Evaluating the performance of Xpert MTB/RIF and its impact on patient level outcomes among individuals attending routine health facilities for HIV care and treatment in Botswana

Abstract

Background: Worldwide, TB is one of the top 10 causes of death and the leading cause from a single infectious agent. Globally, ten percent of TB patients co-infected with HIV and 72% occurred in Africa. Botswana has the second-highest HIV infection rate in the world, with one in five adults infected. The estimated annual incidence of TB was at 385 per 100, 000 population and with a TB/HIV co-infection rate of 60% in 2015 [5]. In Botswana, as in the rest of sub-Saharan African countries, undiagnosed TB or TB diagnosed late in the course of a disease is the leading cause of death among PLHIV regardless of ART status. The ‘End TB strategy’ proposes reaching 95% reduction in the number of TB deaths and 90% reduction in TB incidence rate by 2035 compared to 2015. To achieve such a goal people with active TB disease have to be diagnosed and the new molecular test, Xpert MTB/RIF, is a key. The World Health Organization (WHO) endorsed the use of Xpert MTB/RIF as a molecular test for the detection of TB cases in 2010 to support intensified case finding activities. Countries are shifting from smear microscopy (smear)-based to Xpert MTB/RIF-based tuberculosis (TB) diagnostic algorithms. Following the WHO recommendation, the Botswana Ministry of Health and Wellness (MoHW) in 2011 implemented Xpert MTB/RIF at peripheral clinics [referred as point-of-care, (POC)] and centralized laboratories that provided a unique opportunity for to this doctoral study. The Xpert MTB/RIF Package Roll out Evaluation Study (XPRES), evaluating the accuracy, impact and operational challenges of the GeneXpert use for TB case finding among HIV infected persons, formed the basis for this PhD. Three sub-studies were nested in the XPRES study, evaluating Xpert MTB/RIF implementation in Botswana. The aim of this PhD was to evaluate the performance of Xpert MTB/RIF in the diagnosis of TB when implemented routinely at a POC and centralized laboratory. The five objectives achieved the above aim through: (1) To compare the performance Xpert MTB/RIF when implemented under routine conditions at peripheral clinics versus centralized laboratory (Study #1). (2) To determine the impact of Xpert MTB/RIF on patient-level outcomes (Study #2). (3) To determine diagnostic and therapeutic factors affecting patient-level outcomes among individuals diagnosed with Xpert MTB/RIF (Study #2). (4) To determine the effect of appearance (quality) and volume (quantity) of sputum on Xpert MTB/RIF test result (Study #3). (5) To determine the prevalence of Mycobacteria species from sputum samples collected from patients enrolled in the Xpert MTB/RIF Package Rollout Evaluation Study (Study #4 and 4a). Additionally, we conduct systematic review and meta-analysis on the impact of diagnostic and therapeutic on treatment outcome, comparing Xpert MTB/RIF and smear (Study #5) which is related with objective 2. Methods: This PhD achieved through five objectives and used retrospective approaches as part of the parent study, the XPRES Trial. Peripheral clinic versus centralized laboratory-based Xpert MTB/RIF performance: experience gained from a pragmatic trial in Botswana: From August 2012 through November 2014. GeneXpert instruments (GeneXpert) deployed in nine centralized laboratories and four point-ofcare (POC) peripheral clinics in a phased approach. Clinicians and laboratorians were trained on the four-symptom TB screening algorithm and Xpert MTB/RIF testing. We documented our experience with staff training and GeneXpert performance across the sites. Test results were extracted from GeneXpert software; unsuccessful tests were analyzed in relation to testing sites and trends over time. Tuberculosis treatment outcomes among people living with HIV diagnosed by Xpert MTB/RIF versus smear in Botswana: From August 2012 - November 2014, PLHIV initiating antiretroviral therapy at 22 HIV care and treatment centers in Botswana were enrolled and underwent systematic screening for TB. GeneXpert instruments were deployed following a stepped-wedge design in 13 sites from November 2012 - June 2013 to serve the centers. National smear and Xpert MTB/RIF intensified case finding algorithms were followed: initially symptomatic patients were evaluated by testing sputum samples using a smear (smear arm) and after GeneXpert instruments were installed, by testing with an Xpert (Xpert arm). TB treatment outcomes identified as unfavorable (death, failure or loss-to-follow-up) and favorable (cure, treatment completion) were compared between patients with drug-sensitive TB diagnosed by smear and Xpert, using cox proportional hazard ratio adjusting for intra-site correlation. The Effect of sputum quality and volume on the yield of bacteriologically-confirmed TB by Xpert MTB/RIF and smear: From August 2012 to November 2014, all people living with HIV were recruited at 22 clinics. For patients screened positive using the four TB symptoms, their sputa were tested by Xpert MTB/RIF and smear. Laboratorians assessed and recorded sputum appearance and volume. The yield of bacteriologically-positive sputum was evaluated using Xpert MTB/RIF and smear, likelihood-ratios were calculated. Higher-than-expected prevalence of non-tuberculous mycobacteria in HIV setting in Botswana: Implications for diagnostic algorithms using Xpert MTB/RIF assay: All PLHIV patients presenting for HIV care and treatment services at 22 clinical sites in Botswana were offered screening for TB and recruited. Patients who had >1 TB symptom were asked to submit sputa for Xpert MTB/RIF and culture. Culture growth was identified as Non-tuberculous mycobacteria (NTM) and Mycobacterium tuberculosis complex (MTBC) using the SD-Bioline TB Ag MPT64 Kit and Ziehl Neelsen microscopy. NTM and MTBC isolates underwent species identification by the Hain GenoType CM and AS line probe assays. Treatment-outcomes, diagnostic and therapeutic impact: Xpert MTB/RIF versus smear systematic review and meta-analysis: Citations (2000-2016) reporting treatment-outcomes of patients diagnosed by Xpert MTB/RIF compared to smear were selected from PubMed, Scopus and conference abstracts. We conducted a systematic review and meta-analysis. Favorable (cured, completed) and unfavorable (failure, death, loss-to-follow-up) outcomes were pooled for meta-analysis; we reviewed numbers of TB cases diagnosed, time to treatment and empiric treatment. The Mantel-Haenszel method with a fixed-effect model was used; I2 was calculated to measure heterogeneity. Results: This PhD comprised of five studies (1-5) and the results presented accordingly as described in the methods section: In evaluating the performance of Xpert MTB/RIF, during 276 instrument-months of operation a total of 3,630 tests were performed, of which 3,102 (85%) were successful with interpretable results. Of all 3,630 Xpert MTB/RIF tests, 528 (15%) were unsuccessful; of these 361 (68%) were classified as “error”, 119 (23%) as “invalid” and 48 (9%) as “no result”. Cumulative incidence of unsuccessful test was higher at POC (17%, 95% confidence interval, CI: 11-25%) than centralized laboratory-based GeneXpert instruments (14%, 95% CI: 11-17%; p=0.14), though the difference was not statistically significant. The total number of recorded error codes was 385 and the most common reasons were related to sample processing (211; 55%). For the TB treatment outcomes study, a total of 256 [199 per 2985 person-years and 57 per 1582 person-years of follow-up, in Xpert and smear arm, respectively, adjusted incidence rate ratio, 9.07, 95% confidence interval (CI) 4.70-17.48, P<0.001] were diagnosed and were treated for drug-sensitive TB. TB treatment outcomes as favorable or unfavorable were available for 203/256 (79.3%); 157 were in Xpert and 46 in smear arm. Unfavorable outcome was 10/46 (21.7%) for smear and was 21/157 (13.4%) for Xpert arm, adjusted hazard ratio, 1.40, 95% CI: 0.75-2.26, p=0.27. Between smear and Xpert arm, respectively, the median number of days (Interquartile Range, IQR) from sputum collection to TB treatment were 22 (3-51) and 6 (2-17), p=0.005; empiric treatment was 39/57 (68.4%) versus 97/199 (48.7%), aOR, 2.28, 95% CI: 1.24–4.20, p=0.011); and microbiologically-confirmed TB was 18/43 (41.9%) versus 102/173 (59.0%), aOR, 2.00, 95% CI: 1.01–3.96, p=0.048). Thirty-six (14%) of 256 patients received TB treatment despite a positive culture only for NTM and 34 of 36 were also negative by Xpert MTB/RIF. For the sputum quality evaluation, among 6,041 patients enrolled 2,296 were presumptive TB, 1,305 (56.8%) had >1 sputa collected and 644/1,305 (49.3%) had both Xpert MTB/RIF and smear results. Since >1 sputa collected from 644 patients 954 sputa were tested by Xpert MTB/RIF and smear. Bacteriologically-positive sputum was two-fold higher with Xpert MTB/RIF 11.4% versus smear 5.3%, p<0.001. Sputum appearance and quantity were not predictive of bacteriologically-positive results, except the volume of 2ml to < 3ml, tested by Xpert MTB/RIF LR+=1.26 (95% CI, 1.05–1.50). The NTM study included 1940 patients who submitted >1 sputum specimen and 427 (22 %) patients had ≥1 positive-culture result identified phenotypically for mycobacterial growth. Of these, 247 and 180 patients were identified as having NTM and MTBC isolates, respectively. Of the 247 patients identified with isolates containing NTM; 19 were later excluded as not having NTM based on additional genotypic testing. Among the remaining 408 patients, 228 (56%, 95% confidence interval, 46-66%) were NTM. M. intracellulare was the most common isolated (47.8%). Other NTMs commonly associated with pulmonary disease included M. malmoense (3.9%), M. avium (2.2%), M. abscessus (0.9%) and M. kansasii (0.4%). After excluding NTM isolates that were non-speciated and M. gordonae 154 (67.5%) of the NTM isolates were potential pathogens. For the systematic review, from 13 citations, 43,594 TB patients were included and 4,825 were with known TB treatment-outcome. From the pooled analysis, the unfavorable outcome among those diagnosed using Xpert MTB/RIF compared with smear was 20.2%, 541/2,675 versus 21.9%, 470/2,150, risk-ratio 0.92, 95% Confidence Interval, 0.82-1.02. Statistical heterogeneity was low (I2=0.0%, p=0.91). Compared to smear, Xpert MTB/RIF was reported to be superior in increasing the number of TB patients diagnosed (2/9 citations), increasing bacteriologicallyconfirmed TB (7/9 citations), reducing empiric treatment (3/5 citations), reducing time to diagnosis (2/3 citations), and reducing time to treatment initiation (1/5 citations). Conclusions: From this PhD we demonstrated that Xpert MTB/RIF implementation was feasible in Botswana. With the introduction of Xpert MTB/RIF in the Botswana TB program setting, we presented operational challenges and improvements achieved with a more sensitive molecular test in the diagnosis of TB patients. However, such improvement in detection of TB was not extended to patient-level treatment outcomes. Several factors, including issues around health system, human capacity, laboratory capacity, inadequate TB diagnostic algorithms and quality of sputum specimen were uncovered. Xpert MTB/RIF introduction was successful in the Botswana setting. There were more unsuccessful tests at the POC compared to the centralized laboratory, though the difference was not statistically significant. This suggests that Xpert MTB/RIF performance was unlikely affected when operated by nurses, environmental factors, power supply at the POC nor the centralized laboratory. However, unsuccessful test incidence (15%) in our settings was higher than previously reported and was mostly related to improper sample processing. Ensuring adequate training among Xpert MTB/RIF testing staff is essential to minimize errors. Xpert MTB/RIF implementation, as evidenced both in the treatment outcome study and the systematic review/meta-analysis, though not statistically significant, showed reduced unfavorable outcome compared to conventional smear. Xpert MTB/RIF test yield in bacteriologically-confirmed TB was superior to smear. Sputum quality and quantity, however, were not consistently predictive of bacteriologically-positive results by Xpert MTB/RIF or smear.

In our high HIV prevalent setting, over-half (56%) of the positive sputum cultures from PLHIV, with symptoms suggestive of TB, were NTM. In our study, though we were not able to distinguish between NTM disease and colonization, the study suggests culture and species identification for PLHIV presenting with TB symptoms remains important to facilitate NTM diagnosis and hasten time to appropriate treatment. Since patients with NTM disease present with symptoms similar to those of TB, where the testing capacity for culture and NTM speciation is limited, misdiagnosis, delivery of sub-optimal treatment and outcome may occur. Further research is warranted to identify gaps in the existing health system, and NTM disease as a potential factor affecting treatment outcome.