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5 Detecting Drug Resistance and Strengthening Laboratory Capacity Key Messages • ew diagnostic technologies require both performance assess- N ments and quality assurance to ensure that they are performing at optimal levels. • The ideal test is not the most inexpensive but the most cost-effective. • he Supranational Reference Laboratory Network has played a T key role in determining the extent of drug resistance by conducting representative surveys in broad regions, assessing the quality of TB programs, and informing policy decisions. • he establishment of national reference laboratories, intermedi- T ate reference laboratories, and laboratories in medical colleges, together with plans for dozens more laboratories for liquid plus molecular testing, will increase laboratory capacity. Successful treatment of MDR TB requires knowing which treatments will work. Today, most diagnoses of MDR TB are made in culture, but this method can take months to produce results, during which time drug- resistant TB patients can continue to infect other people if they are receiv- ing inadequate treatment. Many new diagnostic technologies now being developed are faster and more accurate, but all technologies involve trade- offs between advantages and disadvantages. Four workshop presenters discussed the next generation of TB diagnostics and strengthening of labo- ratory capacity. Their presentations addressed the advantages and disadvan- 59
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60 DRUG-RESISTANT TUBERCULOSIS IN INDIA tages of new technologies compared with current methods, the performance testing and quality assurance needed for new technologies, the history and current responsibilities of the Supranational Reference Laboratory Net- work, and the contributions of FIND and the EXPAND-TB program to the scaling up of laboratory capacity in India. DIAGNOSIS OF DRUG-RESISTANT TB1 As discussed earlier, the lack of diagnostic capacity has been a crucial barrier to the treatment of MDR TB. Today, however, at least 20 new diag- nostic technologies are in different stages of development, and expanding laboratory capacity has become a global priority. Ideally, DST should have at least the following characteristics: • high intra- and interlaboratory reproducibility; • short turnaround time; • ability to distinguish between high and low levels of resistance; • practicality and affordability; • minimal investment and costs for consumables; • minimal labor time; and • applicability to both first- and second-line drugs. Culture methods, which are regarded as the gold standard, are still widely used today, but have limitations, including • long turnaround time; • failure to provide precise identification of species; • possibility of negative cultures for patients on treatment; • laborious testing procedures, including standardization of critical concentrations and establishment of appropriate inoculum size; and • issues concerning the stability of the drug in different culture media, the reliability of results, and quality assurance. Several rapid assays measure resistance directly from clinical speci- mens. For example, a meta-analysis showed that the nitrate reductase assay (NRA) has a pooled sensitivity for detection of isoniazid and rifampicin resistance of 94 percent and 96 percent, respectively (Bwanga et al., 2009). The same analysis found a pooled sensitivity for the microscopic observa- tion drug susceptibility (MODS) assay of 92 percent and 96 percent, respec- 1This section is based on the presentation of Camilla Rodrigues, Consultant Clinical Mi- crobiologist and Chair of Infection Control, Hinduja Hospital.
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61 DETECTING DRUG RESISTANCE tively. This method has the additional benefit of accurate case detection and simultaneous identification of MDR. Finally, thin layer agar (TLA) culture has sensitivity, specificity, and predictive values of 100 percent for isoniazid and rifampicin resistance (Robledo et al., 2008). Other new methods, such as the mycobacteria growth indicator tube (MGIT), detect metabolic activity or products. With these methods, the time to detection is 10-14 days, compared with 3–4 weeks or more with other methods. Universally accepted standards for critical concentrations are needed to ensure the reproducibility of DST on second-line drugs. One study, for example, found that 21 supranational reference laboratories had different critical concentrations as a result of variations in testing systems and media (Kim et al., 2004). Quality assurance is therefore a critical factor (Rodrigues et al., 2008). Colorimetric redox indicator (CRI) assays, which are fast and inexpen- sive, have good sensitivity for isoniazid and rifampicin but do not perform as well for ethambutol and pyrazinamide, said Camilla Rodrigues, Con- sultant Clinical Microbiologist and Chair of Infection Control, Hinduja Hospital. They also raise concerns about biosafety and containment. Phage-based technologies were extremely promising when they first appeared; however, that initial promise has faded in recent years. Improved phage technology is currently being developed in the United States and South Africa, and has again raised the hope of this technology being used for both rapid diagnosis and DST. Lysis with mycobacteriophages is fairly sensitive for rifampicin and isoniazid resistance but has limitations in such areas as analytical sensitivity and application to smear-negative patients (Krishnamurthy et al., 2002). Finally, the detection of specific mutations has advanced through the rapid development of genotypic hardware. This technique has proven most effective with rifampicin but might be less successful with other drugs—for example, isoniazid and the fluoroquinolones, where M.tb. has more muta- tions that code for drug resistance. Molecular DST includes DNA sequencing, polymerase chain reaction (PCR) single-strand conformational polymorphism (SSCP), solid phase hybridization assays, real-time formats, and microarrays. The advantages of molecular methods are • rapid provision of results; • high sensitivity; • good performance characteristics; • direct application to clinical specimens; • less biohazard risk; • feasibility of automation and high throughput; and • ability to target genomically based resistance.
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62 DRUG-RESISTANT TUBERCULOSIS IN INDIA However, molecular methods also have limitations: • polyresistance due to the effects of multiple genes; • requirements for infrastructure, experienced staff, and funding; • risk of false-positive tests, especially with methods that target only known or described mutants; and • silent mutations, or infections with more than one strain. DNA sequencing is the most informative molecular method, but it is so labor-intensive and expensive that it cannot be used routinely. Other methods are good at detecting specific mutations, but they also can be labor-intensive and somewhat dependent on the person who performs the test. Real-time formats and microarrays are promising but expensive. Microfluidic technologies may help close the gap between needs and capabilities, especially to the extent that they permit integration of different technologies. For example, GeneXpert MTB/RIF is a self-contained, closed, fully integrated, and automated platform that provides results for detection of rifampicin resistance within 2 hours. The test has a sensitivity almost equal to that of culture, requires minimal hands-on technical time, and can be used in laboratories with considerably less biohazard risk. In a recent study (Boehme et al., 2010), GeneXpert MTB/RIF outperformed smear tests, yielding a relative increase in case detection and accurately ruling out MDR TB. Even in countries with little MDR TB, there is value to using GeneXpert to diagnose TB as it is much more sensitive than microscopy and faster than and as sensitive as culture tests. According to Rodrigues, the only disadvantages are that it is expensive and has a relatively low positive predictive value in areas with low MDR TB prevalence. According to Rodrigues, the ideal test for TB would have the following features: • provision of rapid results; • ability to obtain results directly from the specimen with the sensi- tivity of culture; • simultaneous detection of resistance; • on-demand availability; • capability for single-patient testing; • ease of use; • reproducibility; • robustness; and • low cost. Rodrigues stressed that the TB program in India needs to define priori- ties; strengthen national laboratory capacity with a tiered network at the
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63 DETECTING DRUG RESISTANCE subdistrict, district, regional, and reference laboratory levels; work toward universal access; and leverage molecular methods with culture methods, since no single test can stand alone. Validation, quality assurance, and quality control should be an integral part of the system, said Rodrigues. Finally, Rodrigues cited two particular clinical challenges. The first is simultaneous infections with different strains, which can lead to conflict- ing test results. The second is the need to be able to distinguish between immune reconstitution inflammatory syndrome (IRIS) and MDR TB in culture-negative patients undergoing treatment. QUALITY ASSURANCE CONSIDERATIONS IN THE DEVELOPMENT OF NEW DIAGNOSTICS2 To determine whether a diagnostic test is accurate, observed Thomas Shinnick, Associate Director for Global Laboratory Activities, Division of Tuberculosis Elimination, CDC, its performance must be comparable to that of a gold standard. Molecular tests also must be subject to quality assurance to ensure that their performance is being maintained. Two key properties of any test are accuracy and precision. Accuracy measures validity, while precision measures reliability or reproducibility. The ideal test is both accurate and precise, so that it yields the right answer every time. With a standard phase II or phase III diagnostic design, estimation of accuracy involves defining a gold standard; ensuring the quality of the test- ing; recruiting consecutive patients in whom the test is indicated (that is, in whom disease is suspected); performing the gold standard test on separate groups with and without disease; performing the experimental test on all subjects and classifying them as test positives or test negatives; and devel- oping a 2–by–2 table to calculate sensitivity, specificity, predictive values, likelihood ratios, and diagnostic odds ratios. With respect to molecular tests for drug resistance, a variety of pheno- typic tests can serve as the gold standard, including liquid or solid culture and clinical outcomes. Of course, said Shinnick, these gold standards are in actuality “bronze or silver standards. They all have their own problems, and so we are always going to have to be looking at analyzing discordant or discrepant results to understand our test and how it is performing.” When discrepant or discordant results occur, several possibilities must be considered. The first is the possibility that errors resulted from mislabel- ing or laboratory cross-contamination. The possibility of a mixed sample also must be considered. The limit of detection of a molecular test is a fac- 2 This section is based on the presentation of Thomas Shinnick, Associate Director for Global Laboratory Activities, Division of Tuberculosis Elimination, CDC.
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64 DRUG-RESISTANT TUBERCULOSIS IN INDIA tor as well. Will a test detect only 10 percent drug-resistant strains when other tests are designed to detect 1 percent drug-resistant strains? To exam- ine these possibilities, a patient sample or isolate can be tested repeatedly, target genes can be resequenced, or the minimal inhibitory concentration can be gauged using liquid or solid media. Clinical outcomes are another way of understanding a test’s performance. Both molecular and conventional tests require quality assurance, emphasized Shinnick. In particular, molecular tests need to be subject to both internal quality controls and external quality assurance. Molecular tests typically involve the use of negative controls, with every test being run to assess the possible contamination of reagents. They also involve the use of positive controls—isolates with particular features used to show that a reagent is behaving as it should. It is important to keep the positive controls away from the test samples to avoid contamination. External quality assurance ensures that a test is performing the same way in all laboratories. Methods used for external quality assurance include sending an isolate with a known mutation to a laboratory to determine whether the right answer has been obtained; comparing results of another molecular or phenotypic test with the test results; or using laboratory performance indicators, such as the level of agreement among results of molecular or phenotypic tests, the concordance between different samples from the same patient, or the percentage of indeterminate results. The accuracy of a test determines its value to the clinician. One way of measuring a test’s accuracy is by calculating its sensitivity and specific- ity, which are the rates of the test’s true positives and true negatives. For clinicians, the most important pieces of information are the likelihood ratio and diagnostic odds ratio, which are essentially inverses of each other. The likelihood ratio denotes whether the test is more likely to be positive in a diseased than in a nondiseased person. Tests with high likelihood ratios give clinicians more information about how to proceed with treatment. Shinnick concluded by saying that the ideal test is not the most inex- pensive but the most cost-effective. Even a relatively expensive test, such as GeneXpert, can be cost-effective when it rapidly identifies MDR TB patients. The patient is treated sooner and has better outcomes, and the community is protected against the transmission of drug-resistant organisms. THE SUPRANATIONAL REFERENCE LABORATORY NETWORK3 When the international Supranational Reference Laboratory Network was initiated in 1964, it consisted of 16 laboratories with strong commit- 3 This section is based on the presentation of Nagamiah Selvakumar, Scientist G, National Institute for Research in Tuberculosis.
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65 DETECTING DRUG RESISTANCE ments to national TB programs. In general, there was an extreme scarcity of good laboratories at the time. Initially, 11 of the laboratories were in Europe and 2 in India. The network now includes 2 laboratories in the African region, 5 in the Ameri- cas, 1 in the Middle East, 11 in Europe, 5 in the Western Pacific, and 2 in South Asia. One of the latter two is the National Institute for Research in Tuberculosis, Chennai, and the other is in Bangkok. At the outset, the laboratories periodically collected information on drug resistance in their regions, said Nagamiah Selvakumar, Scientist G, National Institute for Research in Tuberculosis. They had commitments to cover at least two countries other than those in which they were located. They also agreed to ensure the quality of drug resistance surveys by retest- ing isolates. They participated in annual external quality assessment pro- grams with the coordinating center in Antwerp, Belgium. If possible, they conducted operational research to generate data and provide information to inform policy decisions. Today the Supranational Reference Laboratory Network determines drug resistance globally through representative surveys in broad regions. It seeks to differentiate between previously treated and untreated cases while continuing to assess the quality of TB programs. It also continues to provide information to inform policy decisions and enhance laboratory capacity. The network has produced four global reports, the first in 1997 and the most recent in 2008, based on data from an increasing number of labo- ratories and countries. The network estimates the prevalence of TB, MDR TB, and XDR TB. It also identifies “hot spots” for drug-resistant TB. For example, a recent national survey in China found that 5.7 percent of new TB cases and 26 percent of previously treated cases were MDR. Subnational data from Tajikistan revealed that 16 percent of new cases of TB and 62 percent of previously treated cases were MDR. Overall, based on quality- controlled data from 114 countries, MDR TB occurred in 3.6 percent of incident TB cases in 2008 (WHO, 2010c).4 The data also show that 58 countries had reported XDR TB as of March 2010, with approximately 25,000 cases emerging every year. Over- all, an estimated 5.4 percent of MDR TB cases are XDR. Another important function of the Supranational Reference Laboratory Network is to conduct external quality assessments to ensure that the data being generated are reliable. The coordinating laboratory in Antwerp sends a panel of 30 cultures to the supranational reference laboratories every year. The cultures have different combinations of resistance and have been clini- cally well validated. In recent rounds of testing, 16 of the 27 supranational 4 See footnote 4 in Chapter 1 and the updated WHO (2011a) report on TB control for more information on global estimates of MDR TB.
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66 DRUG-RESISTANT TUBERCULOSIS IN INDIA reference laboratories consistently performed better than other laboratories being assessed (Van Deun et al., 2011). The objectives of external quality assessment are to standardize techniques, validate methods, and improve the precision of reporting. As an example of the activities of a supranational reference labora- tory, Selvakumar described those of the National Institute for Research in Tuberculosis, Chennai. It supports not just India, but also Sri Lanka, the Maldives, and, until recently, North Korea (which is now supported by the laboratory in Bangkok). It develops protocols and conducts retesting and panel testing for the external quality assurance program. It also conducts DST for suspected cases of MDR TB. It facilitates international train- ing programs on laboratory diagnosis of MDR TB and quality assurance microscopy. And it participates in WHO meetings and serves as a consul- tant to other Southeast Asian countries. In the 16 rounds of quality assurance that have been completed at the National Institute for Research in Tuberculosis, Chennai, efficiency has been acceptable for all first-line drugs except rifampicin, for which results were indiscriminate in two rounds. In the last two rounds, DST has been conducted with second-line drugs, with acceptable efficiency being found for ofloxacin, kanamycin, amikacin, and capreomycin. The National Institute for Research in Tuberculosis, Chennai, con- ducted the first statewide drug resistance survey for Gujarat and will be conducting surveys in 2011–2013 for Tamil Nadu and Rajasthan. It moni- tors India’s other national reference laboratories, intermediate reference laboratories, regional medical research centers, and private laboratories. The Supranational Reference Laboratory Network has made substan- tial contributions to policy making. The first and second global reports provided information on the MDR TB problem and identified the hot spots in the Soviet Union and China, contributing to the origins of the DOTS- Plus program and the GLC. The third report suggested that previously treated cases and HIV status be included in drug resistance surveys. That report also pointed to the unreliability of second-line DST. This information helped WHO’s Global Laboratory Initiative (GLI) formulate guidance for the development of 150 national reference laboratories and almost 8,000 advanced diagnostic centers, and improved the training of hundreds of thousands of microbiologists and technologists. The National Institute for Research in Tuberculosis, Chennai, also provides information to inform policy decisions. For example, it has recom- mended having two sputum samples for diagnosis and treating the presence of bacilli in a single smear as an indicator of TB. It also has shown that the sensitivity of diagnoses can be seriously affected when sputum samples are transported in cetyl-pyridinium chloride (CPC) solution, implemented a lot
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67 DETECTING DRUG RESISTANCE quality assurance system, and developed software for analysis of retesting and panel testing results. According to Selvakumar, the Supranational Reference Laboratory Net- work still has important limitations: • Not all the high-burden countries have national reference laboratories. • Many national reference laboratories lack the resources and exper- tise needed to conduct drug resistance surveys. • Only the results of panel testing are known, but the results of retesting are needed to ensure the quality of testing. • External quality testing for the LPA needs to be considered because intermediate resistance to rifampicin has emerged as a problem. • The role of operational research in national reference laboratories or supranational reference laboratories is not clearly defined. • Other challenges include inadequate staffing, the threat of contrac- tual staff leaving the laboratory, excessive workloads, and inad- equate laboratory infrastructure and staff training. EXPANDING LABORATORY CAPACITY IN INDIA FOR THE DIAGNOSIS OF DRUG-RESISTANT TB5 FIND was created in 2003 to drive the development and implementa- tion of accurate and affordable diagnostic tests that are appropriate to patient care in low-resource settings, said Neeraj Raizada, Medical Officer, FIND. FIND’s India office was established in 2007 and had two broad initial projects. One was to conduct an LPA demonstration project and a liquid culture, DST, and rapid speciation laboratory preparedness study. The other was to complete demonstration projects on several new technolo- gies, including light-emitting diode (LED)-based fluorescent microscopy and cartridge-based nucleic acid amplification testing (NAAT). Data from these two projects were presented regularly to the RNTCP and to the DOTS-Plus committee. The data contributed to the RNTCP’s laboratory scale-up plan and to the endorsement of the LPA, liquid culture, and rapid speciation tests under the EXPAND-TB and Global Fund projects. The RNTCP’s laboratory scale-up plan called for introducing the LPA in 43 laboratories and liquid culture, DST, and rapid speciation in 33 laboratories (see Chapter 2). The LPA will be the primary diagnostic tool, with follow-up using solid or liquid culture. The turnaround time will be 3 days, compared with the baseline of 4.5 months. Laboratory capacity is expanding to 12,000 LPAs annually, compared with a previous capacity to conduct 5,000 cultures and drug susceptibility tests. The plan is to be 5 This section is based on the presentation of Neeraj Raizada, Medical Officer, FIND.
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68 DRUG-RESISTANT TUBERCULOSIS IN INDIA implemented in a phased manner, with the LPA being introduced into 12 laboratories in 2010–2011, 14 laboratories in 2011–2012, and 17 laborato- ries in 2012–2013. The implementation of the RNTCP laboratory scale-up plan is being funded by various sources. The EXPAND-TB project was initiated in India in March 2010 with UNITAID and has three implementing partners: FIND, the STOP TB Part- nership GLI, and the WHO GDF. A major initiative has been the Global Fund Round 9 project, in which FIND is implementing the laboratory component. The laboratory component complements the EXPAND-TB project in the introduction of rapid diagnostics. Support also comes from the RNTCP and from National Rural Health Mission and state funds. The specific operational objectives of the EXPAND-TB project are to accelerate and expand access to quality-assured new diagnostics; leverage price reductions for diagnostic tools, instruments, reagents, and supplies; foster a greater number of suppliers of new TB diagnostics, thereby having an impact on market dynamics and achieving a further cost reduction for rapid diagnostics; and improve the case detection and management of TB and MDR TB. The program is supporting the development of a preidenti- fied list of approved equipment and consumables, including LPA and liquid culture equipment. It also is supporting 40 laboratories for LPA equipment and consumables, and 31 laboratories for liquid culture equipment and consumables. The Global Fund Round 9 project further supports these laboratories in the implementation of the national laboratory scale-up plan through human resources, equipment, on-site technical support, and long- term mentoring and provides funds for human resource development and related costs for on-site technical support. Introducing LPA testing, very broadly, involves five steps: 1. establishing two to three clean rooms, including a hybridization room, an amplification room, and a master mix room; 2. supplying equipment and consumables for LPA; 3. training of both laboratory and field staff; 4. establishing LPA proficiency through a mechanism approved by the National Laboratory Committee; and 5. creating mechanisms for rapid transportation of patient specimens and reporting of results. According to Raizada, introducing liquid culture is more challenging than introducing the LPA. It involves the establishment of a biosafety level 3 laboratory along with air-handling and cooling units. Training and pro- ficiency testing are necessary. Also, MGIT requires an uninterrupted sup- ply of electricity, which in turn requires power backup at each laboratory because of the erratic power supply in some parts of India.
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69 DETECTING DRUG RESISTANCE As of April 2011, 28 districts in India representing a population of 46 million had screened 2,658 suspected cases of MDR TB in the previous year. For these cases, 92 percent of LPA results were available at the time of data collection, and 8 percent of test results were still pending. The inva- lidity rate in the uncontrolled field setting was 8 percent. Forty percent of samples were diagnosed with drug-resistant TB. The average time between collection of specimen from the field, transportation to the LPA laboratory, and testing and reporting of results was 7 days. Meanwhile, backup testing with Löwenstein-Jensen medium returned results on only 52 percent of the samples. The strengths of the Indian laboratory scale-up experience thus far have been • strong coordination at all levels in implementing the drug-resistant TB response plan; • quarterly national-level meetings by the Central TB Division, with the participation of the national reference laboratories and all implementing partners; • strong coordinated efforts in human resource development; • proactive program leadership at the central and state levels; • fast-track budget mobilization at the state level; and • contributions of WHO and RNTCP consultants in addressing field problems. Key challenges have included • dealing with delays in the development of an online management information system; • ensuring an uninterrupted supply of second-line drugs; • maintaining laboratory proficiency despite drug shortages; and • delivering treatments as volume increases and turnaround times decrease. POTENTIAL INNOVATIONS AND ACTION ITEMS Through the presentations provided in this session and the subsequent discussions, individual workshop speakers and presenters noted key innova- tions and action items. They include the following: • Priorities for all aspects of the TB program in India are needed, including for diagnosis, laboratory capacity, treatment, and infec- tion control.
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70 DRUG-RESISTANT TUBERCULOSIS IN INDIA • A strengthened tiered network at the subdistrict, district, regional, and reference laboratory levels is essential. • Because no single test can stand alone, molecular methods should be leveraged with culture methods. • Fostering a greater number of suppliers of new TB diagnostics would affect market dynamics and achieve a further cost reduction for rapid diagnostics.