THE YIELD OF GENEXPERT IN PULMONARY TUBERCULOSIS&RIFAMPICIN RESISTANCE by supervised by Dr.Mohammed A.Ali Shahatha Professor Kasim M.Sultan

introduction

Tuberculosis (TB) is a leading public health problem worldwide causing ~9 million active disease cases and~2 million deaths annually. Delayed diagnosis and incomplete or improper treatment of TB patients leads to evolution of drug-resistant strains of Mycobacterium tuberculosis, including multidrug- resistant(MDR) and extensively drug-resistant TB (XDRTB). The agent being the tubercle bacilli, a susceptible person as a host and an environment which allows the bacilli to survive and transfer from one host to another. Drug-resistant strains of M. tuberculosis arise from spontaneous chromosomal mutations at predictable low frequency.

Drug-Resistant Tuberculosis Or by misusedrug such as monotherapy or the addition of single drugs to failing regimens, results in the e mergence of resistant mutants (acquired resistance). Transmission of such resistant strains to another person may result in infection and eventually disease (primary resistance) Drug-Resistant Tuberculosis The current standard for first-line drug-susceptibility testing is an automated liquid culture system, which requires 4 to 13 days for results. The World Health Organization (WHO) recommends that standard drug-susceptibility testing be performed at the same time that the Xpert MTB/RIF assay is performed to confirm rifampin resistance and the susceptibility of the M. tuberculosis isolate to other drugs.

Because First-line antituberculosis treatment often fails in patients with rifampin-monoresistant or multidrug-resistant tuberculosis & conventional susceptibility testing can require many weeks to complete. There is thus an urgent need to develop a rapid, simple, and accurate assay to assess drug resistance in M. tuberculosis Which is called Xpert MTB/RIF assay. This automated system employs real time PCR and molecular beacon probes to determine the presence of M. tuberculosis complex DNA as well as rpo B gene mutations conferring rifampin ( RIF) resistance rapidly and with high accuracy of both smear positive and smear negative samples.

Rifampicin resistance is particularly amenable to rapid molecular detection since >95% of all rifampicin resistant strains contain mutations localized within the 81 bp core region of the rpoBgene which encodes the active site of the enzyme .In addition, the rpoB core region is flanked by Mycobacterium tuberculosis- specific DNA sequences. Thus, it is possible to test for M. tuberculosis and for rifampicin resistance.

The GeneXpert unit that has been tested for the Xpert MTB/RIF cartridge has four modules, with a capacity to perform 15 to 20 tests in one working day. Results are available for each test in less than 2 hours and modules are independent so that each individual test can be started independently.

Considerations of GeneXpert

1.Primary considerations. 1.All persons living with HIV who have signs symptoms of TB. 2.Those seriously ill and suspected of having TB regardless of HIV status. 3.Those with unknown HIV status presenting with strong clinical evidence of HIV infection in HIV prevalent settings. 2.Secondary considerations In low-burden settings, pre-test screening strategies should be considered to optimise Xpert MTB/RIF efficiency and cost,such as CXR. Molecular tests, including Xpert MTB/RIF, are not suitable for patient monitoring as these tests also detect DNA from non-viable bacilli. Patients whose diagnosis of TB is confirmed by Xpert MTB/RIF and who have rifampicin susceptible TB disease should be monitored during treatment with sputum smear microscopy.

Advantages of Xpert MTB/RIF For these patients, sputum smear microscopy should be performed at completion of the intensive phase of treatment, five months into treatment and at the end of treatment as per WHO guidelines. Advantages of Xpert MTB/RIF 1.Deliver a highly accurate results in less than 2 hours 2. Simultaneous detection of both MTB and rifampicin resistance, a marker for as MDR strains,up to 95% of rifampicin resistance strains are INH resistance. 3. Unprecedented sensitivity for detecting MTB even in smear negative, culture positive specimens. 4. By using sealed disposable cartridges, this new MTB/RIF test apparently overcomes the problem of cross -contamination, in which the products from previous assays cause false-positive results that considered An Achilles heel of polymerase chain reaction tests

Disadvantages of Xpert MTB/RIF 1. A single MTB/RIF assay may be insufficient for ‘‘ruling-out’’ TB, although a second test for each patient increased sensitivity to 62% MTB/RIF test has intermediate sensitivity, better than smear microscopy but less than broth-culture, risking false-negative results . 2. limited shelf-life of the diagnostic cartridges about 6 months. 3. The need for specific operating temperature which is currently limited to a maximum of 30°C. 4. The need for specific store temperature Xpert MTB/RIF cartridges and the specimen reagent should be stored at 2-28˚C 5.humidity. 6. Requirement for stable electrical power and even short term interruption of power may cause indeterminate or incorrect results. 7. The need for annual servicing and calibration of each machine that costs amounts to 1,800 USD (calibration – 1,400 USD [4 modules].

materials&methods

This is primarily retrospective laboratory-based study, evaluation of 56 patients suspected of having TB from clinical point of view part of them found to be positive by direct examination, other part found to be positive by specimen culture. Gene Xpert MTB/RIF instrument , utilizes molecular beacon technology to detect DNA sequences amplified in a hemi- nested rt- PCR assay Five different nucleic acid hybridization probes are used in the same multiplex reaction . Each probe is complementary to a different target sequence within the rpo Bgene of rifampicin-susceptible M. tuberculosis and is labeled with a different colored fluorophore.

molecular beacon technology The assay utilizes five differently colored molecular beacons, each of which binds to a different target segment within the rpoB core region Each molecular beacon was designed to be so specific that it does not bind to its target if the target sequence differs from the rifampin-susceptible sequence by as little as a single from the rifampin-susceptible sequence by as little as a single nucleotide substitution. Since molecular beacons fluoresce only when they are bound to their targets, the absence of any one of the five colors in the assay indicates that thebacilli in the sample are rifampin resistant.

Results

In this retrospective study the patients was divided into 3 sub groups : The 1st group selected according to clinical features(fever.cough,weightloss,night sweat). 2 .The 2nd group subjected to sputum examination (sputum direct microscopical examination ,sputum culture). 3. The 3rdgoup subjected to bronchoaleviolar lavage(direct specimen microscopical examination ,specimen culture)

In this retrospective study tuberculosis seems to affect slightly predominantly male. Male to female ratio (1.3:1) Total Female male Gender 56 25 31

Genexpert Sputum examination Of (56) samples processed( 29) sample yielded M.Tuberculosis by sputum direct microscopical examination wheres only (20 )samples were positive by culture of sputum, The Xpert™ MTB/RIF assay detected (41) patients positive M. Tuberculosis, (36)patients were RIF susceptibility positive Genexpert Sputum examination Type of lab. Exam. RIF resistance M. Tuberculosis Sputum culture Direct microscopical examination No. of patients 36 41 20 29

All of smear- positive, culture-positve (19/19) 100% & ( 5/6 ) 83% of smear- negatine, culture –positive specimens were identified by the direct Xpert test as having TB DNA. The last case unidentified by Genexpert which is smear negative is considerer to be as a false negative according to result of direct smear microscopical examination which considered as a primary diagnostic test although the highly sensitivity of Genexpert in dignosing live tubercle bacilli ,additionally it diagnose the non-viable one with the same efficiency, such a feature considered an Achilles tendon of this test. This study revealed 2 cases ; smear positive, Genexpert positive to be as rifampicin resistant while the other 36 cases are sensitive to rifampicin. There are 4 false-positive results , both smear- and culture-negativethat were collected from the patients who were strongly clinically suspected of M. tuberculosis..,

Sensitivity & specificity were caliculated according to the following formulae: Sensitivity=no. of true positive/no. of true positive+no. of false negative.=no. of true positive/total no. of sick individual in population. Specificity=no. of true negative/no. of true negative+no. of false positive .=no. of true negative/total no. of well individual in population Sensitivity&specificity in comparision of of Genxpert smear positive ,culture positive sputum specimen were 87%,95% respectively. Sensitivity & specificity of Genexpert comparision of smear negative,culturepositve 85%,100% respectivel In general view,Compared to the culture results of sputum , sensitivity of Genexpert assay was( 96%) while specificity was (80%).

Genexpert + results 19 5 6 4 Type of sputum examination Direct smear + Culture examination+ 5 6 Direct smear- Culture examination 4 Culture examination

Regarding BAL results Direct microscopical examination revealed (5) positive results while pecimen culture reaveled( 4) positive cases. All of smear positive,culture positive(3/3) 100%, 1 case smear negative,culture negative found Genexpert positive ( false positive ) &1 case smear negative ,culture positive , found to be negative by Genexpert. Sensitivity&specificity ofGenxpert in comparision of smear positive ,culture positive BAL specimen were 100%,100% respectively Sensitivity&specificity of Genxpert in comparision of smear negative,culturepositve 75%,100% respectively. In general ,comparedto,the culture result of BAL the sensitivity result was (100%),while specificity was(75%).

1 Total results 3 _ 4 5 Genexpert + Type of BAL examination Direct smear + Culture examination+ _ 1 Direct smear- Culture examination- 4 5 Total

discussion

The results that are obtained from the assay indicate whether a patient is infected with M.Tubercle & whethere the tubercle bacilli is present in the sample, and whether the bacilli are rifampin resistant. Other studies performed by chang et al, who found the sensitivity& specificity 75%,93% respectively,another study by Lesley Scott ,who found , sensitivity & specificity for sputum positive,culture positive specimen about 95%,95% respectively, Sputum negative, culture positive specimen 80%,90% respectively. In another study done by Catharina C. Boehme & MTB/RIF test was 97.6%. The sensitivity was 99.8% for smear- and culture-positive cases and 90.2% for smear- negative, culture-positive cases, with no significant variation in overall sensitivity across sites .

Recommendations

Genexpert indicated in the following Xpert MTB/RIF should be used as the initial diagnostic test in individuals suspected of having MDR-TB or HIV-associated TB. (Strong recommendation)   2. Xpert MTB/RIF may be considered as a follow-on test to microscopy in settings where MDR-TB or HIV is of lesser concern, especially in further testing of smear-negative specimens.

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