Diagnostic predictive values of the Hain genotype MTBDRsl assay in mycobacterial strains isolated from Sudan

Introduction hain GenoType MTBDRsl is nucleic acid amplification assay based on reverse hybridization with specific oligonucleotide probes on nitrocellulose strips. MTBDRsl identifies M. tuberculosis complex and detects resistance to fluoroquinolone, second line injectable drugs and ethambutol evident as mutations of gyrA, rrs and embB genes respectively. This study aimed to evaluate the diagnostic performance of the Hain GenoType MTBDRsl Assay using 1% proportion method on LJ medium as gold standard. Methods a total of 52 rifampicin resistant (RR) isolates were tested for second line drug sensitivity by 1% proportion method and by MTBDRsl assay. Results two strains were identified as mycobacteria other than tuberculosis MOTT and the rest were Mycobacterium tuberculosis complex MTBC. Five of the MTBC isolates (5/50; 10%) showed resistance to at least one second line drug and one isolate (1/50; 2%) was XDR. XDR strain was concordantly detected by the two methods. One of two Kanamycin-resistant isolates showed discordant results. Ofloxacin showed one false positive and one false negative result. Most discrepancies were detected with Ethambutol. The sensitivity, specificity, positive and negative predictive values were respectively as follows: Ethambutol (63.3.4%, 85.7%, 94.4% and 62%), for Kanamycin (67%, 100%, 100% and 97.9%), for Amikacin and Capreomycin (100%, 100%, 100% and 100%), for Ofloxacin (75%, 97.5%, 75% and 97.8%). For XDR isolate the values were 100%, 100%, 100% and 100% respectively. Conclusion MTBDRsl showed high specificity and negative predictive values making it acceptable and time-saving for early presumptive detection of resistance to second-line drugs in Sudan.


Introduction
Drug resistant TB has emerged as a significant global public health problem and a great challenge for TB control. Drug resistant TB has resulted from drug misuse and/or drug mismanagement. In 2014 WHO estimate 480 000 multi drug resistant (MDR) cases; Most of these cases are in India, China, Russian Federation and South Africa [1]. Extensively drug-resistant TB (XDR-TB) had been reported by 105 countries by 2015. The magnitude of XDR-TB is not small (9.7% of MDR-TB) in addition XDR-TB is an important killer of TB patients [1,2]. Most species of mycobacteria grow slowly with generation time of 18 to 24 hours; resulting in delay of TB diagnosis by conventional methods [3]. Molecular techniques reducing turnaround time (TAT) required to detect and identify M. tuberculosis to 1-2 days instead of several weeks in the conventional methods [4]. Extensively drug-resistant tuberculosis (XDR-TB) is defined as multi drug resistant tuberculosis MDR-TB; resistance of rifampicin and isoniazid with additional resistance to any fluoroquinolone and to at least one of three injectable drugs used for TB treatment: capreomycin, kanamycin, or amikacin [5]. In recent years several molecular methods have been developed for the diagnosis of tuberculosis and detection of drug resistance, including line probe assays [6]. Line probe assays (LPAs) are not new; nevertheless Hain Lifescience (Hain Lifescience GmbH, Germany) developed two kits of line probe assay for the detection of drug resistance, the genotype MTBDRplus and MTBDRsl [7] the later is one of the few commercially available molecular tests for detection of second line drugs [8]. MTBDRplus simultaneously detects MDR and the MTBDRsl detect the resistance of aminoglycosides/cyclic peptides, fluoroquinolones, and ethambutol through detection of mutations in the relevant genes [7]. In 2015, the Hain Lifescience developed the commercially available version 2.0 of the MTBDRsl assay and recently recommended by WHO [1]; to date version 2.0 MTBDRsl is not supplied in Sudan. To our knowledge; in Sudan second line drug resistance and XDR susceptibility testing has never been studied before this report.

Methods
Isolation of mycobacteria: Lowenstein-Jensen medium (L.J.) containing glycerol and Lowenstein-Jensen supplemented with pyruvate were used for isolation of mycobacteria from sputum specimens. Petroff method for decontamination (4% NaOH for 20 minutes) was used to homogenize and decontaminate sputum specimens.
Isolates identification: isolates were firstly identified phenotypically according to their reaction with ZN staining method, growth rate, colonial morphology and pigment production and by their susceptibility to Para nitro benzoic acid (PNB). Then isolates were identified genetically by the hybridization of isolates DNA Amplicons with TUB probe incorporated on validity zone within the Hain GenoType MTBDRsl strips.

Results
Hain Genotype MTBDRsl showed significantly reduced sensitivity for Kanamycin (p = 0.04) and Ofloxacin (p = 0.00; Odds ratio: 135; Relative risk: 34.5) at 67% and 75% respectively (  [15,20]. XDR: the majority of MDR (90%, 45/50) were susceptible to the all second line anti-tuberculous drugs. MTBDRsl showed very high performance (p=0.02) in detecting XDR with sensitivity of (100%) and specificity of (100%). Positive and negative predictive values were (100%) and (100%). The isolate was concordantly detected by the two methods Table1. To our knowledge; in Sudan second line drug resistance and XDR susceptibility testing has never been studied before this report. The low frequency of XDR-TB within the study cohort is within the reported frequency that was reported from many countries (0.8-15.2%) [19]. but is also this study constrained by the limited sample size. Moreover; this may reflect the imperfect reference standard currently available for this drug [17,20].  Study ensures the quality of the service that is delivered by the local health system.

Competing interests
The authors declare no competing of interests.     Page number not for citation purposes 7