
Principle:
The commonly nonpathogenic, slow-growing scotochromogens and nonphotochromogens possess a lipase that splits Tween 80 (trade name for a detergent polyoxyethylene sorbitan monooleate) into oleic acid and polyoxyethylated sorbitol, whereas pathogenic species do not. This modifies the optical characteristic of the test solution from straw yellow to pink.
Tween 80 Hydrolysis Test
Tween 80 Hydrolysis Test Results:
Positive result is recorded when the liquid, not the cells, turns from [?]. [?] usually turns positive within 24 hours. Read again at 3, 5 and 10–12 days. Record results and discard positives. Discard all tubes at 12 days.
light orange to pink or red
M. kansasii
Principle:
The enzyme arylsulfatase is present in most mycobacteria. The rate at which the arylsulfatase enzyme breaks down phenolphthalein disulfate into phenolphthalein (which forms a red color in the presence of sodium bicarbonate) and other salts helps to differentiate certain strains of mycobacteria.
Arylsulfatase Test
The 3-day test is particularly useful for identifying the potentially pathogenic rapid growers M. fortuitum and M. chelonae. Slowgrowing M. marinum and M. szulgai are positive in the 14-day test.
Arylsulfatase Test
Arylsulfatase Test Results:
(+) -
(-) -
Red color
No red color
- Principle:
Some mycobacteria are able to convert ferric
ammonium citrate to iron oxide. After growth of
the isolate appears on an egg-based medium
slant, rusty-brown colonies appear in a positive
reaction upon the addition of 20% aqueous
solution of ferric ammonium citrate. The test is
most useful is distinguishing M. chelonae which is
generally negative, from other rapid growers,
which are positive.
Iron Uptake Test
Iron Uptake Test
Results:
(+) - The color of a truly positive iron uptake test will be [?]
very dark rust
Principle:
Pyrazinamidase is an enzyme that deaminates pyrazinamide (PZA) to form pyrazinoic acid, which produces a red band in the culture medium after the addition of freshly prepared 1% ferrous ammonium sulfate. The deamination of pyrazinamide in PZA substrate medium within 4 days is a useful phenotypic characteristic by which M. marinum (positive) can be differentiated from M. kansasii (negative) and by which weakly niacinpositive strains of M. bovis (negative) can be distinguished from M. tuberculosis complex (positive).
Pyrazinamidase Test
Pyrazinamidase Test Results:
(+) - After 4 hours of the addition of [?], examine the tubes for a pink band in the reagent layer on the surface of the agar
1% ferrous ammonium sulfate
• CXRs are useful tools to aid diagnosis of TB when the TB disease cannot be confirmed with bacteriological diagnostic tools.
Chest X-rays
• However, it has low specificity and does not differentiate drug-susceptible TB (DS-TB) from drug resistant TB (DR-TB).
Chest X-rays
• TST is a basic screening tool for TB infection when a physician has doubts in making a clinical diagnosis of TB in children.
Tuberculin skin test (TST)
• It involves intradermal injection of (mycobacterial antigens) to trigger a delayed hypersensitivity reaction among those previously infected. A positive tuberculin test indicates that an individual has been infected in the past. It does not imply that active disease or immunity to disease is present.
Tuberculin skin test (TST)
tuberculin; is a glycerol extract of the tubercle bacillus.
Mycobacterial antigen
is a filtrate of glycerol broth culture concentrated by evaporation in water bath
OT (Old/Original Tuberculin)
is purified by precipitation of OT with trichloroacetic acid (TCA), and the one that is presently used in TST. WHO-recommended TST tests are either five tuberculin units (TU) of tuberculin-purified protein derivative (PPD-S) or 2 TU of tuberculin PPD RT23, which give similar reactions in children infected with MTB.
PPD (Purified Protein Derivative)
- It is performed by injecting intradermally 0.1 ml of tuberculin PPD into the inner surface of the forearm. The injection should be made with a tuberculin syringe, with the needle bevel facing upward.
Mantoux test
- When placed correctly, the injection should produce a pale elevation of the skin (a wheal) 6 to 10 mm in diameter.
Mantoux test
- The skin test reaction should be read between 48 and 72 hours after administration. Inspect the site for area of induration and palpate (It should be hard, dense and raised).
Mantoux test
- Measure the diameter (in mm) of the area of induration. Note: erythema (redness) should not be measured
Mantoux test
- An induration of > 5 mm in children with immunosuppressed conditions, such as HIV or severe malnutrition,
TST-positive
- An induration of >10 mm in other children regardless of BCG vaccination status.
TST-positive
- Previous BCG vaccination
- Infection with nontuberculosis mycobacteria
- Technical errors: Incorrect method of TST administration, incorrect interpretation of reaction,
- incorrect bottle of antigen used
False-Positive Reactions
Cutaneous anergy, i.e., the inability to react to skin tests because of a weakened immune
- system)
- Recent TB infection (within 8-10 weeks of exposure)
- Very old TB infection (many years)
- Very young age (less than 6 months old)
- Recent live-virus vaccination (e.g., measles and smallpox)
- Overwhelming TB disease
- Some viral illnesses (e.g., measles and chicken pox)
False-Negative Reactions
- Uses a small “button” (a round plastic head) that has four to six short metal tines coated with tuberculin antigen.
- The tines are pressed into the skin (usually on the inner side of the forearm), forcing the antigens into skin.
Tine test
- Similar to tine test but uses gun device is used to inject the tuberculin antigen from 6 spring-released needle points.
Heaf test
- The tuberculin is scratched on the skin.
Von Pirquet test
- A cloth soaked in tuberculin is applied to the surface of the skin.
Vollmer patch test
- Tuberculin is mixed with lanolin to make an ointment and then rubbed on the skin
Moro percutaneous test
diagnosis of leprosy is most commonly based on
clinical signs and symptoms, and slit skin smear examination
is a leprosy skin test to determine what type of leprosy the patient has. Now, molecular methods (e.g., PCR) are available for identification of M. leprae.
Lepromin test
- scrapings with a scalpel blade from skin (earlobes, elbows, and knees) or nasal mucosa; take from the most active lesion (raised and red, usually at the edge).
Slit skin smear (SSS)
not required for diagnosis but can support a diagnosis of leprosy and rule out other diseases
Skin biopsy
- Pinch the site tight.
- Incise.
- Scrape and collect material.
- Smear on slide.
- Air-dry and fix.
Slit skin smear method
singly, in parallel bundles, or in globular masses.
Typical acid-fast bacilli
- NOT diagnostic of exposure to or infection with M. leprae because it can be positive for any mycobacterial infections.
Lepromin test
- It is a prognostic test of an individual’s capability to develop cell-mediated immunity (a delayed-type hypersensitivity reaction) to M. leprae.
Lepromin test
- It involves intradermal injection of inactivated (heat killed) M. leprae. The injection site is examined after 1 2 days, and if there’s 3-4 weeks of injection for redness, swelling, or other skin changes.
Lepromin test
occurs within the first 2 days
Fernández reaction
occurs within 3-4 weeks. It can help to predict the evolution of the indeterminate lesion
Mitsuda reaction
If the Mitsuda test is negative, the patient will usually go on to [?], and if the Mitsuda test is positive, the patient will more likely develop [?]
lepromatous leprosy
tuberculoid leprosy
AFB (SSS): Few
TUBERCULOID LEPROSY
AFB (SSS): Numerous
LEPROMATOUS LEPROSY
LEPROMIN TEST: Strongly positive
TUBERCULOID LEPROSY
LEPROMIN TEST: Negative
LEPROMATOUS LEPROSY
MTB detected; rifampicin resistance not detected
T
MTB detected; rifampicin resistance detected
RR
MTB detected; rifampicin resistance indeterminate
TI
MTB not detected
N
Invalid/no result/error
I
