Laboratory tests in Genitourinary Tuberculosis

Screening test Tuberculin test Evaluate the presence of existing cellular response to MTBC Tuberculin is sterile suspension of protein extracted from cultures of M tuberculosis Given intradermally into volar aspect of forearm. After 48-72 hrs delayed hypersensitivity will cause induration at site of injection in prior immune patients. A positive skin test supports diagnosis of TB, but a negative skin test does not necessarily exclude extra-pulmonary manifestation. This is especially true in cases of GUTB

Interferon-Gamma Release Assays IGRAs are blood tests that measure the level of IFN-γ (a surrogate of cellular immune reactivity) produced in response to MTBC-specific antigens Two IGRA available QuantiFERON-TB Gold In-Tube test (QFT-GIT)- TB Platinex. T-SPOT. TB test.

Interferon-Gamma Release Assays QFT-GIT T-SPOT.TB assay Whole blood is collected in three specialized test tubes, one containing the MTBC antigens ESAT-6, CFP-10, and TB7.7, and two controls (negative and positive). The blood is incubated directly in the collection tubes for 16 to 24 hours. Plasma is then separated and IFN-γ is measured using enzyme-linked immunosorbent assay (ELISA). In the, peripheral blood mononuclear cells are separated from whole blood and then incubated with ESAT-6 and CFP-10 in wells coated with antibodies that capture IFN-γ. Enzyme-linked immunospot (ELISPOT) assay is used to detect an increase in the number of cells (appearing as spots in each test well) that secrete IFN-γ in relation to a negative control. Spots are manually counted

Detection of TB Direct approach- Detection of Mycobacteria or its products Indirect approach- Measurement of humoral and cellular responses of host against tuberculosis.

Direct Approach Microscopy Simplest and most rapid method of detecting acid fast bacilli in clinical specimen by Ziehl Neelson staining. The limitation is that it requires atleast 5 x 1000 bacilli per ml

Urine for AFB microscopy: Ideally 5 or atleast 3 consecutive morning samples are to be examined. Classically ‘ Sterile pyuria in acidic urine

Microscopy Result can be influenced by type of specimen, thickness of smear, extent of discolouration, type of counter stain used and experience of examiner. Concentration of urine sample by cytocentrifugation enhances the sensitivity.

ZN Staining Make a thin smear of the material for study and heat fix at 65-75 C. Pour carbol fuschin over smear and heat gently and allow it to stand for 5 minutes. Rinse smears with water until no color appears in the effluent. Pour 20% sulphuric acid, wait for one minute and keep on repeating this step until the slide appears light pink in color (15-20 sec). Wash well with clean water. Cover the smear with methylene blue or malachite green stain for 1–2 minutes. Wash off the stain with clean water. Wait for smear to air-dry. Examine the smear microscopically, using the 100x oil immersion objective.

ZN staining reporting

Advantage of microscopy Simple, inexpensive Easy to perform, read and detect bacilli Results reported within hours Epidemiological indicator for NTCP

2.Urine Culture Gold standard test for Diagnosis Best sample is first void urine The different mediums used are 1) Egg based:Lowenstein Jensen's medium(4-6 weeks) 2) Agar based: Middle brook 7H -10 or 11 agar(3-5 weeks) 3) Liquid based detection system- Kirchners or Middlebrook 7H9 broth The specimens are incubated at 37degrees under 5% CO2 Current guidelines recommend culturing on atleast on 1 solid media and concurrenly on 1 liquid media to maximize yield

Advantages of Culture Bacterial growth can be identified visually or by automated detection of its metabolism. Provides a definitive diagnosis of TB. Significantly increases the number of cases identified (30– 50%) when compared with microscopy.

Disadvantages of culture More complex and expensive. Takes longer time. Needs appropriate lab infrastructure & bio-safety precautions.

Newer Methods Microcolony detection on solid media Septicheck AFB media MODS(Microscopic observation of in broth culture) BACTEC 460 radiometric system, BACTEC MGIT 960 system MB/BacT system ESP II culture system

Microcolony detection Sample cultured with Middlebrook 7H11 agar and examined microscopically. In less than7 days microcolonies appears. Less expensive and requires half the time than conventional culture.

Septi-Chek: This is a biphasic medium system consisting of an enriched selective 7H9 broth and a paddle with nonselective Middlebrook 7H11 agar on one side and with two sections on other side- one with NAP(para Nitro a-Acetyl amino b hydroxypropiophenone) and egg containing agar, second with chocolate agar for detection of contamination This system has also been found to be quite useful for rapid detection of growth of mycobacteria.

Septi-chek This method requires 3 weeks This method has the potential to expedite processing, obviate CO2 incubation requirement and facilitate early detection of positive cultures

BACTEC System It is based on generation of radioactive carbon dioxide(quantitative) from substrate palmitic acid. The system gives results of culture and Antimycobacterial Drug Susceptibility Test (AST) within 10-14 days. Inclusion of NAP (beta nitro alpha acetylamine beta hydroxy propiophenone) helps in distinguishing M.tuberculosis (inhibited) from other mycobacteria.

BACTEC More recently a non-radiometric BACTEC MGIT 960 TB system has been introduced, which is better automated with no issues of radioactive disposal.

Advantages of BACTEC Test Early results Superior than LJ medium for isolation and identification of mycobacterium Even in case of contamination the medium can be decontaminated to recover mycobacteria Accurate drug suseptibility testing

MGIT (Mycobacteria growth indicator tube): Automated system for growth and detection of mycobacteria with a capacity to incubate and continuously monitor 960 MGIT every 60 min for increase in fluorescence. This system helps in early detection (7- 12 days) of mycobacterial growth and has been reported to be useful for drug susceptibility testing

MGIT Test is Rapid, accurate and cost effecive method for high volume laboratory.

MB/BacT: Non radiometric continuous monitoring system with computerized database management. The system is based on colorimetric detection of CO2. It takes average 13.7 days for detection. This has been reported to be useful for drug susceptibility testing of M.tuberculosis

ESP II culture system Non radiometric automated continuous monitoring system based on pressure change within the headspace above the broth culture medium in a sealed bottle i.e. either gas production or consumption due to microbial growth.

Rapid diagnostic tests Analysis of lipid profile DNA probes Ribosomal r RNA based probe Gene amplification method for direct detection of M Tuberculosis sequences from clinical specimens

i.Analysis of lipid profiles: Mycobacteria have characteristic lipid profiles. These lipid profiles can be analysed by Gas chromatography, mass spectrum and HPLC or HP-TLC13 and quick identification of mycobacterial isolates can be done.

(ii) DNA probes: Based on information about specific gene sequences well defined oligonucleotide probes for identification of various clinically relevant mycobacteria have been developed and are readily available. These include probes for identification of M. tuberculosis, M. avium and several other mycobacteria. When used along with BACTEC, Septi-Chek, MGIT it helps in rapidly confirming the diagnosis within 1 to 2 days

(iii) Ribosomal rRNA based probes: Ribosomal RNA gene region has been extensively explored for designing systems for ribosomal DNA fingerprinting and for development of probes/as well as gene amplification assays. These probes target rRNA, ribosomal DNA, spacer and flanking sequences. These probes were earlier radiolabeled but have now been developed into chemiluminescent techniques. rRNA targeting probes are 10-100 fold more sensitive than DNA targeting.

Classified as those based on 1.polymerase chain reaction (PCR) (iv) Gene amplification methods for direct detection of M.tuberculosis sequences from clinical specimens: Classified as those based on 1.polymerase chain reaction (PCR) 2.isothermal amplification reactions. Highly sensitive and under optimum conditions may detect 1-10 organisms. Useful role in early confirmation of diagnosis in paucibacillary extra-pulmonary forms of tuberculosis.

DNA amplifications by PCR Invented by Kary Mullis in 1987. It detects mycobacterial DNA in clinical specimens as a replacement for culture and shortens diagnostic time to 24 hours. The technique cdmprises of specific replication ofthe target DNA (DNA extracted from a clinical specimen) until there is sufficient to detect by standard hybridization techniques. This replication is achieved by using synthetic oligonucleotide primers which are complementary to each strand of the target double-stranded DNA sequences. This process leads to a million-fold amplification of target DnA through multiple cycles of denaturation, annealing and polymerization resulting in an exponential increase in the number of copies of the target.

PCR It speeds the detection of MTBC providing results within 1-2 days. Also detects cases with low bacillary loads where culture fails. Sensitivity 87%-96% Cannot be used to monitor the response to treatment

Real time PCR Hybridisation of amplified nucleic acids with fluorescent-labelled probes spanning DNA regions of interest and monitored inside thermal cyclers. The fluorescent signal increases in direct proportion to the amount of amplified product in the reaction tube.

PCR Amplifies a single copy or a few copies of a piece of DNA, generating thousands to millions of copies of a particular DNA sequence. Denaturation (DNA melting): Two strands of the DNA double helix are physically separated at a high temperature.

PCR Annealing step: Temperature is lowered and the two DNA strands become templates for DNA polymerase to selectively amplify the target DNA. Extension/elongation step Final elongation Final hold

PCR The specimens is centrifuged in a 50 ml sterile centrifuge tubes at 5,000 rpm for 20 min. Supernatant was discarded gently and the sediment was taken for PCR as well as for AFB smear preparation. 200 microlitre of the sediment was taken for PCR. In the remaining sediment equal volume of decontamination solution was added and was incubated at room temperature for 20 min for TB culture

PCR DNA was extracted with AuPrep DNA extraction kit by silica column based method. PCR was performed for the amplification of MPB64 gene in Mycobacterium tuberculosis complex using primers Amplification was done on thermal cycler with initial denaturation at 95 0 C for 6 min, denaturation at 95 0 C for 30 s, annealing at 60 0C for 30 s and primer extension at 72 0C for 30 s with 35 rounds of repetitive cycles for denaturation, annealing, extension with final extension at 72 0C for 7 min.

Problems of DNA amplification by PCR LACK OF SPECIFICITY Dead and living organisms cannot be differentiated. False positive results due to contaminated DNA in the specimen which can also be amplified. Its greatest value is in low prevalence populations to detect latent infections.

GeneXpert MTB/RIF Automated, cartridge-based nucleic acid amplification test (NAAT) and detects target sequence in simple or complex samples using Real time PCR. Performed directly on sputum, processed sputum sediment and selected extrapulmonary specimens from adults and children. Capacity to have 1, 2, 4, 16, 48 or 80 independently functioning modules.

Xpert MTB-Advantages Simultaneously detects M. tuberculosis and rifampicin resistance in less than 2 hours. The sensitivity for detecting TB is similar to that of to liquid culture (sensitivity 88%); the specificity is also high (99%). The superior performance of Xpert MTB/RIF in detecting TB over that of microscopy makes it a particularly useful tool for casefinding among people living with HIV

Xpert MTB-Advantages As a tool for detecting rifampicin resistance, Xpert MTB/RIF has a sensitivity of 95% and specificity of 98% when compared with phenotypic reference standards. Biosafety precautions and the training required are minimal. For smear-negative culture-positive TB, the pooled sensitivity of Xpert MTB/RIF has been found to be 68%

Disadvantages A stable uninterruptable electrical supply is needed. The ambient operating temperature of the instrument cannot exceed 30 °C, cartridges must be stored at less than 28 °C.

Disadvantages The modules require annual calibration. Does not eliminate the need for conventional microscopy, culture and DST. In patients who are not at risk for drug resistance but tested positive for Rif resistance, a second Xpert MTB/RIF test should be performed to control for preanalytical and postanalytical errors, and to improve the clinician’s confidence in the diagnosis