Treatment Outcomes of Isoniazid-Resistant (Rifampicin Susceptible) Tuberculosis Patients in Uzbekistan, 2017-2018.

Tuberculosis patients "resistant to isoniazid and susceptible to rifampicin (Hr-TB)" remain neglected, despite a high burden and poor outcomes. The World Health Organization (WHO) recommends a 6 month regimen consisting of levofloxacin, rifampicin, ethambutol, and pyrazinamide (LRZE) to treat Hr-TB. In contrast, Uzbekistan uses a 9 month regimen (LRZE plus a second-line injectable in the first 3 months). We aimed to assess the treatment outcomes of this novel regimen among Hr-TB patients treated in two regions of Uzbekistan (Fergana and Bukhara) in 2017-2018. We conducted a cohort study involving secondary analysis of routine surveillance data. Of 132 Hr-TB patients, 105 (80%) were successfully treated. Death was the predominant unsuccessful outcome (13, 10%) followed by "treatment failure" (10, 8%) and "lost to follow-up" (4, 2%). High treatment success is an indicator of the potential effectiveness of the novel regimen and adds to the limited global evidence on this issue. However, the sample size was small and there was no comparison group. Since the study was conducted in two regions of Uzbekistan only, the findings have limited generalizability. We recommend future research using an adequate sample size and an appropriate study design (randomized controlled trial or prospective cohort with a control group receiving the WHO-recommended regimen).

Universal Access to Xpert MTB/RIF Testing for Diagnosis of Tuberculosis in Uzbekistan: How Well Are We Doing?

As per national guidelines in Uzbekistan, all presumptive tuberculosis patients should be tested using the Xpert MTB/RIF assay for diagnosing tuberculosis. There is no published evidence how well this is being implemented. In this paper, we report on the Xpert coverage among presumptive tuberculosis patients in 2018 and 2019, factors associated with non-testing and delays involved. Analysis of national aggregate data indicated that Xpert testing increased from 24% in 2018 to 46% in 2019, with variation among the regions: 21% in Tashkent region to 100% in Karakalpakstan. In a cohort (January-March 2019) constituted of 40 randomly selected health facilities in Tashkent city and Bukhara region, there were 1940 patients of whom 832 (43%, 95% confidence interval (CI): 41-45%) were not Xpert-tested. Non-testing was significantly higher in Bukhara region (73%) compared to Tashkent city (28%). In multivariable analysis, patient's age, distance between primary health centre (PHC) and Xpert laboratory, diagnostic capacity and site of PHC were associated with non-testing. The median (interquartile range) duration from date of initial visit to PHC to receiving results was 1 (1-2) day in Tashkent city compared to 3 (1-6) days in Bukhara region (p-value < 0.001). While there is commendable progress, universal access to Xpert testing is not a reality yet.

Scaling Up Molecular Diagnostic Tests for Drug-Resistant Tuberculosis in Uzbekistan from 2012-2019: Are We on the Right Track?

Uzbekistan has a large burden of drug-resistant tuberculosis (TB). To deal with this public health threat, the National TB Program introduced rapid molecular diagnostic tests such as Xpert MTB/RIF (Xpert) and line probe assays (LPAs) for first-line and second-line drugs. We documented the scale-up of Xpert and LPAs from 2012-2019 and assessed whether this led to an increase in patients with laboratory-confirmed multidrug-resistant/rifampicin-resistant TB (MDR/RR-TB) and extensively drug-resistant TB (XDR-TB). This was a descriptive study using secondary program data. The numbers of GeneXpert instruments cumulatively increased from six to sixty-seven, resulting in annual assays increasing from 5574 to 107,330. A broader use of the technology resulted in a lower proportion of tests detecting Mycobacterium tuberculosis with half of the positive results showing rifampicin resistance. LPA instruments cumulatively increased from two to thirteen; the annual first-line assays for MDR-TB increased from 2582 to 6607 while second-line assays increased from 1435 in 2016 to 6815 in 2019 with about one quarter to one third of diagnosed patients showing second-line drug resistance. Patient numbers with laboratory-confirmed MDR-TB remained stable (from 1728 to 2060) but there was a large increase in patients with laboratory-confirmed XDR-TB (from 31 to 696). Programmatic implications and ways forward are discussed.

Rapid Tuberculosis Diagnostics Including Molecular First- and Second-Line Resistance Testing Based on a Novel Microfluidic DNA Extraction Cartridge.

Xpert MTB/RIF testing has improved tuberculosis (TB) diagnostics and rifampicin (Rif) resistance testing worldwide. However, it has weaknesses, such as its restriction to Rif resistance testing and the inability to use extracted DNA for further testing. Herein, a holistic diagnostic workflow, including TB detection and resistance testing toward Rif, isoniazid, and important second-line drugs (SLDs), based on a novel microfluidic DNA extraction cartridge (TB-Disk), is presented. DNA from 73 precharacterized sputum samples was extracted with TB-Disk, including 45 clinical and bacteriologically confirmed TB samples, nine TB-negative samples, and 19 sputum samples spiked with twofold dilutions of TB bacteria. The extracted DNA was subjected to further testing with FluoroType MTB (FT-MTB), GenoType MTBDRplus (GT-plus), and GenoType MTBDRsl. A total of 100% (20/20) and 72% (18/25) of smear-positive and smear-negative TB samples were identified as Mycobacterium tuberculosis complex positive. A total of 79% (33/42) of subsequently GT-plus tested samples yielded a valid result. Eight samples were identified as multidrug-resistant TB by GT-plus and further tested for resistance toward SLDs using GenoType MTBDRsl, yielding 75% (6/8) valid results. FT-MTB with cartridge-based DNA extraction (Disk-DNA) and DNA extracted with FluoroLyse yielded similar analytical sensitivities. FT-MTB with Disk-DNA was 100% specific. TB-Disk in combination with FT-MTB enables sensitive TB detection. The Disk-DNA can be further used for screening resistance toward first-line drugs and SLDs.

Comparison of Xpert MTB/RIF with ProbeTec ET DTB and COBAS TaqMan MTB for direct detection of M. tuberculosis complex in respiratory specimens.

BACKGROUND: Nucleic acid amplification assays allow for the rapid and accurate detection of Mycobacterium tuberculosis (MTB) directly in clinical specimens thereby facilitating diagnosis of tuberculosis (TB). With the fully automated Xpert MTB/RIF system (Cepheid) an innovative solution of TB diagnostics has been launched. We performed a direct head-to-head comparison of Xpert MTB/RIF with two widely used commercial assays, ProbeTec ET DTB (DTB) (Becton-Dickinson) and COBAS TaqMan MTB (CTM-MTB) (Roche). METHODS: 121 pre-characterized respiratory specimens (68 culture-positive for MTB complex, 24 culture-positive for non-tuberculous mycobacteria and 29 culture-negative) taken from our frozen specimen bank were tested for the presence of MTB complex by the three assays. RESULTS: Among culture-positive samples (n = 68), overall sensitivity for detection of MTB complex was 74.6%, 73.8%, and 79.1% for Xpert MTB/RIF, CTM-MTB, and DTB, respectively. Within the subgroup of smear-negative TB samples (n = 51) sensitivity was 68% for Xpert MTB/RIF and CTM-MTB and 72% for DTB. Among smear-positive TB samples (n = 17), all (100%) were detected by DTB and 94.1% and 93.3% by Xpert MTB/RIF and CTM-MTB, respectively. Specificity was best for CTM-MTB (100%) and lowest for Xpert MTB/RIF (96.2%) due to misidentification of two NTM samples as MTB complex. CTM-MTB yielded the highest rate of invalid results (4.1%) (0.8% by Xpert MTB/RIF and DTB, respectively). CONCLUSIONS: The direct comparison of Xpert MTB/RIF with CTM-MTB and DTB yielded similar overall performance data. Whereas DTB was slightly superior to Xpert MTB/RIF in terms of sensitivity, at least in the sample collection tested here, CTM-MTB performed best in terms of specificity.

Multi-centre evaluation of the speed-oligo Mycobacteria assay for differentiation of Mycobacterium spp. in clinical isolates.

BACKGROUND: A new DNA line probe assay (Speed-oligo Mycobacteria, Vircell) has been launched for rapid differentiation of Mycobacterium spp. from cultures. Compared to other line-probe assays, Speed-oligo Mycobacteria covers a relatively limited spectrum of species but uses a simpler and faster dip-stick technique. The present multi-centre, multi-country study aimed at evaluating the utility and usability of Speed-oligo Mycobacteria in routine mycobacteriology diagnostics. Results from Speed-oligo Myobacteria were compared to those from Genotype CM (HAIN lifescience, Nehren, Germany), another line-probe assay. METHODS: Speed-oligo Mycobacteria assay was performed in three main steps: 1) DNA extraction from cultured material 2) PCR amplification of the target gene and an internal control and 3) hybridization of the PCR products to specific probes by means of a dip-stick. RESULTS: Two hundred forty-two clinical isolates were recovered from consecutive positive mycobacterial cultures at two German (IML Gauting, Bioscientia Ingelheim), one Czech (KLINLAB Prague), and at a Sudanese (Khartoum) laboratory. All Mycobacterium species covered by the assay were reliably recognized. The rate of false positive results was 1.2% and concerned only the species M. marinum and M. peregrinum. The identification rate, i.e. the proportion of isolates which was correctly differentiated to the level of species or complex by the assay, differed significantly among laboratories being 94.9%, 90.7%, and 75.0% at the study sites IML Gauting, KLINLAB Prague and Bioscientia Ingelheim, respectively. This difference was caused by different spectra of NTM species encountered by the laboratory centres in daily routine diagnostics. CONCLUSIONS: Speed-oligo Mycobacteria assay was proved a rapid and easy-to-perform alternative to conventional line-probe assays. The assay showed excellent sensitivity with regard to identification of genus Mycobacterium and species/complexes covered by the test. However, due to its relatively limited spectrum of taxa, a varying proportion of NTM may not be identified by the assay in daily diagnostics demanding further analyses. The only significant shortcoming in terms of specificity was the misidentification of the clinically relevant species M. marinum.

Evaluation of the hyplex TBC PCR test for detection of Mycobacterium tuberculosis complex in clinical samples.

BACKGROUND: Tuberculosis (TB) is one of the major public health concerns worldwide. The detection of the pathogen Mycobacterium tuberculosis complex (MTBC) as early as possible has a great impact on the effective control of the spread of the disease. In our study, we evaluated the hyplex TBC PCR test (BAG Health Care GmbH), a novel assay using a nucleic acid amplification technique (NAAT) with reverse hybridisation and ELISA read out for the rapid detection of M. tuberculosis directly in clinical samples. RESULTS: A total of 581 respiratory and non-respiratory specimens from our pneumological hospital and the National TB Institute of Uzbekistan were used for the evaluation of the PCR assay. Of these, 292 were classified as TB samples and 289 as non-TB samples based on the results of the TB cultures as reference method. The PCR results were initially used to optimise the cut-off value of the hyplex TBC test system by means of a ROC analysis. The overall sensitivity of the assay was determined to be 83.1%. In smear-positive TB samples, the sensitivity of the hyplex TBC PCR test was estimated to 93.4% versus 45.1% in smear-negative samples. The specificity of the test was 99.25%. Of the two specimens (0.75%) with false-positive PCR results, one yielded a culture positive for non-tuberculous mycobacteria. Based on the assumption of a prevalence of 8% TB positives among the samples in our diagnostic TB laboratory, the positive and negative predictive values were estimated to 90.4% and 98.5%, respectively. CONCLUSIONS: The hyplex TBC PCR test is an accurate NAAT assay for a rapid and reliable detection of M. tuberculosis in various respiratory and non-respiratory specimens. Compared to many other conventional NAAT assays, the hyplex TBC PCR test is in a low price segment which makes it an attractive option for developing and emerging countries with high TB burdens.