Molecular Drug Susceptibility Testing Research Articles

Additional Drug Resistance Patterns among Multidrug-Resistant Tuberculosis Patients in Korea: Implications for Regimen Design.

Detailed information on additional drug resistance patterns of multidrug-resistant tuberculosis (MDR-TB) is essential to build an effective treatment regimen; however, such data are scarce in Korea. We retrospectively analyzed the results of phenotypic drug susceptibility testing (DST) of culture confirmed-TB patients from January 2010 to December 2014 in 7 university hospitals in Korea. MDR-TB was identified among 6.8% (n = 378) of 5,599 isolates. A total of 57.1% (n = 216) of the MDR-TB patients had never been treated for TB. Strains from MDR-TB patients showed additional resistance to pyrazinamide (PZA) (35.7%), any second-line injectable drug (19.3%), and any fluoroquinolone (26.2%). Extensively drug resistant TB comprised 12.4% (n = 47) of the MDR-TB patients. Of 378 MDR-TB patients, 50.3% (n = 190) were eligible for the shorter MDR-TB regimen, and 50.0% (n = 189) were fully susceptible to the 5 drugs comprising the standard conventional regimen (PZA, kanamycin, ofloxoacin, prothionamide, and cycloserine). In conclusion, the proportion of new patients and the levels of additional drug resistance were high in MDR-TB patients. Considering the high levels of drug resistance, the shorter MDR-TB treatment regimen may not be feasible; instead, an individually tailored regimen based on the results of molecular and phenotypic DST may be more appropriate in MDR-TB patients in Korea.

Characterization of rpoB Gene Mutations in Clinical Isolates of Mycobacterium TB

SESSION TITLE: Tuberculosis SESSION TYPE: Original Investigation Poster PRESENTED ON: Wednesday, October 26, 2016 at 01:30 PM - 02:30 PM PURPOSE: 1) to evaluate the phenotypic characteristics of the specific mutation site, 2) to compare the performance of phenotypic and genotypic (molecular) susceptibility test for rifampin (RIF), 3) to define mutation sites which are correlated with RIF resistance levels and rifabutin (RBU) resistance. METHODS: Conventional phenotypic drug susceptibility testing (DST) in Middlebrook 7H10 medium, rpoB gene sequencing for 1kb around RIF resistance determining region (RRDR), and molecular DST (GenoType® MTBDRplus assay, MTBDRplus) were performed for 674 clinical isolates of M. tuberculosis which were obtained from the patients who had failed with first-line therapy. RESULTS: Based on conventional phenotypic DST 502 (74.5%) isolates were resistant to RIF. The rpoB gene sequencing showed 491 isolates possessed 517 mutation sites in the RRDR. The most frequent mutated site was codon 531 (185/517, 35.7%). Among the RIF-resistant isolates having the mutations at codon 533, most of the isolates (89%) showed minimal inhibitory concentration (MIC) lower than 16 ug/ml, suggesting that the mutation at codon 533 may not be associated with a high level of RIF resistance. Moreover, mutations at codon 533 (44/64, 68.8%) were frequently found in RIF-resistant and RBU-susceptible isolates. Sensitivity and specificity of the the rpoB gene sequencing and MTBDRplus were 93.4%, 91.4% and 91.4%, 95.1% respectively. CONCLUSIONS: Mutations at codon 533 were frequently found in clinical isolates of M. tuberculosis which were RIF-susceptible, low level resistant or RBU-susceptible. CLINICAL IMPLICATIONS: Physicians can expect phenotypic RIF- susceptible or RBU-susceptible despite of rpoB gene mutation if the mutation site is codon 533. DISCLOSURE: The following authors have nothing to disclose: Yangjin Jegal, Jihee Jung, Nackmoon Sung No Product/Research Disclosure Information

Executive Summary: Official American Thoracic Society/Centers for Disease Control and Prevention/Infectious Diseases Society of America Clinical Practice Guidelines: Treatment of Drug-Susceptible Tuberculosis.

The American Thoracic Society, Centers for Disease Control and Prevention, and Infectious Diseases Society of America jointly sponsored the development of this guideline for the treatment of drug-susceptible tuberculosis, which is also endorsed by the European Respiratory Society and the US National Tuberculosis Controllers Association. Representatives from the American Academy of Pediatrics, the Canadian Thoracic Society, the International Union Against Tuberculosis and Lung Disease, and the World Health Organization also participated in the development of the guideline. This guideline provides recommendations on the clinical and public health management of tuberculosis in children and adults in settings in which mycobacterial cultures, molecular and phenotypic drug susceptibility tests, and radiographic studies, among other diagnostic tools, are available on a routine basis. For all recommendations, literature reviews were performed, followed by discussion by an expert committee according to the Grading of Recommendations, Assessment, Development and Evaluation methodology. Given the public health implications of prompt diagnosis and effective management of tuberculosis, empiric multidrug treatment is initiated in almost all situations in which active tuberculosis is suspected. Additional characteristics such as presence of comorbidities, severity of disease, and response to treatment influence management decisions. Specific recommendations on the use of case management strategies (including directly observed therapy), regimen and dosing selection in adults and children (daily vs intermittent), treatment of tuberculosis in the presence of HIV infection (duration of tuberculosis treatment and timing of initiation of antiretroviral therapy), as well as treatment of extrapulmonary disease (central nervous system, pericardial among other sites) are provided. The development of more potent and better-tolerated drug regimens, optimization of drug exposure for the component drugs, optimal management of tuberculosis in special populations, identification of accurate biomarkers of treatment effect, and the assessment of new strategies for implementing regimens in the field remain key priority areas for research. See the full-text online version of the document for detailed discussion of the management of tuberculosis and recommendations for practice.

Transmission of Drug-Resistant Leprosy in Guinea-Conakry Detected Using Molecular Epidemiological Approaches.

Molecular drug susceptibility testing was performed on skin biopsies from 24 leprosy patients from Guinea-Conakry for the first time. We identified primary drug resistance in 4 cases and a dapsone-resistant cluster caused by the same strain. Primary transmission of drug-resistant Mycobacterium leprae, including a rifampicin-resistant strain, is reported.

Official American Thoracic Society/Centers for Disease Control and Prevention/Infectious Diseases Society of America Clinical Practice Guidelines: Treatment of Drug-Susceptible Tuberculosis.

The American Thoracic Society, Centers for Disease Control and Prevention, and Infectious Diseases Society of America jointly sponsored the development of this guideline for the treatment of drug-susceptible tuberculosis, which is also endorsed by the European Respiratory Society and the US National Tuberculosis Controllers Association. Representatives from the American Academy of Pediatrics, the Canadian Thoracic Society, the International Union Against Tuberculosis and Lung Disease, and the World Health Organization also participated in the development of the guideline. This guideline provides recommendations on the clinical and public health management of tuberculosis in children and adults in settings in which mycobacterial cultures, molecular and phenotypic drug susceptibility tests, and radiographic studies, among other diagnostic tools, are available on a routine basis. For all recommendations, literature reviews were performed, followed by discussion by an expert committee according to the Grading of Recommendations, Assessment, Development and Evaluation methodology. Given the public health implications of prompt diagnosis and effective management of tuberculosis, empiric multidrug treatment is initiated in almost all situations in which active tuberculosis is suspected. Additional characteristics such as presence of comorbidities, severity of disease, and response to treatment influence management decisions. Specific recommendations on the use of case management strategies (including directly observed therapy), regimen and dosing selection in adults and children (daily vs intermittent), treatment of tuberculosis in the presence of HIV infection (duration of tuberculosis treatment and timing of initiation of antiretroviral therapy), as well as treatment of extrapulmonary disease (central nervous system, pericardial among other sites) are provided. The development of more potent and better-tolerated drug regimens, optimization of drug exposure for the component drugs, optimal management of tuberculosis in special populations, identification of accurate biomarkers of treatment effect, and the assessment of new strategies for implementing regimens in the field remain key priority areas for research. See the full-text online version of the document for detailed discussion of the management of tuberculosis and recommendations for practice.

Detection of katG and inhA mutations to guide isoniazid and ethionamide use for drug-resistant tuberculosis.

Depending on the presence of mutations that determine isoniazid (INH) susceptibility (katG and inhA), Mycobacterium tuberculosis may be susceptible to high doses of INH or ethionamide (ETH). To describe the INH resistance profile and association of katG mutation with previous INH treatment and level of drug resistance based on rapid molecular drug susceptibility testing (DST) in southern Brazil and central Mozambique. Descriptive study of 311 isolates from Ribeirão Preto, São Paulo, Brazil (2011-2014) and 155 isolates from Beira, Mozambique (2014-2015). Drug resistance patterns and specific gene mutations were determined using GenoType(®) MTBDRplus. katG gene mutations were detected in 12/22 (54.5%) Brazilian and 32/38 (84.2%) Mozambican isolates. inhA mutations were observed in 9/22 (40.9%) isolates in Brazil and in 4/38 (10.5%) in Mozambique. Both katG and inhA mutations were detected in respectively 1/22 (5%) and 2/38 (5.2%). The difference in the frequency of katG mutations in Brazil and Mozambique was statistically significant (P = 0.04). katG mutations were present in 68.8% (33/48) of patients previously treated with INH and 31.2% (15/48) of patients without previous INH. This difference was not statistically significant (P = 0.223). INH mutations varied geographically; molecular DST can be used to guide and accelerate decision making in the use of ETH or high doses of INH.

Multidrug-Resistant Tuberculosis in Children: Recent Developments in Diagnosis, Treatment and Prevention

Multidrug-resistant (MDR) tuberculosis (TB) threatens global TB control. Few children with MDR-TB are diagnosed and appropriately treated; this is related to difficulties confirming a diagnosis of TB disease and challenges with current treatment regimens. This review highlights recent developments in the diagnosis, treatment and prevention of MDR-TB in children. Although phenotypic drug susceptibility testing (DST) remains the gold standard, molecular DST methods are facilitating rapid confirmation of MDR-TB. Increased knowledge about correct paediatric dosing of existing drugs and the addition of new and repurposed drugs is improving treatment. Observational studies support the effectiveness of preventing disease in MDR-TB-exposed contacts. Although challenges remain, there have been many exciting developments in the diagnosis and treatment of MDR-TB in children; it is a priority to continue improving MDR-TB care and to improve access for children. Preventive therapy following MDR-TB exposure/infection is a promising strategy under evaluation.

Rapid detection of extensively drug-resistant (XDR-TB) strains from multidrug-resistant tuberculosis (MDR-TB) cases isolated from smear-negative pulmonary samples in an Intermediate Reference Laboratory in India

BackgroundDirect sputum smear microscopy is commonly used for diagnosing tuberculosis (TB). The objectives of the study were first, to determine the recovery of Mycobacterium tuberculosis in smear-negative sputum samples through liquid culture (using MGIT 960) and solid culture (using LJ slant) and second, to screen multidrug-resistant isolates through line probe assay and further third, to identify XDR isolates through MGIT second-line DST from these positive MDR cultures in Delhi region. MethodsIn this study, the sample size was 717 (sputum smear AFB negative and culture positive for M. tuberculosis complex by both solid and liquid culture methods) MDRTB suspects who were enrolled from January 2014 to December 2014 at the Intermediate Reference Laboratory in New Delhi Tuberculosis Centre, New Delhi. Rapid line probe assay was performed on all culture-positive samples, which were direct smear-negative specimens, and LPA-confirmed MDR samples were tested on MGIT 960 second-line DST for identification of XDR strains. ResultsAn overall increase in the culture positivity (9.4%) among these smear-negative cases shows a good sign of recovery from M. tuberculosis infection in these samples. 717 (9.4%) positive cultures (MGIT+LJ) were subjected to line probe assay. Out of these 717 cultures, 9 (1.2%) were confirmed as NTM, 50 (7%) were MDR, 4 (0.6%) were mono-rifampicin resistant and 654 (91.2%) cultures were sensitive to both drugs Rif and Inh, respectively. Out of these 54 (50 MDR +4 mono-RIF resistant) cultures as screened by LPA, 1 (1.8%) was XDR, 10 (18.6%) were mono-ofloxacin resistant and 1 (1.8%) was mono-Kanamycin resistant. Sensitivity to both drugs KAN and OFX was seen in 42 (77.8%) cultures. ConclusionsSince the bacterial load in direct smear-negative suspected MDR samples is less, it is important to recover mycobacteria by rapid liquid culture method in such samples. Initial screening for MDRTB is to be done in such cases by performing rapid molecular genotypic drug susceptibility test such as LPA. Baseline second-line DST is also done to rule out the XDR cases among them for rapid and better management of XDRTB patients.

Diagnostic Molecular Mycobacteriology in Regions With Low Tuberculosis Endemicity: Combining Real-time PCR Assays for Detection of Multiple Mycobacterial Pathogens With Line Probe Assays for Identification of Resistance Mutations

Molecular assays have not yet been able to replace time-consuming culture-based methods in clinical mycobacteriology.Using 6875 clinical samples and a study period of 35months we evaluated the use of PCR-based assays to establish a diagnostic workflow with a fast time-to-result of 1–2days, for 1. detection of Mycobacterium tuberculosis complex (MTB), 2. detection and identification of nontuberculous mycobacteria (NTM), and 3. identification of drug susceptible MTB.MTB molecular-based detection and culture gave concordant results for 97.7% of the specimens. NTM PCR-based detection and culture gave concordant results for 97.0% of the specimens. Defining specimens on the basis of combined laboratory data as true positives or negatives with discrepant results resolved by clinical chart reviews, we calculated sensitivity, specificity, PPV and NPV for PCR-based MTB detection as 84.7%, 100%, 100%, and 98.7%; the corresponding values for culture-based MTB detection were 86.3%, 100%, 100%, and 98.8%. PCR-based detection of NTM had a sensitivity of 84.7% compared to 78.0% of that of culture-based NTM detection. Molecular drug susceptibility testing (DST) by line-probe assay was found to predict phenotypic DST results in MTB with excellent accuracy.Our findings suggest a diagnostic algorithm to largely replace lengthy culture-based techniques by rapid molecular-based methods.

Clinical features, Outcomes and Molecular Profiles of Drug Resistance in Tuberculous Meningitis in non-HIV Patients.

Tuberculous meningitis continues to be a serious problem for physicians because it is difficult to make an early diagnosis and the consequences of delaying treatment are severe. The objective of this study is to provide data for the optimization of diagnostic and timely treatment of tuberculous meningitis. Of the 401 human immunodeficiency virus (HIV)-negative tuberculous meningitis patients in our study, 332 were found to have an impaired blood brain barrier (82.8%). Nearly 17.0% of patients failed to be timely diagnosed. Headache (53.6%) and fever (48.6%) were the most common features, and Computed Tomography/Magnetic Resonance Imaging (CT/MRI) detected 96 patients (23.9%) with abnormal meningeal imaging. Cerebrospinal fluid real-time polymerase chain reaction was positive in 73.8% of the tuberculous meningitis patients, whereas, smears and cultures detected only 6.7% and 5.2%, respectively. Further analysis identified striking differences between drug-resistant and drug-susceptible tuberculous meningitis. Patients with drug resistance correlated with grave prognosis. Tuberculous meningitis diagnosis should overall embody clinical symptoms, laboratory and cerebral imaging findings, and more sensitive diagnostic approaches are still warranted. Our data suggest cerebrospinal fluid polymerase chain reaction for mycobacterial DNA and molecular drug susceptibility testing as routine assays for suspected tuberculous meningitis patients, and observation of the blood brain barrier function could be performed for individual management.

Evaluating Shared Laboratory Services: Detecting Mycobacterium Tuberculosis Complex and Drug Resistance Using Molecular and Culture-Based Methods.

We explored sharing nucleic acid amplification testing (NAAT) for detection of Mycobacterium tuberculosis complex (MTBC) and molecular and phenotypic drug susceptibility testing between two state public health tuberculosis (TB) laboratories, and evaluated turnaround times and cost-effectiveness. From September 1, 2012, to May 30, 2013, the Wisconsin State Laboratory of Hygiene (Wisconsin Lab) submitted specimens to the Microbial Diseases Laboratory of the California Department of Public Health (California Lab) for NAAT and molecular drug susceptibility testing (MDST) by pyrosequencing, and culture-based TB drug susceptibility testing by the BACTEC(TM) MGIT(TM) 960 system. A total of 182 specimens were referred to the California Lab, and 47 TB cases and 12 drug-resistant cases were identified. The average time for specimen transport was two days, which included one day for processing and packaging in the submitting laboratory. The average turnaround time for NAAT was 0.3 days at the Wisconsin Lab and 3.8 days at the California Lab, including time for specimen transport. Turnaround time for culture-based drug susceptibility testing increased by a median of 16 days when specimens were sent to the California Lab, but MDST results were reported in fewer than four days, a median of 22 days sooner than any culture-based drug susceptibility testing results. This study revealed advantages and disadvantages associated with sharing services, and identified opportunities for improvement. Referral of specimens resulted in longer turnaround times for mycobacteriology test results and additional costs for transporting specimens. However, specialized testing such as pyrosequencing may not be available in low TB incidence areas, and these rapid results can have positive effects on patient management and TB control.

Comparative Study between Line Probe Assay by MTBDRplus (Hain PCR) and XpertMTB/RIF Assay (GeneXpert PCR) for the Detection of Mycobacterium Tuberculosis Complex Directly from Clinical Samples and from Cultured Isolates

Background: Tuberculosis remains a major public health problem worldwide. Nucleic acid amplification tests (NAATs) have been introduced to overcome conventional methods’ low sensitivity and diagnostic delays. These rapid tests such as the line probe assay (LPA) and XpertMTB/RIF assay can provide results within hours directly on specimens and thus enable prompt and appropriate treatment, decrease morbidity and mortality, and interrupt transmission. Objectives: The aim of this study was to evaluate the efficiency and reliability of MTBDRplus and XpertMTB/RIF assay for the detection of Mycobacterium tuberculosis complex (MTBC) in smear-positive and smear-negative pulmonary and extrapulmonary specimens in comparison to the gold standard VersaTREK liquid culture system. Methodology and Results: In this study, a total of 130 samples were obtained from tuberculous patients. The percentages of the MTBC detection in AFB-negative samples by Hain PCR was 0.0%,while, the percentages of the MTBC detection in AFB-positive (+1), (+2), (+3) and(+4) samples by Hain PCR were 0.0%,55.6%, 75% and 93.8%. The percentage of the MTBC detection in AFB-negative samples by GeneXpert PCR was higher than those which were detected by Hain PCR (30.4% vs 0.0%) with high significant difference. Mean while, the percentage of the MTBC detection in AFB-positive (+1) samples by GeneXpert PCR was higher than those which were detected by Hain PCR (100% vs 0.0%) with high significant difference. The percentage of the MTBC detection in total AFB-positive samples by GeneXpert PCR was higher than those which were detected by Hain PCR (90.5% vs 54.8%) with high significant difference. The sensitivity, specificity and accuracy of Gene Xpert PCR were 64.9%,0.0% and 56.9% while, they were40.4%,100% and 47.6% by Hain PCR. The degree of detection of MTBC by GeneXpertPCR was correlated with the results of AFB staining with high significant difference. Conclusion : our findings showed the superior sensitivity of the Xpert MTB/RIF compared to the MTBDRplus in the detection of MTBC. However, the molecular drug susceptibility testing results should always be confirmed by phenotypic methods.

Clinical implications of molecular drug resistance testing for Mycobacterium tuberculosis: a TBNET/RESIST-TB consensus statement.

The emergence of drug-resistant strains of Mycobacterium tuberculosis is a challenge to global tuberculosis (TB) control. Although culture-based methods have been regarded as the gold standard for drug susceptibility testing (DST), molecular methods provide rapid information on mutations in the M. tuberculosis genome associated with resistance to anti-tuberculosis drugs. We ascertained consensus on the use of the results of molecular DST for clinical treatment decisions in TB patients. This document has been developed by TBNET and RESIST-TB groups to reach a consensus about reporting standards in the clinical use of molecular DST results. Review of the available literature and the search for evidence included hand-searching journals and searching electronic databases. The panel identified single nucleotide mutations in genomic regions of M. tuberculosis coding for katG, inhA, rpoB, embB, rrs, rpsL and gyrA that are likely related to drug resistance in vivo. Identification of any of these mutations in clinical isolates of M. tuberculosis has implications for the management of TB patients, pending the results of in vitro DST. However, false-positive and false-negative results in detecting resistance-associated mutations in drugs for which there is poor or unproven correlation between phenotypic and clinical drug resistance complicate the interpretation. Reports of molecular DST results should therefore include specific information on the mutations identified and provide guidance for clinicians on interpretation and on the choice of the appropriate initial drug regimen.

A Model of Shared Mycobacteriology Testing Services: Lessons Learned.

The need for public health laboratories (PHLs) to prioritize resources has led to increased interest in sharing diagnostic services. To address this concept for tuberculosis (TB) testing, the New York State Department of Health Wadsworth Center and the Rhode Island State Health Laboratories assessed the feasibility of shared services for the detection and characterization of Mycobacterium tuberculosis complex (MTBC). We assessed multiple aspects of shared services including shipping, testing, reporting, and cost. Rhode Island State Health Laboratories shipped MTBC-positive specimens and isolates to Wadsworth Center. Average turnaround times were calculated and cost analysis was performed. Testing turnaround times were similar at both PHLs; however, the availability of conventional drug susceptibility testing (DST) results for Rhode Island primary specimens and isolates were extended by approximately four days of shipping time. An extended molecular testing panel was performed on every specimen submitted from Rhode Island State Health Laboratories to Wadsworth Center, and the total cost per specimen at Wadsworth Center was $177.12 less than at Rhode Island State Health Laboratories, plus shipping. Following a mid-study review, Wadsworth Center provided testing turnaround times for detection (same day), species determination of MTBC (same day), and molecular DST (2.5 days). The collaboration between Wadsworth Center and Rhode Island State Health Laboratories to assess shared services of TB testing highlighted a successful model that may serve as a guideline for other PHLs. The provision of additional rapid testing at a lower cost demonstrated in this study could potentially improve patient management and result in significant cost and resource savings if used in similar models across the country.

Reduced turn-around time for Mycobacterium tuberculosis drug susceptibility testing with a proportional agar microplate assay

Multidrug-resistant tuberculosis is a major issue worldwide; however, accessibility to drug susceptibility testing (DST) is still limited in developing countries, owing to high costs and complexity. We developed a proportion method on 12-well microplates for DST. The assay reduced the time to results to <12 days and <10 days when bacterial growth was checked with the naked eye or a microscope, respectively. Comparison with the Canetti–Grosset method showed that the results of the two assays almost overlapped (kappa index 0.98 (95% CI 0.91–1.00) for isoniazid, rifampicin, streptomycin; and kappa index 0.92 (95% CI 0.85–0.99) for ethambutol). The sequencing of genes involved in drug resistance showed similar level of phenotype–genotype agreement between techniques. Finally, measurement of the MICs of rifampicin and ethambutol suggests that the currently used critical ethambutol concentration should be revised, and that the current molecular drug susceptibility tests for rifampicin need to be re-evaluated, as in vitro rifampicin-sensitive isolates could harbour drug resistance-associated mutation(s).

Multidrug-Resistant Tuberculosis in Patients for Whom First-Line Treatment Failed, Mongolia, 2010-2011.

In Ulaanbaatar, Mongolia, multidrug-resistant tuberculosis (MDR TB) was diagnosed for more than a third of new sputum smear–positive tuberculosis patients for whom treatment had failed. This finding suggests a significant risk for community-acquired MDR TB and a need to make rapid molecular drug susceptibility testing available to more people.

Scaling up multidrug-resistant tuberculosis care in China.

Drug-resistant tuberculosis threatens to undermine advances in tuberculosis control worldwide. Multidrug-resistant tuberculosis (MDRTB), defined as resistance to isoniazid and rifampicin, requires treatment for up to 24 months with expensive diagnostics and second-line drugs. Fewer than 15% of patients diagnosed with tuberculosis worldwide receive drug susceptibility testing (DST); only 20% of the estimated 480?000 new cases of MDRTB annually are treated appropriately.1 Of these, cure rarely exceeds 65%. Those on ineffective therapy risk acquiring additional resistance and spreading drug-resistant tuberculosis to others. In recent years, new molecular diagnostics and antituberculosis drugs promise an opportunity to improve outcomes for patients with drug-resistant tuberculosis, leading the WHO to prioritise universal access to prevention, diagnosis, and treatment of MDRTB.1 However, identification of viable models for coordinating and financing scale-up of such services remains a major challenge for most middle-income and low-income countries. After the widespread implementation of directly observed therapy, short course (DOTS), tuberculosis prevalence and mortality have decreased greatly in China.2 However, MDRTB remains a formidable problem for China with an estimated 100 000 incident cases annually, a fifth of the global burden.3 In The Lancet Global Health, Renzhong Li and colleagues report4 on an intervention to provide a comprehensive programme of universal access to MDRTB care, which might offer a model of improved care for other high-burden countries. The programme was implemented in four cities and involved provision of a comprehensive diagnostic, care, and financial package, including financing for specimen shipment, resistance testing, costs of patient transportation and nutritional supplements, and a subsidy for directly observed therapy. It emphasised close communication between treatment facilities and improved care linkages. Diagnosis consisted of the use of a rapid molecular diagnostic (Genechip Capitabio) for isoniazid and rifampicin DST and training of centralised staff in molecular and conventional DST. Treatment was tailored to confirmed degree of tuberculosis resistance, per WHO guidelines. The financial scheme imposed a cap of US$4644 per treated case, with patients' expenses capped at 10% of the total, through creative use of government insurance funds and project resources. The results were impressive,4 with a 90% decrease in time from testing to treatment initiation and substantial increases in the number of diagnosed patients, use of appropriate regimen, and retention at 6 months, largely due to major decreases in deaths and defaults. The findings from Li and colleagues4 add to the mounting evidence that the prioritisation of investments in operational coordination improvements is crucial for better outcomes of MDRTB.4, 5, 6 Specifically, they show that a faster time to diagnosis has both clinical and cost benefits: elimination of hospital stay preceding diagnosis, which reduces potential nosocomial transmission of MDRTB along with hospital expenses, and faster initiation of MDRTB treatment. The investigators note that the National Center for Tuberculosis Control and Prevention will scale-up features of the programme nationwide, including free testing for MDRTB with molecular diagnostics and the standard care package for MDRTB. This investment would seem to be wise. An estimate of the national scale-up cost suggests an initial price tag of $500 million annually, or roughly double domestic spending on tuberculosis in 2011.7 This number is a substantial figure, but only about 0·2% of annual public health spending in China (an amount considered low by international standards) and 80% of the government's yearly outlay on HIV.8, 9, 10 Moreover, extrapolating the mortality effect shown in Li and colleagues' study,4 MDRTB-related deaths could be reduced by roughly 80%, with up to 20?000 deaths averted in the first year alone. To maximise the chances of success, additional elements of the programme should be considered. The molecular diagnostic used in the study's package4 only performs on smear-positive tuberculosis cases, which were just 28% of those diagnosed with tuberculosis. A molecular diagnostic such as Xpert MTB/RIF (Cepheid) or MTBDRplus (Hain Lifescience GmbH), which can be done on smear-negative or culture-positive specimens, could be incorporated to capture all MDRTB cases and avoid initiating patients with smear-negative samples on ineffective therapy.11 47 (29%) of the 163 patients with MDRTB tested for additional drug resistance had extensively (XDR) or pre-XDR tuberculosis discovered a median of 45–55 days after start of MDRTB treatment, delaying initiation of an effective regimen. As rapid second-line DSTs become available, they should be incorporated into diagnostic schemes to ensure the most effective treatment for all patients. Further benefits could be gained from integrating HIV testing and treatment, as the authors acknowledge.4 Comprehensive, high-quality treatment models are challenging logistically and financially, demanding commitment at all levels and implementers with considerable technical expertise. This study offers a model with potential for achieving advances in tuberculosis treatment relatively swiftly, while highlighting important policy issues that merit attention. As improved diagnostics and drugs for MDRTB become available, the relevance of such a complete, integrated approach to diagnosis, treatment, and cost to scale-up MDRTB care will only continue to grow.

Effect of a comprehensive programme to provide universal access to care for sputum-smear-positive multidrug-resistant tuberculosis in China: a before-and-after study.

China has a quarter of all patients with multidrug-resistant tuberculosis (MDRTB) worldwide, but less than 5% are in quality treatment programmes. In a before-and-after study we aimed to assess the effect of a comprehensive programme to provide universal access to diagnosis, treatment, and follow-up for MDRTB in four Chinese cities (population 18 million). We designated city-level hospitals in each city to diagnose and treat MDRTB. All patients with smear-positive pulmonary tuberculosis diagnosed in Center for Disease Control (CDC) clinics and hospitals were tested for MDRTB with molecular and conventional drug susceptibility tests. Patients were treated with a 24 month treatment package for MDRTB based on WHO guidelines. Outpatients were referred to the CDC for directly observed therapy. We capped total treatment package cost at US$4644. Insurance reimbursement and project subsidies limited patients' expenses to 10% of charges for services within the package. We compared data from a 12 month programme period (2011) to those from a retrospective survey of all patients with MDRTB diagnosed in the same cities during a baseline period (2006-09). 243 patients were diagnosed with MDRTB or rifampicin-resistant tuberculosis during the 12 month programme period compared with 92 patients (equivalent to 24 per year) during the baseline period. 172 (71%) of 243 individuals were enrolled in the programme. Time from specimen collection for resistance testing to treatment initiation decreased by 90% (from median 139 days [IQR 69-207] to 14 days [10-21]), the proportion of patients who started on appropriate drug regimen increased 2·7 times (from nine [35%] of 26 patients treated to 166 [97%] of 172), and follow-up by the CDC after initial hospitalisation increased 24 times (from one [4%] of 23 patients to 163 [99%] of 164 patients). 6 months after starting treatment, the proportion of patients remaining on treatment increased ten times (from two [8%] of 26 patients to 137 [80%] of 172), and 116 (67%) of 172 patients in the programme period had negative cultures or clinical-radiographic improvement. Patients' expenses for hospital admission after MDRTB diagnosis decreased by 78% (from $796 to $174), reducing the ratio of patients' expenses to annual household income from 17·6% to 3·5% (p<0·0001 for all comparisons between baseline and programme periods). However, 36 (15%) patients did not start or had to discontinue treatment in the programme period because of financial difficulties. This comprehensive programme substantially increased access to diagnosis, quality treatment, and affordable treatment for MDRTB. The programme could help China to achieve universal access to MDRTB care but greater financial risk protection for patients is needed. Bill & Melinda Gates Foundation.

Target product profile of a molecular drug-susceptibility test for use in microscopy centers.

Current phenotypic testing for drug resistance in patients with tuberculosis is inadequate primarily with respect to turnaround time. Molecular tests hold the promise of an improved time to diagnosis. A target product profile for a molecular drug-susceptibility test (DST) was developed on the basis of a collaborative effort that included opinions gathered from researchers, clinicians, policy makers, and test developers on optimal clinical and operational characteristics in settings of intended use. In addition, the current diagnostic ecosystem and the diagnostic development landscape were mapped. Molecular DSTs for detecting tuberculosis in microscopy centers should ideally evaluate for resistance to rifampin, fluoroquinolones, isoniazid, and pyrazinamide and enable the selection of the most appropriate treatment regimen. Performance characteristics of DSTs need to be optimized, but compromises can be made that depend on the trade-off between a false-positive result and a false-negative result. The operational requirements of a test will vary depending on the site of implementation. However, the most-important considerations pertain to quality control, maintenance and calibration, and the ability to export data. This target product profile defines the needs as perceived by the tuberculosis stakeholder community and attempts to provide a means of communication with test developers to ensure that fit-for-purpose DSTs are being developed.

Innovations in Tuberculosis Diagnostics: Progress and Translational Challenges

Despite the long and hard battle against tuberculosis (TB), WHO estimated that 9 million people developed the disease in 2013, and nearly 1.5 million people died of TB (World Health Organization, 2014). To make matters worse, drug-resistance is a growing threat, and 3 out of 9 million TB cases are either not diagnosed, or not notified to TB control programs. But there is some good news from the perspective of new tool introduction. Slowly but surely, the landscape of TB technologies is changing (Pai and Schito, 2015). We now have a variety of new TB diagnostics, including rapid molecular tests (e.g. Xpert MTB/RIF, Cepheid Inc., USA) for detection as well as drug susceptibility testing (DST) (UNITAID, 2014). We also have new TB drugs (e.g. bedaquiline and delamanid) on the market, and new TB drug regimens are expected within the next 2–3 years. These are major, exciting developments in the fight against a very ancient scourge. This article reviews the current best diagnostic tools available for TB diagnosis and monitoring, and describes the most important gaps, and translational challenges for developing innovative products that can meet the needs (Table 1). Table 1 Unmet needs in TB diagnosis and monitoring. As shown in the Table, there are critical unmet needs that range from a simple, triage test for use in the community, to DST tools that can detect a range of mutations for several important drugs that will make up future drug regimens (Denkinger et al., 2015a, Denkinger et al., 2015b). For the next-generation DST tools, a big translational challenge is the paucity of good data on the correlation of mutations with phenotypic DST results and clinical outcomes and the association with cross-resistance (Solomon et al., 2015). This is particularly important to make sure that we have companion diagnostics for emerging TB drug regimens (Denkinger et al., 2015b). The translational challenges associated with DST are reviewed elsewhere (Solomon et al., 2015). For the development of rapid triage tests, non-sputum based tests for active TB, highly predictive LTBI tests, and an accurate test for cure, we need validated biomarkers. Although considerable efforts are being made to identify biomarkers that can meet some of these needs, progress has been slow, and the translational challenges have been reviewed elsewhere (Wallis et al., 2010). Increased investments are necessary to support biomarker discovery, validation, and translation into clinical tools. Unfortunately, a recent analysis of the TB R&D funding landscape by Treatment Action Group showed a big gap between investment needed and actual expenditure on R&D. Donors, governments, and members of the Stop TB Partnership will need to device creative strategies to plug this gap. While the TB diagnostics R&D space has managed to attract over 50 companies and product developers, they will require technical and funding support to overcome the translational challenges shown in Table 1. Organizations such as Foundation for Innovative New Diagnostics (FIND), Geneva, Bill and Melinda Gates Foundation, World Health Organization, UNITAID, Global Laboratory Initiative, Stop TB Partnership's New Diagnostics Working Group, Critical Path Institute, PATH, McGill International TB Centre, and several academic partners have worked together to produce several reports that are of great relevance, including a technology and market landscape report, a needs assessment study, a consensus report on target product profiles of highest priority, a series of market analyses, and a series of articles which outline the characteristics of the next-generation assays, and translational challenges for product development. All of these are available on a website (www.tbfaqs.org) created to provide answers to the most frequently asked questions by TB product developers. Hopefully, these collective efforts will result in a more robust pipeline of tools that can overcome the translational challenges, and push the agenda towards the goal of TB elimination.