Real-time polymerase chain reaction as an alternative method for diagnosis of multidrug-resistant tuberculosis: can it stand alone in this concern?

Background Delays in diagnosing multidrug-resistant tuberculosis (MDR-TB) are responsible for higher tuberculosis morbidity and mortality and its subsequent transmission. Molecular assays such as real-time PCR (RT-PCR) used to identify drug resistance in Mycobacterium tuberculosis are more rapid than standard drug susceptibility testing. Objectives The aim of this study was to evaluate the diagnostic performance of the Anyplex MTB/MDR RT-PCR assay in detecting MDR-TB strains. Patients and methods Sputum samples were collected from 29 patients with symptoms and radiological findings suggestive of active pulmonary tuberculosis, with at least one of three sputum smear samples showing acid-fast bacilli and/or sputum culture isolates positive for M. tuberculosis. The results obtained by RT-PCR were compared with those obtained by the Mycobacterium growth indicator tube SIRE method. Results M. tuberculosis was confirmed in 29 specimens. Only six cases determined as MDR-TB were obtained by Mycobacterium growth indicator tube SIRE. For detection of rifampicin-resistant and isoniazid-resistant strains, the RT-PCR assay yielded a sensitivity of 62.5 and 66.66% and specificity of 80 and 95%, respectively. The overall sensitivity of that assay was 64.2% and specificity was 88.88%. Conclusion RT-PCR is an easy and reliable assay for rapid detection of MDR-TB in clinical specimens. However, RT-PCR should be followed by a culture method to increase the overall sensitivity of that assay.


Introduction
In recent years, the emergence of drug-resistant strains, defined as resistance to at least isoniazid (INH) and rifampicin (RIF), has been an important problem and is threatening the control of tuberculosis (TB) [1]. Therefore, the rapid detection of drug resistance is essential to begin effective therapies and protect the community from TB.
Drug resistance in Mycobacterium tuberculosis is due to mutations in genes or promoters of genes activating the drug or encoding the drug targets, which are detectable in the majority of drug-resistant isolates [2]. Ninety-five percent of mutations associated with RIF resistance occur mainly in an 81-bp RIFresistance-determining region of the rpoB gene [3]. Mutations associated with INH resistance occur mainly in the katG gene (codon 315), the inhA gene and regulatory region, and the ahpC regulatory region [4].
Several molecular methods have been previously described for drug susceptibility testing (DST) of M. tuberculosis, including real-time PCR (RT-PCR), for simultaneous detection of M. tuberculosis and its drug susceptibility [5].
A key advantage of genotypic drug susceptibility assays over phenotypic assays is the shorter time required for the assay, with genotypic assays requiring just hours to complete in contrast to phenotypic tests that can take weeks. Because of the need to grow the organism, phenotypic methods require weeks of culture, during which the patient may be treated with the wrong antibiotics, resulting in poorer treatment outcome or the transmission of resistant strains [6].
This study aimed to evaluate the diagnostic efficacy of RT-PCR in the detection of RIF-resistant and INH-resistant M. tuberculosis strains. The drug susceptibility obtained with the Mycobacterium growth indicator tube (MGIT) SIRE served as the gold standard for comparison with that obtained with the Anyplex II MTB/multidrug-resistant (MDR) RT-PCR assay.
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Patients and methods M. tuberculosis isolates were obtained from 29 patients attending the outpatient clinic of the Department of Chest in 1 year, selected from those presenting with symptoms and radiological findings suggestive of active pulmonary TB, with at least one of three sputum smear samples showing acid-fast bacilli and/or sputum culture isolates positive for M. tuberculosis.

Specimen collection
Three early morning sputum specimens on 3 consecutive days were collected from each patient [7]. The specimens were transported to the TB lab. of the Microbiological Unit in the Department of Clinical Pathology, Faculty of Medicine, Assiut University, and were screened by direct Ziehl-Neelsen smear for acid-fast bacilli, and further processing of the specimens was performed.

Sputum processing
The specimens were decontaminated and liquefied by 2% N-acetyl-l-cysteine NaOH according to the manufacturer's instructions [8]. Finally, after the last centrifugation step, the processed samples were used for Ziehl-Neelsen smear preparation (concentration method), as inoculum for culture on MGIT and for DNA extraction. Anyplex II MTB/MDR detection assay Sputum specimens were examined by the Anyplex II MTB/MDR detection assay for the simultaneous detection of M. tuberculosis and its resistance to firstline anti-TB drugs (INH and RIF) using the CFX96 RT-PCR System (Bio-Rad). It covers six mutations causing INH resistance in the katG gene and inh A promoter region, and 15 mutations causing RIF resistance in the rpoB gene.

DNA extraction
A part of the processed samples was subjected to DNA extraction using the Epicenter kit according to the manufacturer's instructions with the DNA extraction solution included in the Anyplex II MTB/MDR detection kit from Seegene System (Seegene, Anyplex RT-PCR, Taewon) [10].

Real-time polymerase chain reaction
Each sample was tested in two separate reactions (MTB/NTB and MTB/MDR) according to the manufacturer? s instructions. One reaction was used for the amplification and detection of MTB in sputum, and the second was used for detection of multidrug-resistant tuberculosis (MDR-TB) of the MTB-positive sample.
The wild-type control was designed to exhibit the same result pattern with a drug-susceptible M. tuberculosis sample, and the drug-resistant result of unknown samples was analyzed on the basis of the result of the wild-type control.

Statistical analysis
The results of the antimicrobial susceptibility test and RT-PCR assay were analyzed using the computer software SPSS (statistical package for the social science, version 10.999; SPSS Inc., Chicago, Illinois, USA).

Results
The study was carried out on sputum samples from 29 patients (17 male and 12 female) with an age range of 19-75 years.
RT-PCR carries the advantages of simultaneous detection of M. tuberculosis (Table 1) and its susceptibility, with availability of the results within 1 working day in comparison with 12-14 days when MGIT SIRE is used (Table 2).
MGIT SIRE revealed that 20% (6 out of 29 cases) of the isolated TB bacilli were MDR-TB bacilli. Out of 29 specimens that were tested with MGIT SIRE and RT-PCR susceptibility tests, nine (31%) specimens were determined resistant to INH with RT-PCR, and eight of these specimens were in agreement with RT-PCR when tested with the MGIT SIRE. Only one specimen was determined genotypically resistant and phenotypically susceptible ( Table 3).
As regards RIF susceptibility results, five (17.2%) out of 29 specimens were determined resistant to RIF with RT-PCR and MGIT SIRE. Only one specimen was determined genotypically susceptible and phenotypically resistant ( Table 3).
The sensitivity and specificity of the RT-PCR susceptibility test for detecting INH resistance using the MGIT SIRE test as reference were 62.5 and 81%, respectively.
The sensitivity and specificity of the RT-PCR susceptibility test for detecting RIF resistance using the MGIT SIRE test as reference were 66.66 and 95%, respectively.
Overall, this assay showed a sensitivity of 64.2% and specificity of 88.88% for the detection of MDR strains.

Discussion
Globally, in 2015, 3.5% of new and 20.5% of previously treated TB cases were estimated to have had MDR-TB [11]. However, patients are often not expeditiously diagnosed, resulting in the delay of appropriate treatment as well as poorer treatment outcomes for patients and the propagation and spread of MDR-TB [6].
Conventional methods for DST of MDR-TB require between 2 and 5 weeks. Implementation of easy and rapid methods for DST is thus of paramount importance to limit the spread of drug-resistant TB [12].
The Anyplex II MTB/MDR RT-PCR assay has been proposed as an alternative approach to detect drugresistant strains, as the results obtained with the conventional susceptibility methods of M. tuberculosis come too late to influence a timely decision on patient management [13].
The use of MGIT SIRE for MDR-TB identification gave results of 20, 20.68, and 27.58% for overall MDR, RIF resistance, and INH resistance, respectively. Similar to our results, El-Sayed Zaki and Hassanin [14] reported that the rate of MDR-TB was 20%, whereas the rates of resistance to RIF and INH in Egypt were 20 and 20%, respectively. A resistance rate of 14.8% for MDR-TB, 16.3% for RIF, and 20.7% for INH were reported by Abdullal [15].
In the current study, 29 specimens were tested with both methods, the MGIT SIRE and Anyplex MTB/ MDR assay, for detection of RIF and INH resistance. Overall, we reported six specimens that were determined as MDR strains with MGIT SIRE. The Anyplex II MTB/MDR RT-PCR assay showed that four specimens out of the six MDR strains were compatible with the MGIT SIRE results, whereas two specimens out of the six MDR strains were determined as false-negative results (susceptible by RT-PCR, resistant by MGIT SIRE), therefore   Goncalves et al. [13] reported that the sensitivity of the RT-PCR assay in detecting INH resistance was 55% and that for detecting RIF resistance was 99%. The specificity of both tests was 100%. Another study by ElFeky and ElShimy [11] reported that the sensitivity and specificity for detection of INH resistance were 83.3 and 100%, respectively.
The differences in results in the different studies could be explained by the geographic and genetic variation in the distribution of drug-resistant strains of M. tuberculosis; different methods used to perform the phenotypic susceptibility testing, difference in the year of M. tuberculosis isolation, and the use of different sizes of sample collections might partially explain the present findings.
In conclusion, RT-PCR is rapid, specific, and a technically affordable molecular technique for detection of MDR-TB in clinical specimens. The optimal approach for DST of M. tuberculosis will likely involve both conventional methods (culture) and a molecular method (RT-PCR) to identify resistant strains missed by RT-PCR assay when used alone. This approach should facilitate the adequate management of MDR-TB and limit the extent and severity of MDR-TB transmission and infection.

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Conflicts of interest
There are no conflicts of interest.