Michael Hoelscher

Advances in tuberculosis diagnostics: the Xpert MTB/RIF assay and future prospects for a point-of-care test

Rapid progress has been made in the development of new diagnostic assays for tuberculosis in recent years. New technologies have been developed and assessed, and are now being implemented. The Xpert MTB/RIF assay, which enables simultaneous detection of Mycobacterium tuberculosis (MTB) and rifampicin (RIF) resistance, was endorsed by WHO in December, 2010. This assay was specifically recommended for use as the initial diagnostic test for suspected drug-resistant or HIV-associated pulmonary tuberculosis. By June, 2012, two-thirds of countries with a high tuberculosis burden and half of countries with a high multidrug-resistant tuberculosis burden had incorporated the assay into their national tuberculosis programme guidelines. Although the development of the Xpert MTB/RIF assay is undoubtedly a landmark event, clinical and programmatic effects and cost-effectiveness remain to be defined. We review the rapidly growing body of scientific literature and discuss the advantages and challenges of using the Xpert MTB/RIF assay in areas where tuberculosis is endemic. We also review other prospects within the developmental pipeline. A rapid, accurate point-of-care diagnostic test that is affordable and can be readily implemented is urgently needed. Investment in the tuberculosis diagnostics pipeline should remain a major priority for funders and researchers.

Operational Feasibility of Using Loop-Mediated Isothermal Amplification for Diagnosis of Pulmonary Tuberculosis in Microscopy Centers of Developing Countries

ABSTRACT The characteristics of loop-mediated isothermal amplification (LAMP) make it a promising platform for the molecular detection of tuberculosis (TB) in developing countries. Here, we report on the first clinical evaluation of LAMP for the detection of pulmonary TB in microscopy centers in Peru, Bangladesh, and Tanzania to determine its operational applicability in such settings. A prototype LAMP assay with simplified manual DNA extraction was evaluated for accuracy and ease of use. The sensitivity of LAMP in smear- and culture-positive sputum specimens was 97.7% (173/177 specimens; 95% confidence interval [CI], 95.5 to 99.9%), and the sensitivity in smear-negative, culture-positive specimens was 48.8% (21/43 specimens; CI, 33.9 to 63.7%). The specificity in culture-negative samples was 99% (500/505 specimens; CI, 98.1 to 99.9%). The average hands-on time for testing six samples and two controls was 54 min, similar to that of sputum smear microscopy. The optimal amplification time was 40 min. No indeterminate results were reported, and the interreader variability was 0.4%. Despite the use of a single room without biosafety cabinets for all procedures, no DNA contamination was observed. The assay was robust, with high end-point stability and low rates of test failure. Technicians with no prior molecular experience easily performed the assay after 1 week of training, and opportunities for further simplification of the assay were identified.

Feasibility, accuracy, and clinical effect of point-of-care Xpert MTB/RIF testing for tuberculosis in primary-care settings in Africa: a multicentre, randomised, controlled trial

BACKGROUND The Xpert MTB/RIF test for tuberculosis is being rolled out in many countries, but evidence is lacking regarding its implementation outside laboratories, ability to inform same-day treatment decisions at the point of care, and clinical effect on tuberculosis-related morbidity. We aimed to assess the feasibility, accuracy, and clinical effect of point-of-care Xpert MTB/RIF testing at primary-care health-care facilities in southern Africa. METHODS In this pragmatic, randomised, parallel-group, multicentre trial, we recruited adults with symptoms suggestive of active tuberculosis from five primary-care health-care facilities in South Africa, Zimbabwe, Zambia, and Tanzania. Eligible patients were randomly assigned using pregenerated tables to nurse-performed Xpert MTB/RIF at the clinic or sputum smear microscopy. Participants with a negative test result were empirically managed according to local WHO-compliant guidelines. Our primary outcome was tuberculosis-related morbidity (measured with the TBscore and Karnofsky performance score [KPS]) in culture-positive patients who had begun anti-tuberculosis treatment, measured at 2 months and 6 months after randomisation, analysed by intention to treat. This trial is registered with Clinicaltrials.gov, number NCT01554384. FINDINGS Between April 12, 2011, and March 30, 2012, we randomly assigned 758 patients to smear microscopy (182 culture positive) and 744 to Xpert MTB/RIF (185 culture positive). Median TBscore in culture-positive patients did not differ between groups at 2 months (2 [IQR 0-3] in the smear microscopy group vs 2 [0·25-3] in the MTB/RIF group; p=0·85) or 6 months (1 [0-3] vs 1 [0-3]; p=0·35), nor did median KPS at 2 months (80 [70-90] vs 90 [80-90]; p=0·23) or 6 months (100 [90-100] vs 100 [90-100]; p=0·85). Point-of-care MTB/RIF had higher sensitivity than microscopy (154 [83%] of 185 vs 91 [50%] of 182; p=0·0001) but similar specificity (517 [95%] 544 vs 540 [96%] of 560; p=0·25), and had similar sensitivity to laboratory-based MTB/RIF (292 [83%] of 351; p=0·99) but higher specificity (952 [92%] of 1037; p=0·0173). 34 (5%) of 744 tests with point-of-care MTB/RIF and 82 (6%) of 1411 with laboratory-based MTB/RIF failed (p=0·22). Compared with the microscopy group, more patients in the MTB/RIF group had a same-day diagnosis (178 [24%] of 744 vs 99 [13%] of 758; p<0·0001) and same-day treatment initiation (168 [23%] of 744 vs 115 [15%] of 758; p=0·0002). Although, by end of the study, more culture-positive patients in the MTB/RIF group were on treatment due to reduced dropout (15 [8%] of 185 in the MTB/RIF group did not receive treatment vs 28 [15%] of 182 in the microscopy group; p=0·0302), the proportions of all patients on treatment in each group by day 56 were similar (320 [43%] of 744 in the MTB/RIF group vs 317 [42%] of 758 in the microscopy group; p=0·6408). INTERPRETATION Xpert MTB/RIF can be accurately administered by a nurse in primary-care clinics, resulting in more patients starting same-day treatment, more culture-positive patients starting therapy, and a shorter time to treatment. However, the benefits did not translate into lower tuberculosis-related morbidity, partly because of high levels of empirical-evidence-based treatment in smear-negative patients. FUNDING European and Developing Countries Clinical Trials Partnership, National Research Foundation, and Claude Leon Foundation.

New antituberculosis drugs, regimens, and adjunct therapies: needs, advances, and future prospects

About 1·3 million people died of tuberculosis in 2012, despite availability of effective drug treatment. Barriers to improvements in outcomes include long treatment duration (resulting in poor patient adherence and loss of patients to follow-up), complex regimens that involve expensive and toxic drugs, toxic effects when given with antiretroviral therapy, and multidrug resistance. After 50 years of no antituberculosis drug development, a promising pipeline is emerging through the repurposing of old drugs, re-engineering of existing antibacterial compounds, and discovery of new compounds. A range of novel antituberculosis drugs are in preclinical development, several phase 2 and 3 trials are underway, and use of adjunct therapies is being explored for drug-sensitive and drug-resistant tuberculosis. Historical advances include approval of two new drugs, delamanid and bedaquiline. Combinations of new and existing drugs are being assessed to shorten the duration of therapy and to treat multidrug-resistant tuberculosis. There has also been progress in development of new antituberculosis drugs that are active against dormant or persister populations of Mycobacterium tuberculosis. In this Review, we discuss recent advances in antituberculosis drug discovery and development, clinical trial designs, laboratory methods, and adjunct host-directed therapies, and we provide an update of phase 3 trials of various fluoroquinolones (RIFAQUIN, NIRT, OFLOTUB, and REMoxTB). We also emphasise the need to engage the community in design, implementation, and uptake of research, to increase international cooperation between drug developers and health-care providers adopting new regimens.

Evaluation of the Xpert MTB/RIF Assay for the Diagnosis of Pulmonary Tuberculosis in a High HIV Prevalence Setting

RATIONALE Xpert MTB/RIF is a novel automated molecular diagnostic recently endorsed by the World Health Organization. However, performance-related data from high HIV prevalence settings are limited. OBJECTIVES The impact of sample-related factors on performance and the significance of Xpert MTB/RIF-positive culture-negative discordance remain unclear. METHODS Xpert MTB/RIF was evaluated using single archived spot-sputum samples from 496 South African patients with suspected TB. Mycobacterium tuberculosis culture positivity and phenotypic resistance to rifampicin served as reference standards. MEASUREMENTS AND MAIN RESULTS Overall, Xpert MTB/RIF detected 95% (95% confidence interval [CI], 88-98%; 89 of 94) of smear-positive culture-positive cases and the specificity was 94% (91-96%; 320 of 339). The sensitivity in smear-negative cases was 55% (35-73%; 12 of 22) when the analysis was restricted to 1 ml of unprocessed sputum and culture time-to-positivity of less than or equal to 28 days. Compared with smear microscopy (n=94), Xpert MTB/RIF detected an additional 17 cases (n=111) representing an 18% (11-27%; 111 vs. 94) relative increase in the rapid TB case detection rate. Moreover, compared with smear microscopy, the inclusion of Xpert MTB/RIF-positive culture-negative TB cases (ruled-in by an alternative diagnostic method) resulted in the detection of a further 16 cases (n=127), thus significantly increasing the rapid TB case detection rate to 35% (95% CI, 26-45%; 94 to 111 vs. 94 to 127; P<0.01), the overall specificity to 99.1% (97-100%; 320 of 323; P<0.001), and sensitivity in smear-negative TB to 60% (P=0.12). Performance strongly correlated with smear status and culture time-to-positivity. In patients infected with HIV compared with patients uninfected with HIV Xpert MTB/RIF showed a trend to reduced sensitivity (P=0.09) and significantly reduced negative predictive value (P=0.01). The negative predictive value for rifampicin resistance was 99.4%. CONCLUSIONS XpertMTB/RIF outperformed smear microscopy, established a diagnosis in a significant proportion of patients with smear-negative TB, detected many highly likely TB cases missed by culture, and accurately ruled out rifampicin-resistant TB. Sample-specific factors had limited impact on performance. Performance in patients infected with HIV, especially those with advanced immunosuppression, warrants further study.

Drug-resistant tuberculosis: time for visionary political leadership

Two decades ago, WHO declared tuberculosis a global emergency, and invested in the highly cost-effective directly observed treatment short-course programme to control the epidemic. At that time, most strains of Mycobacterium tuberculosis were susceptible to first-line tuberculosis drugs, and drug resistance was not a major issue. However, in 2013, tuberculosis remains a major public health concern worldwide, with prevalence of multidrug-resistant (MDR) tuberculosis rising. WHO estimates roughly 630 000 cases of MDR tuberculosis worldwide, with great variation in the frequency of MDR tuberculosis between countries. In the past 8 years, extensively drug-resistant (XDR) tuberculosis has emerged, and has been reported in 84 countries, heralding the possibility of virtually untreatable tuberculosis. Increased population movement, the continuing HIV pandemic, and the rise in MDR tuberculosis pose formidable challenges to the global control of tuberculosis. We provide an overview of the global burden of drug-resistant disease; discuss the social, health service, management, and control issues that fuel and sustain the epidemic; and suggest specific recommendations for important next steps. Visionary political leadership is needed to curb the rise of MDR and XDR tuberculosis worldwide, through sustained funding and the implementation of global and regional action plans.

Preferential infection and depletion of Mycobacterium tuberculosis–specific CD4 T cells after HIV-1 infection

HIV-1 preferentially infects M. tuberculosis-specific CD4+ T cells due to their increased production of IL-2.

CD8 T-Cell Recognition of Multiple Epitopes within Specific Gag Regions Is Associated with Maintenance of a Low Steady-State Viremia in Human Immunodeficiency Virus Type 1-Seropositive Patients

ABSTRACT The importance of HLA class I-restricted CD8 T-cell responses in the control of human immunodeficiency virus (HIV) infection is generally accepted. While several studies have shown an association of certain HLA class I alleles with slower disease progression, it is not fully established whether this effect is mediated by HIV-specific CD8 T-cell responses restricted by these alleles. In order to study the influence of the HLA class I alleles on the HIV-specific CD8 T-cell response and on viral control, we have assessed HIV-specific epitope recognition, plasma viral load, and expression of HLA class I alleles in a cohort of HIV-seropositive bar workers. Possession of the HLA class I alleles B5801, B8101, and B0702 was associated with a low median viral load and simultaneously with a broader median recognition of Gag epitopes compared to all other HLA alleles (twofold increase) (P = 0.0035). We further found an inverse linear relationship between the number of Gag epitopes recognized and the plasma viral load (R = −0.36; P = 0.0016). Particularly, recognition of multiple epitopes within two regions of Gag (amino acids [aa] 1 to 75 and aa 248 to 500) was associated with the maintenance of a low steady-state viremia, even years after acute infection.

Tuberculosis Diagnostics and Biomarkers: Needs, Challenges, Recent Advances, and Opportunities

Tuberculosis is unique among the major infectious diseases in that it lacks accurate rapid point-of-care diagnostic tests. Failure to control the spread of tuberculosis is largely due to our inability to detect and treat all infectious cases of pulmonary tuberculosis in a timely fashion, allowing continued Mycobacterium tuberculosis transmission within communities. Currently recommended gold-standard diagnostic tests for tuberculosis are laboratory based, and multiple investigations may be necessary over a period of weeks or months before a diagnosis is made. Several new diagnostic tests have recently become available for detecting active tuberculosis disease, screening for latent M. tuberculosis infection, and identifying drug-resistant strains of M. tuberculosis. However, progress toward a robust point-of-care test has been limited, and novel biomarker discovery remains challenging. In the absence of effective prevention strategies, high rates of early case detection and subsequent cure are required for global tuberculosis control. Early case detection is dependent on test accuracy, accessibility, cost, and complexity, but also depends on the political will and funder investment to deliver optimal, sustainable care to those worst affected by the tuberculosis and human immunodeficiency virus epidemics. This review highlights unanswered questions, challenges, recent advances, unresolved operational and technical issues, needs, and opportunities related to tuberculosis diagnostics.

Tuberculosis comorbidity with communicable and non-communicable diseases: integrating health services and control efforts

Recent data for the global burden of disease reflect major demographic and lifestyle changes, leading to a rise in non-communicable diseases. Most countries with high levels of tuberculosis face a large comorbidity burden from both non-communicable and communicable diseases. Traditional disease-specific approaches typically fail to recognise common features and potential synergies in integration of care, management, and control of non-communicable and communicable diseases. In resource-limited countries, the need to tackle a broader range of overlapping comorbid diseases is growing. Tuberculosis and HIV/AIDS persist as global emergencies. The lethal interaction between tuberculosis and HIV coinfection in adults, children, and pregnant women in sub-Saharan Africa exemplifies the need for well integrated approaches to disease management and control. Furthermore, links between diabetes mellitus, smoking, alcoholism, chronic lung diseases, cancer, immunosuppressive treatment, malnutrition, and tuberculosis are well recognised. Here, we focus on interactions, synergies, and challenges of integration of tuberculosis care with management strategies for non-communicable and communicable diseases without eroding the functionality of existing national programmes for tuberculosis. The need for sustained and increased funding for these initiatives is greater than ever and requires increased political and funder commitment.