Diagnosis of opportunistic infections: HIV co-infections tuberculosis

Scott, Lesley; da Silva, Pedro; Boehme, Catharina C.; Stevens, Wendy; Gilpin, Christopher M.

Current Opinion in HIV & AIDS: March 2017 - Volume 12 - Issue 2 - p 129–138
doi: 10.1097/COH.0000000000000345
HIV AND DIAGNOSTICS: Edited by Wendy Stevens

Purpose of review: Tuberculosis (TB) incidence has declined ∼1.5% annually since 2000, but continued to affect 10.4 million individuals in 2015, with 1/3 remaining undiagnosed or underreported. The diagnosis of TB among those co-infected with HIV is challenging as TB remains the leading cause of death in such individuals. Accurate and rapid diagnosis of active TB will avert mortality in both adults and children, reduce transmission, and assist in timeous decisions for antiretroviral therapy initiation. This review describes advances in diagnosing TB, especially among HIV co-infected individuals, highlights national program's uptake, and impact on patient care.

Recent findings: The TB diagnostic landscape has been transformed over the last 5 years. Molecular diagnostics such as Xpert MTB/RIF, which simultaneously detects Mycobacterium tuberculosis (MTB) resistance to rifampicin, has revolutionized TB control programs. WHO endorsed the use of Xpert MTB/RIF in 2010 for use in HIV/TB co-infected patients, and later in 2013 for use as the initial diagnostic test for all adults and children with signs and symptoms of pulmonary TB. Line probe assays (LPAs) are recommended for the detection of rifampicin and isoniazid resistance in sputum smear-positive specimens and mycobacterial cultures. A second-line line probe assay has been recommended for the diagnosis of extensively drug-resistant (XDR)-TB Assays such as the urine lateral flow (LF)-lipoarabinomannan (LAM), can be used at the point of care (POC) and have a niche role to supplement the diagnosis of TB in seriously ill HIV-infected, hospitalized patients with low CD4 cell counts of less than 100 cells/μl. Polyvalent platforms such as the m2000 (Abbott Molecular) and GeneXpert (Cepheid) offer potential for integration of HIV and TB testing services. While the Research and Development (R&D) pipeline appears to be rich at first glance, there are actually few leads for true POC tests that would allow for earlier TB diagnosis or rapid, comprehensive drug susceptibility testing, especially when considering the very high attrition rates observed between biomarker discovery and product market entry.

Summary: In this review, we describe diagnostic strategies specifically for HIV and TB co-infected individuals. Molecular diagnostics in particular within the past 5 years have revolutionized and ‘disrupted’ this field. They lend themselves to integration of services with platforms capable of polyvalent testing. Impact on patient care is, however, still debatable. What has been highlighted is the need for health system strengthening and for true POC testing that can be used in active case finding.

aDepartment of Molecular Medicine and Haematology, Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, Gauteng, South Africa

bNational Priority Programs, National Health Laboratory Service, Johannesburg, Gauteng, South Africa

cFoundation for Innovative New Diagnostics, Geneva

dGlobal TB Program, WHO, Geneva, Switzerland

Correspondence to Lesley Scott, Department of Molecular Medicine and Haematology, Faculty of Health Sciences, University of the Witwatersrand, 7 York Road, Parktown, Johannesburg, Gauteng, South Africa. Tel: +27 11 489 8567; fax: +27 11 489 5812; e-mail: lesley.scott@wits.ac.za

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In 2010, it was true to report that TB curative drugs had not changed in 50 years, TB control programs were weak, treatment regimens were lengthy, and medications toxic. There was limited attention to infection control, inadequate investment in R&D and with the HIV epidemic all served to make Mycobacterium tuberculosis (MTB) disease an increasing global threat [1]. Five years on, we see the global incidence of TB slowly decreasing at around 1.5% per annum but still well below the 4–5% annual reduction in incidence needed to meet the first milestones (35% reduction in the number of TB deaths; 20% reduction in TB incidence) of the End TB strategy, set for 2020 [2]. WHO has recommended the use of a standardized shorter multidrug-resistant tuberculosis (MDR)-TB regimen of 9–12 months for the majority of patients (excluding pregnant women) with pulmonary MDR/rifampicin-resistant (RR)-TB that is not resistant to second-line drugs. WHO recommendations for the use of two new drugs, notably bedaquiline and delamanid, have helped to improve outcomes for patients with MDR/XDR-TB. Since 2010, WHO has endorsed the use of several new diagnostic technologies such as Xpert MTB/RIF (Cepheid, Sunnyvale, California, USA), molecular line probe assays (Hain LifeSciences, Nehren, Germany and Nipro Corporation, Osaka, Japan) for the diagnosis of MDR-TB and XDR-TB, TB-loop-mediated isothermal amplification assay (TB-LAMP) (Eiken, Tokyo, Japan), and LF-LAM (Alere Inc., Waltham, Massachusetts, USA).

In 2016, WHO reports the status of the TB epidemic with 10.4 million new incident cases [3]. TB remains the leading infectious disease causing mortality with an estimated 1.4 million TB deaths in 2015, and an additional 0.4 million deaths resulting from TB disease among people living with HIV. An estimated 11% of TB patients are co-infected with HIV and high rates (3.9% of new TB cases and 21% of previously treated cases) of MDR-TB of which 9.5% have XDR-TB represent a major public health crisis [4]. Of note, 80% estimated incident TB cases are reported from 22 high-burden countries, with those in sub-Saharan Africa bearing the brunt of dual HIV and TB epidemics.

TB [including drug-resistant (DR)-TB] is still the leading cause of death among HIV co-infected individuals. This is evident from autopsy studies, of which a systematic review and meta-analysis of 36 eligible studies [5] reported a pooled prevalence of 39.7% [confidence interval (CI) 32.4–47%] in adults. This varied by world region: 63% in South Asia, 43% in sub-Saharan Africa, and 27% in the Americas. A study in South Africa (SA) from the North West province [6▪], which has a high (13%) HIV prevalence, reported a quarter of home deaths had evidence of undiagnosed TB disease, emphasizing the burden of TB in the community and underlining the fatal consequences of delayed TB diagnosis and treatment [7].

Diagnosis of TB is particularly difficult among HIV co-infected individuals who may have atypical, nonspecific clinical presentation, and more often (24–61%) smear-negative disease [8] with less cavitary lesions (due to impairment of granuloma formation [9]), along with higher rates of extrapulmonary TB (EPTB) [10,11▪,12]. Sputum-based diagnosis is therefore less sensitive among HIV co-infected patients, resulting in more smear-negative TB disease leading to more empiric treatment among those at greatest risk of disease [13]. This requires caution after the REMEMBER (Reducing Early Mortality and Early Morbidity by Empiric Tuberculosis (TB) Treatment Regimens) trial illustrated that empiric TB therapy did not reduce mortality at 24 weeks compared with isoniazid preventive therapy in adult outpatients with advanced HIV disease initiating antiretroviral therapy (ART) [14▪▪].

The emergence of MDR-TB and XDR-TB further highlights the need for sensitive and timely diagnosis. A meta-analysis performed by Mesfin et al.[15] confirmed the association between MDR-TB and HIV, with the odds of having MDR-TB among HIV-positive cases being 24% higher. However, recent studies show HIV co-infection not to be a direct driver for the emergence and transmission of resistant strains [16▪]. As mechanistic mathematical modeling approaches show [17▪] the vast majority (up to 80% in underresourced settings) of MDR-TB is due to transmission and not acquisition, a change in dogma regarding DR acquisition versus strain transmission is being called for [18]. It is critical too to understand that MDR-TB strains are as equally transmissible as drug-susceptible [19]. As Van Rie and Warren [18] further highlight, transmission of MDR-TB drives the epidemic in high-burden settings, and the TB epidemic can be contained by implementation of active case finding with rapid TB detection and drug resistance detection (at least for rifampicin) for all people with signs and symptoms of TB as the greatest number of MDR-TB cases will be among newly diagnosed TB cases [20▪].

TB remains one of the top 10 leading causes of death in children (WHO reports 170 000 deaths in 2015) and of concern is ∼57% children diagnosed with and treated for TB are HIV-infected in high-burden countries [21▪]. In South Africa it is estimated that children younger than 14 years account for 15–20% total TB burden. Diagnosing childhood TB is challenging as the most appropriate specimen to collect depends on age and clinical presentation. Specimens (especially sputum) are also paucibacillary [22], often of poor quality and quantity. A study of ART programs showed sputum smear microscopy and chest X-ray where available was only used in 86% and 52% of TB diagnoses, respectively [23▪]. Although WHO recommends Xpert MTB/RIF testing for children, Xpert MTB/RIF (where available) was only used in 8% and culture in 17% cases [24]. Furthermore, a study in Johannesburg showed 67% sputum collected from children younger than 14 years (median age 24 months) was below the required volume for Xpert MTB/RIF testing [25]. Overall, this highlights the need for strengthening the capacity for diagnosis, proactive screening for TB and MDR-TB in inpatient settings and the community [26]. In addition, HIV co-infected individuals require ART scale up, continuous monitoring, and collaboration between HIV and TB control programs. This will require task shifting [27] and integration of services for adults and children [23▪]. Screening pregnant women for HIV and TB may also improve access to care [28], and healthcare providers are encouraged to increase competency in linkage to care and integration, which will also enhance prevention in young infants and children [23▪].

The poor sensitivity of smear microscopy (38–69%) in HIV-infected individuals has been well described [29], where the presence of 5000–10 000 bacteria are required for visual detection [30], compared with liquid culture, which remains the gold standard at the lowest limit of detection of MTB of ∼10–100 cfu/ml [30,31]. The contrast, however, is a poor (yet affordable and easy to perform in a standard laboratory) test that yields a result in less than 24 h compared with a sensitive test that could take longer than 6 weeks [if drug susceptibility testing (DST) is included] and becomes less clinically relevant, and requires a biosafety laboratory environment and skilled operation. The desired diagnostic needs to be fast, accurate, affordable, and capable of being performed in the household or community, and on a range of specimen types (sputum, urine, stool), simultaneously with HIV diagnosis and monitoring. This is becoming a reality through molecular technology specifically for HIV co-infected individuals [11▪]. WHO recommends that the Xpert MTB/RIF assay is used as the initial diagnostic for TB for all adults and children with signs and symptoms of TB and especially where the burden of HIV is high and high rates of DR-TB are suspected. Another desirable component becoming available on several of the molecular instruments is the ability to perform more than one type of test on the same platform. Xpert HIV-1 Qualitative test for early infant diagnosis is now prequalified for use by WHO and provides an opportunity for integration of TB and HIV diagnosis, and extend testing services closer to POC. This would also be the case for the Xpert HIV-1 Quantitative test for HIV viral load monitoring [32], once approved, to improve patient access and impact on the 90/90/90 goals.

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The Xpert MTB/RIF (Cepheid) is a cartridge-based molecular test that is fully automated only requiring addition of reagent buffer to liquefy and inactivate any TB bacilli present in a clinical specimen [33–35]. Results are reported within 2 h, and include the detection of rifampicin resistance conferring mutations (RIF). The limit of detection of MTB complex (MTBC) with Xpert MTB/RIF on clinical specimens is 150 cfu/ml [34]. Figure 1 simplistically outlines the overall performance of Xpert MTB/RIF with specific reference to performance among HIV-infected individuals.

In summary, Xpert MTB/RIF performs well on adult respiratory specimens compared with culture reference, but less well in sputum smear-negative specimens. This is similar among childhood specimens, with somewhat increased performance of Xpert MTB/RIF in HIV-infected children. Performance of Xpert MTB/RIF is good on lymph node and other tissue specimens and cerebrospinal fluid, with greater sensitivity in HIV-infected individuals’ lymph node tissue. Xpert MTB/RIF is known to have poorer performance in pleural fluid irrespective of smear status.

The impact of Xpert MTB/RIF on patient care has, to date, been described in 33 studies from 22 countries, including 10 sub-Saharan African countries. Of these, 20 studies (refer to Table 1) discuss the impact of the Xpert MTB/RIF in HIV-infected populations: Xpert MTB/RIF increases detection of TB and dramatically reduces treatment initiation times for DR-TB. The placement of GeneXpert instruments at treatment facilities and at POC facilities results in shortened treatment initiation times than centralized testing. Changes in empiric treatment practice however varies, and overall impact on mortality has not been shown, and in fact studies undertaken to measure it were underpowered [65▪▪].

The Foundation for Innovative New Diagnostics and collaborating partners is currently undertaking a multicenter study in eight countries of the much anticipated Xpert Ultra (Cepheid) test, which promises to reduce the limit of detection of MTB in sputum to the realm of liquid culture (10–100 cfu/ml) [66]. Much has been learnt from the implementation of Xpert MTB/RIF as summarized in Table 2 and includes [67,68] cost and forecast models [69,70], program interfacing [71], socioeconomic trends [72], and quality assessment [73]. Additional innovations, being reported for the first time for possible TB control (currently, developed for GeneXpert instruments), are remote connectivity [74,75▪]. Cepheid's C360 is a web-based software that remotely connects all instruments and centrally collects result run information. This, together with a laboratory information system allows for central program and laboratory monitoring on test and module performance (potentially in real time), with its further application in TB control [76].

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Line probe assays

GenoType MTBDRplus, (Hain LifeSciences) was the first commercial line probe assay (LPA) recommended for use by WHO in 2008 [77]. It remains the most widely studied LPA. Further data have since been published on the use of LPAs and newer versions of LPA technology have since been developed: Hain Genotype MTBDRplus version 2 [78–81]; and the Nipro NTM+MDRTB detection kit developed by the Nipro Corporation (Japan) DR-TB [82▪]. These newer LPAs aim to improve sensitivity for MTBC detection and to simultaneously detect resistance to rifampicin and isoniazid. These LPAs are recommended for use in regional or centralized high-throughput laboratories for the rapid detection of rifampicin and isoniazid resistance in sputum-smear-positive specimens and from mycobacterial cultures. The tests are not recommended for use on sputum smear-negative specimens [83]. LPA require laboratory-trained personnel skilled in PCR, to perform the assay in well managed laboratories. LPA identify drug resistance through manual extraction of MTBC DNA and PCR amplification of the resistance hotspot regions in the rpoB, inhA, and katG genes. The impact of expanded testing using the LPA in South Africa resulted in a substantial increase in the proportion of new cases identified as MDR-TB, and although the time to treatment was reduced, it still took 2 months [84].

A second-line line probe assay (SL-LPA) for the detection of resistance to second-line anti-TB drugs – MTBDRsl assay (Hain LifeSciences), incorporates probes to detect mutations within genes (gyrA and rrs) for version 1.0 [85] and, in addition, gyrB and the eis promoter for version 2.0 [86–88], which are associated with resistance to the class of fluoroquinolones or the second-line injectable agents. The presence of mutations in these regions does not necessarily imply resistance to all the drugs within that class. Although specific mutations within these regions may be associated with different levels of resistance (i.e., different minimum inhibitory concentrations) to each drug within these classes, the extent of cross-resistance is not completely understood. WHO recommends the use of SL-LPA as an initial test to detect resistance directly on sputum from patients diagnosed with resistance to RIF or MDR-TB [88].

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Loop-mediated isothermal amplification assay

Loop-mediated isothermal amplification (LAMP) is a unique, temperature-independent technique for amplifying DNA that is simple to use, providing a visual display that is easy to read. TB-LAMP does not require sophisticated instrumentation and can be used at a peripheral health center level, given biosafety requirements similar to microscopy. A meta-analysis of 10 studies reported a sensitivity of 80% (78–83%) and specificity of 96% (95–97%) to diagnose pulmonary TB [89]. One of the datasets included in the meta-analysis stood out in terms of its findings: the study conducted in Malawi among individuals with cough (44% HIV positivity) reported a sensitivity of 65% (48–79%), specificity of 100% (98–100%), similar performance (P = 0.132) to Xpert, but lower performance compared with concentrated fluorescent smear microscopy with duplicate reading (P = 0.02) [90]. TB-LAMP, however, provides better results than sputum smear microscopy, detecting 15% more patients with pulmonary TB, if performed in all persons presenting with signs and symptoms. If used as an add-on test after microscopy has been performed, more than 40% increase in TB cases were detected among those with smear-negative results compared with other rapid tests that have been recommended by WHO in recent years.

TB-LAMP only detects TB [therefore only suitable for testing of patients at low risk of multidrug-resistant TB (MDR-TB)], and therefore should not replace Xpert MTB/RIF, which simultaneously detects TB and rifampicin resistance. TB-LAMP may be a plausible alternative in settings with low prevalence of HIV and low prevalence of drug resistance, especially where environmental conditions (unstable electricity, temperature, humidity, excessive dust [91]) and possible cost limit access to implementation of Xpert MTB/RIF. The test does not detect drug resistance. It can be performed outside of conventional laboratories but requires training of healthcare staff, similar to the training needed for performing sputum smear microscopy [92].

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Lipoarabinomannan assay

LF-LAM is a lateral flow assay requiring 60 μl urine and visual reading of band intensity compared with the manufacturer's supplied reference line on a card to report a result within 25 min, making it applicable to identify active TB at POC, but will require a good quality framework to ensure accuracy. WHO only recommends its use in HIV-positive hospitalized individuals, whose CD4 cell count is < 100 cells/μl [93], and for seriously ill persons irrespective of their CD4 cell count [94]. A meta-analysis in this population reports the LF-LAM used to diagnose TB with a pooled sensitivity of 56% (41–70%) and pooled specificity 90% (81–95%). Combining Xpert MTB/RIF testing of urine with urine LF-LAM improved overall TB diagnostic sensitivity to 75% (61–87%) and specificity to 93% (81–97%) with the added advantage of Xpert MTB/RIF simultaneously detecting susceptibility to RIF [95▪]. Peter et al.[96▪▪] showed that POC LF-LAM reduced mortality at 8 weeks in hospitalized patients.

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Abbott RealTime MTB and MTB-RIF/INH assays

The m2000 (Abbott Molecular, Chicago, Illinois, USA) platform is widely used for centralized HIV viral load testing. The platforms’ flexible, automated extraction and closed real-time PCR systems (testing 93 specimens/8 h day), lend itself to other molecular assays such as the Abbott RealTime MTB (amplification and detection of IS6110 and protein antigen B) and MTB-RIF/INH (similar region detection to those reported by MTBDRplus) assays (Abbott Molecular) for qualitative detection of MTBC [97,98▪]. Similar performance to Xpert MTB/RIF in high TB and HIV settings has been noted (Scott LE, unpublished data), and with the added advantage of reporting RIF and INH susceptibility simultaneously [99▪]. The m2000 platform has full connectivity functionality, and training and quality management systems are in place in many HIV and TB high-burden countries. Placement for TB testing would be similar to HIV viral load testing, therefore lending itself to integration of HIV and TB laboratory services. This principle of platform integration is not new to molecular testing services and is now also being investigated by Cepheid to provide HIV viral load testing (Xpert HIV-1) on their GeneXpert platform. Therefore, integration of HIV and TB services is not only patient centric now but platform and testing service centric too.

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The development pipeline for diagnosing active (including drug resistant) TB appears rich from a molecular diagnostics perspective [100], including a large focus on whole genome sequencing and next-generation sequencing [101,102▪]. The aim is to improve sensitivity, speed, ease of use, ability to discriminate TB from other inflammatory or autoimmune diseases and identify subclinical TB in HIV infection [103]. However, very few candidate assays are in the R&D pipeline for true point-of-care tests in rapid diagnostic test format, with disappointing results from biomarker research [104]. There are molecular platforms in the pipeline that will get us closer to patients, but it remains unclear whether test implementation would be cost-effective [105▪,106▪]. GeneXpert Omni (Cepheid) may address the criticism of GeneXpert, which requires a laboratory infrastructure (e.g., because of the need for continuous and stable electrical supply) and has limited utility for community testing. The anticipated launch of GeneXpert Omni in 2017 does not leave much time to address issues for implementers of regulatory assurance, quality control and maintenance, staff resources, logistic support, and cost [107]. Mobile phone and thus platform connectivity may be a particularly challenging field for countries to address. Future evaluation studies will also require broader design to assess impact of TB diagnostics and more attention paid to analyses in methodology studies [108]. This too will apply to evaluation of high throughput centralized testing platforms (e.g., m2000) that will require flexibility around the informed consent process required for trials to match the platforms daily testing throughput.

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The last few years have seen improvements in the integration of TB and HIV diagnosis and care. New polyvalent platforms should ease integration from a diagnostic standpoint. The major gaps today are true POC testing for early and active case detection and universal rapid DST. Although new tests have transformed TB control and acted as catalyst for change, impact is lower than anticipated. The linkage to care must be optimized to fully capitalize on the potential of new TB diagnostics [109]. Innovation and support is needed not only in the form of new tests, but more importantly for the strengthening of healthcare and delivery services to improve the cascade of care. Only with a comprehensive approach will we be able to achieve the sustainable development goals.

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The authors thank Lara Noble for her assistance with the literature review, referencing and editing.

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Financial support and sponsorship

L.S. is supported by funding received from the South African Medical Research Council with funds received from the South African National Department of Health, and the UK Medical Research Council, with funds received from the UK Government's Newton Fund under the UK/South Africa Newton Fund #015NEWTON TB, and by the National Institute of Allergy and Infectious Diseases of the National Institutes of Health under Award Number R21AI116015. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

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Conflicts of interest

The authors have no conflicts of interest.

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Papers of particular interest, published within the annual period of review, have been highlighted as:

▪ of special interest

▪▪ of outstanding interest

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45. Lusiba JK, Nakiyingi L, Kirenga BJ, et al. Evaluation of Cepheid's Xpert MTB/Rif test on pleural fluid in the diagnosis of pleural tuberculosis in a high prevalence HIV/TB setting. PLoS One 2014; 9:e102702.
46. Lawn SD, Kerkhoff AD, Burton R, et al. Rapid microbiological screening for tuberculosis in HIV-positive patients on the first day of acute hospital admission by systematic testing of urine samples using Xpert MTB/RIF: a prospective cohort in South Africa. BMC Med 2015; 13:192.
47. Manabe YC, Zawedde-Muyanja S, Burnett SM, et al. Rapid improvement in passive tuberculosis case detection and tuberculosis treatment outcomes after implementation of a bundled laboratory diagnostic and on-site training intervention targeting mid-level providers. Open Forum Infect Dis 2015; 2:ofv030.
48. Al-Darraji HA, Abd Razak H, Ng KP, et al. The diagnostic performance of a single GeneXpert MTB/RIF assay in an intensified tuberculosis case finding survey among HIV-infected prisoners in Malaysia. PLoS One 2013; 8:e73717.
49. Yoon C, Cattamanchi A, Davis JL, et al. Impact of Xpert MTB/RIF testing on tuberculosis management and outcomes in hospitalized patients in Uganda. PLoS One 2012; 7:e48599.
50. Lorent N, Kong C, Kim T, et al. Systematic screening for drug-resistant tuberculosis with Xpert((R)) MTB/RIF in a referral hospital in Cambodia. Int J Tuberc Lung Dis 2015; 19:1528–1535.
51. van Kampen SC, Tursynbayeva A, Koptleuova A, et al. Effect of introducing Xpert MTB/RIF to test and treat individuals at risk of multidrug-resistant tuberculosis in Kazakhstan: a prospective cohort study. PLoS One 2015; 10:e0132514.
52▪. Cox HS, Daniels JF, Muller O, et al. Impact of decentralized care and the Xpert MTB/RIF Test on rifampicin-resistant tuberculosis treatment initiation in Khayelitsha, South Africa. Open Forum Infect Dis 2015; 2:ofv014.

Placement of Xpert MTB/RIF impacts on patient care; decentralization decreases time to treatment.

53. Moyenga I, Roggi A, Sulis G, et al. The impact of Xpert(R) MTB/RIF depends on service coordination: experience in Burkina Faso. Int J Tuberc Lung Dis 2015; 19:285–287.
54. Auld SC, Moore BK, Kyle RP, et al. Mixed impact of Xpert((R)) MTB/RIF on tuberculosis diagnosis in Cambodia. Public health action 2016; 6:129–135.
55▪. Hanrahan CF, Clouse K, Bassett J, et al. The patient impact of point-of-care vs. laboratory placement of Xpert((R)) MTB/RIF. Int J Tuberc Lung Dis 2015; 19:811–816.

Empiric treatment is common when Xpert MTB/RIF is not available at POC.

56. Van Den Handel T, Hampton KH, Sanne I, et al. The impact of Xpert((R)) MTB/RIF in sparsely populated rural settings. Int J Tuberc Lung Dis 2015; 19:392–398.
57. Balcha TT, Sturegard E, Winqvist N, et al. Intensified tuberculosis case-finding in HIV-positive adults managed at Ethiopian health centers: diagnostic yield of Xpert MTB/RIF compared with smear microscopy and liquid culture. PLoS One 2014; 9:e85478.
58. Cox HS, Mbhele S, Mohess N, et al. Impact of Xpert MTB/RIF for TB diagnosis in a primary care clinic with high TB and HIV prevalence in South Africa: a pragmatic randomised trial. PLoS Med 2014; 11:e1001760.
59. Durovni B, Saraceni V, van den Hof S, et al. Impact of replacing smear microscopy with Xpert MTB/RIF for diagnosing tuberculosis in Brazil: a stepped-wedge cluster-randomized trial. PLoS Med 2014; 11:e1001766.
60. Theron G, Peter J, Dowdy D, et al. Do high rates of empirical treatment undermine the potential effect of new diagnostic tests for tuberculosis in high-burden settings? Lancet Infect Dis 2014; 14:527–532.
61▪. Mupfumi L, Makamure B, Chirehwa M, et al. Impact of Xpert MTB/RIF on antiretroviral therapy-associated tuberculosis and mortality: a pragmatic randomized controlled trial. Open Forum Infect Dis 2014; 1:ofu038.

Centralized Xpert vs. FM does not impact on patient mortality and empirical treatment is still common.

62▪. Churchyard GJ, Stevens WS, Mametja LD, et al. Xpert MTB/RIF versus sputum microscopy as the initial diagnostic test for tuberculosis: a cluster-randomised trial embedded in South African roll-out of Xpert MTB/RIF. Lancet Global Health 2015; 3:e450–e457.

Xpert does not decrease patient mortality, indicating that linkage to care must be improved.

63. Hanrahan CF, Selibas K, Deery CB, et al. Time to treatment and patient outcomes among TB suspects screened by a single point-of-care xpert MTB/RIF at a primary care clinic in Johannesburg, South Africa. PLoS One 2013; 8:e65421.
64. Menzies NA, Cohen T, Lin HH, et al. Population health impact and cost-effectiveness of tuberculosis diagnosis with Xpert MTB/RIF: a dynamic simulation and economic evaluation. PLoS Med 2012; 9:e1001347.
65▪▪. Auld AF, Fielding KL, Gupta-Wright A, Lawn SD. Xpert MTB/RIF- why the lack of morbidity and mortality impact in intervention trials? Trans R Soc Trop Med Hyg 2016; 110:432–444.

The possible reasons for the low impact of Xpert MTB/RIF on mortality and morbidity is discussed, with recommendations for future trials to inform Xpert MTB/RIF use in resource-limited settings.

66. Jones M, Chakravorty S, Simmons M, et al. MTB-RIF Ultra – design and analytical performance of a second generation GeneXpert assay (Poster 0475). ECCMID 2016; 10 April 2016; Amsterdam, The Netherlands.
67. Qin ZZ, Pai M, Van Gemert W, et al. How is Xpert MTB/RIF being implemented in 22 high tuberculosis burden countries? Eur Respir J 2015; 45:549–554.
68. Raizada N, Sachdeva KS, Sreenivas A, et al. Catching the missing million: experiences in enhancing TB & DR-TB detection by providing upfront Xpert MTB/RIF testing for people living with HIV in India. PLoS One 2015; 10:e0116721.
69. Schnippel K, Meyer-Rath G, Long L, et al. Diagnosing Xpert MTB/RIF negative TB: impact and cost of alternative algorithms for South Africa. South Afr Med J 2013; 103:101–106.
70. South African National AIDS Council. South African HIV and TB Investment Case Phase 1 Reference Report. South Africa: South African National AIDS Council; 2016. Available from: http://sanac.org.za/wp-content/uploads/2016/03/1603-Investment-Case-Report-LowRes-18-Mar.pdf. Accessed 20 December 2016
71. Houben RM, Lalli M, Sumner T, et al. TIME Impact - a new user-friendly tuberculosis (TB) model to inform TB policy decisions. BMC Med 2016; 14:56.
72. Uplekar M. Implementing the end TB strategy: well begun will be half done. Indian J Tuberc 2015; 62:61–63.
73. Scott L, Albert H, Gilpin C, et al. Multicenter feasibility study to assess external quality assessment panels for Xpert MTB/RIF assay in South Africa. J Clin Microbiol 2014; 52:2493–2499.
74. Theron G, Jenkins HE, Cobelens F, et al. Data for action: collection and use of local data to end tuberculosis. Lancet 2015; 386:2324–2333.
75▪. Andre E, Isaacs C, Affolabi D, et al. Connectivity of diagnostic technologies: improving surveillance and accelerating tuberculosis elimination. Int J Tuberc Lung Dis 2016; 20:999–1003.

Connectivity of the GeneXpert instruments to a central database allows for national TB surveillance.

76. Stevens WS, Cunningham B, Cassim N. Persing DH, et al. Cloud-based surveillance, connectivity, and distribution of the GeneXpert analyzers for diagnosis of tuberculosis (TB) and multiple-drug-resistant TB in South Africa. Molecular microbiology: diagnostic principles and practice ASM Press, 3rd ed.Washington DC, USA:2016.
77. World Health Organisation. Molecular line probe assays for rapid screening of patients at risk of multidrug-resistant tuberculosis (MDR-TB). Geneva, Switzerland: World Health Organisation; 2008.
78. Barnard M, Gey van Pittius NC, van Helden PD, et al. The diagnostic performance of the GenoType MTBDRplus version 2 line probe assay is equivalent to that of the Xpert MTB/RIF assay. J Clin Microbiol 2012; 50:3712–3716.
79. Crudu V, Stratan E, Romancenco E, et al. First evaluation of an improved assay for molecular genetic detection of tuberculosis as well as rifampin and isoniazid resistances. J Clin Microbiol 2012; 50:1264–1269.
80. Matabane MM, Ismail F, Strydom KA, et al. Performance evaluation of three commercial molecular assays for the detection of Mycobacterium tuberculosis from clinical specimens in a high TB-HIV-burden setting. BMC Infect Dis 2015; 15:508.
81. Bai Y, Wang Y, Shao C, et al. GenoType MTBDRplus assay for rapid detection of multidrug resistance in Mycobacterium tuberculosis: a meta-analysis. PLoS One 2016; 11:e0150321.
82▪. Nathavitharana RR, Hillemann D, Schumacher SG, et al. Multicenter noninferiority evaluation of Hain GenoType MTBDRplus version 2 and Nipro NTM+MDRTB Line Probe Assays for Detection of Rifampin and Isoniazid Resistance. J Clin Microbiol 2016; 54:1624–1630.

New evidence for the use of LPA for first line resistance testing.

83. World Health Organisation. The use of molecular line probe assays for the detection of resistance to isoniazid and rifampicin. 2016; Geneva, Switzerland: World Health Organisation, Available from: http://apps.who.int/iris/bitstream/10665/250586/1/9789241511261-eng.pdf. Accessed 20 December 2016.
84. Hanrahan CF, Dorman SE, Erasmus L, et al. The impact of expanded testing for multidrug resistant tuberculosis using genotype [correction of genotype] MTBDRplus in South Africa: an observational cohort study. PLoS one 2012; 7:e49898.
85. Theron G, Peter J, Richardson M, et al. The diagnostic accuracy of the GenoType((R)) MTBDRsl assay for the detection of resistance to second-line antituberculosis drugs. Cochrane Database Syst Rev 2014; 10: CD010705.
86. Brossier F, Guindo D, Pham A, et al. Performance of the new version (v2.0) of the GenoType MTBDRsl test for detection of resistance to second-line drugs in multidrug-resistant Mycobacterium tuberculosis complex strains. J Clin Microbiol 2016; 54:1573–1580.
87. Tagliani E, Cabibbe AM, Miotto P, et al. Diagnostic performance of the new version (v2.0) of GenoType MTBDRsl assay for detection of resistance to fluoroquinolones and second-line injectable drugs: a multicenter study. J Clin Microbiol 2015; 53:2961–2969.
88. World Health Organisation. The use of molecular line probe assays for the detection of resistance to second-line antituberculosis drugs. Geneva, Switzerland: World Health Organisation; 2016.
89. Yuan LY, Li Y, Wang M, et al. Rapid and effective diagnosis of pulmonary tuberculosis with novel and sensitive loop-mediated isothermal amplification (LAMP) assay in clinical samples: a meta-analysis. J Infect Chemother 2014; 20:86–92.
90. Nliwasa M, MacPherson P, Chisala P, et al. The sensitivity and specificity of loop-mediated isothermal amplification (LAMP) assay for tuberculosis diagnosis in adults with chronic cough in Malawi. PLoS One 2016; 11:e0155101.
91. World Health Organisation. The use of loop-mediated isothermal amplification (TB-LAMP) for the diagnosis of pulmonary tuberculosis: policy guidance. Geneva, Switzerland: World Health Organisation; 2016. Available from: http://apps.who.int/iris/bitstream/10665/249154/1/9789241511186-eng.pdf. Accessed 20 December 2016
92. Gray CM, Katamba A, Narang P, et al. Feasibility and operational performance of tuberculosis detection by loop-mediated isothermal amplification platform in decentralized settings: results from a multicenter study. J Clin Microbiol 2016; 54:1984–1991.
93. World Health Organisation. The use of lateral flow urine lipoarabinomannan assay (LF-LAM) for the diagnosis and screening of active tuberculosis in people living with HIV policy guidance. Geneva, Switzerland: World Health Organisation Press; 2015.
94. Lawn SD. Point-of-care detection of lipoarabinomannan (LAM) in urine for diagnosis of HIV-associated tuberculosis: a state of the art review. BMC Infect Dis 2012; 12:103.
95▪. Shah M, Hanrahan C, Wang ZY, et al. Lateral flow urine lipoarabinomannan assay for detecting active tuberculosis in HIV-positive adults. Cochrane Database Syst Rev 2016; 5:CD011420.

Systematic review regarding the use of LAM in HIV-infected populations.

96▪▪. Peter JG, Zijenah LS, Chanda D, et al. Effect on mortality of point-of-care, urine-based lipoarabinomannan testing to guide tuberculosis treatment initiation in HIV-positive hospital inpatients: a pragmatic, parallel-group, multicountry, open-label, randomised controlled trial. Lancet 2016; 387:1187–1197.

While LAM has little impact on outpatients with suspected TB, it decreases mortality at 8 weeks in hospitalized patients with severe immune suppression.

97. Chen JH, She KK, Kwong TC, et al. Performance of the new automated Abbott RealTime MTB assay for rapid detection of Mycobacterium tuberculosis complex in respiratory specimens. Eur J Clin Microbiol Infect Dis 2015; 34:1827–1832.
98▪. Kostera J, Leckie G, Tang N, et al. Analytical and clinical performance characteristics of the Abbott RealTime MTB RIF/INH Resistance, an assay for the detection of rifampicin and isoniazid resistant Mycobacterium tuberculosis in pulmonary specimens. Tuberculosis 2016; 101:137–143.

First publication involving the Abbott MTB RIF/INH assay.

99▪. Hofmann-Thiel S, Molodtsov N, Antonenka U, Hoffmann H. Evaluation of the Abbott RealTime MTB and RealTime MTB INH/RIF assays for direct detection of Mycobacterium tuberculosis complex and resistance markers in respiratory and extra-pulmonary specimens. J Clin Microbiol 2016; 54:3022–3027.

First independent publication on the Abbott MTB RIF/INH assay.

100. Bates M, Zumla A. The development, evaluation and performance of molecular diagnostics for detection of Mycobacterium tuberculosis. Expert Review Molec Diagn 2016; 16:307–322.
101. Abubakar I, Lipman M, McHugh TD, Fletcher H. Uniting to end the TB epidemic: advances in disease control from prevention to better diagnosis and treatment. BMC Med 2016; 14:47.
102▪. Witney AA, Cosgrove CA, Arnold A, et al. Clinical use of whole genome sequencing for Mycobacterium tuberculosis. BMC Med 2016; 14:46.

The clinical potential of WGS of TB in the future will impact patient care.

103. Haas CT, Roe JK, Pollara G, et al. Diagnostic ’omics for active tuberculosis. BMC Med 2016; 14:37.
104. Pai M, Behr M, Dowdy D, et al. Tuberculosis. Nat Rev 2016. 216076.
105▪. Houben RM, Menzies NA, Sumner T, et al. Feasibility of achieving the 2025 WHO global tuberculosis targets in South Africa, China, and India: a combined analysis of 11 mathematical models. Lancet Global Health 2016; 4:e806–e815.

Scale-up of multiple interventions is necessary to meet the 2025 global TB targets.

106▪. Menzies NA, Gomez GB, Bozzani F, et al. Cost-effectiveness and resource implications of aggressive action on tuberculosis in China, India, and South Africa: a combined analysis of nine models. Lancet Global Health 2016; 4:e816–e826.

Cost-effectiveness strategies and resource requirements must be optimized to each country.

107. Drain PK, Garrett NJ. The arrival of a true point-of-care molecular assay-ready for global implementation? Lancet Global Health 2015; 3:e663–e664.
108. Schumacher SG, Sohn H, Qin ZZ, et al. Impact of molecular diagnostics for tuberculosis on patient-important outcomes: a systematic review of study methodologies. PLoS One 2016; 11:e0151073.
109. Furin J, Akugizibwe P, Ditiu L, et al. No one with HIV should die from tuberculosis. Lancet 2015; 386:e48–e50.

drug resistance; HIV/TB care; implementation; molecular TB diagnostics; TB control; Xpert MTB/RIF

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