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4 Diagnosis John Ridderhof of the GLI provided an overview of the state of TB diagnosis around the world and highlighted major gaps. Because of the worldwide shortage of laboratories capable of detecting drug resistance, a mere 5 percent of all MDR TB cases are detected. Table 4-1 shows results of a WHO survey of those countries with a high burden of TB. WHO recommends at least one culture laboratory per 5 million population and one facility capable of conducting drug susceptibility testing (DST) per 10 million. Ridderhof argued that, while these numbers may be adequate for the United States, they are inadequate for high-burden countries. Further- more, as the survey results indicate, actual capacity is far less than even these recommended levels. Many countries lack a national reference labora- tory to perform some of the most basic surveillance, and only a handful of countries meet the original recommended standard for culture laboratories. While there is additional capacity in the private sector and in some research institutions, it is generally not available to public health providers. The laboratories that are available are distributed disproportionately around the world; there is a particularly acute lack of supranational laboratories in sub-Saharan Africa. However, it is expected that the GLI will provide techni- cal assistance and proficiency testing programs to help build this capacity. ACTuAL NEED Current global capacity allows for the conduct of approximately 10 million culture tests for the diagnosis of TB, although much of that capacity is centered in developed countries. In a recent effort led by WHO, it was 

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 THREAT OF DRug-RESISTANT TubERCulOSIS TAbLE 4-1 Laboratory Capacity in High-Burden Countries, 2006 No. of Culture No. of DST National Laboratories Laboratories Reference per 5 Million per 10 Million Country Laboratory Population Population India Yes 0.03 0.07 China Yes 1.4 2.7 Indonesia No 0.9 1.8 South Africa Yes 1.3 2.7 Nigeria No 0.0 0.0 Bangladesh Yes 0.1 0.2 Ethiopia Yes 0.1 0.1 Pakistan No 0.1 0.2 Philippines Yes 0.2 0.3 Congo Yes 0.1 0.2 Russian Federation No 34 68 Vietnam Yes 1.0 2.1 Kenya Yes 0.3 0.5 Tanzania Yes 0.4 0.8 Uganda Yes 0.5 1.0 Brazil Yes 5.1 10 Mozambique Yes 0.2 0.5 Thailand Yes 5.1 10 Myanmar Yes 0.2 0.4 Zimbabwe Yes 0.4 0.8 Cambodia Yes 1.1 2.1 Afghanistan No 0.2 0.4 NOTE: DST = drug susceptibility testing. SOURCE: Ridderhof, 2008 (based on data from WHO, 2008a, and Stop TB data from Mohamed Abdel Aziz, private communication; used with permission). estimated that the actual need is at least 60 million culture tests (Weyer et al., 2007). There is also a critical need for enhanced DST capacity. To meet these needs, hundreds or thousands of new laboratories would have to be instituted around the globe. Considering just the centralized facilities needed to conduct molecular procedures for initial screening, many new facilities would be required. At least a $1 billion investment in laboratory capacity is necessary according to Ridderhof, not counting the training and systems that would have to be implemented in the facilities. There is general agreement among partner organizations, including U.S. government agencies, that an expanded global effort is needed to address this issue. At the same time, a significant coordination challenge exists because so many different implementing organizations are involved. Within PEPFAR, for example, at least 20 U.S.-based organizations are developing laboratory capacity in Africa. The result can be duplication of effort and conflicting recommendations from multiple technical assistance consultants.

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 DIAgNOSIS DIAGNOSTIC QuALITy If patients are smear positive, at least 95 percent of these-smear positive specimens should also have a positive culture since the culture test is more accurate than microscopy (Kent and Kubica, 1985). But the experience of the Japanese TB Institute in working with national reference laboratories in a number of countries indicates extremely low yields, and these results were obtained using older, more forgiving methods, such as solid culture. A number of factors—for example, transport problems—could explain these results. Historically, however, there has been limited quality assurance for drug resistance surveillance. CuRRENTLy AvAILAbLE DIAGNOSTICS Although the development of line probe assays1 and subsequent WHO approval have created a promising diagnostic tool for TB, scaling up the capacity to implement this tool widely will be challenging. Ridderhof out- lined key strategic priorities for overcoming these challenges. The first is to establish a country-specific roadmap to determine the sequence of events for strengthening laboratory capacity and coordinating all of the implementing partners so they have a common strategic plan. The second is to develop the human resources, both consultants and qualified individuals at the country level, needed to direct and implement additional capacity. These resources will be especially important for implementing methodologies more complex than microscopy, such as molecular procedures, cultures, and DST. The third priority is to focus on improving biosafety. Infection control is an issue not just in the clinic (see Chapter 3), but also in the laboratory. Grow- ing evidence indicates high rates of transmission in laboratories performing cultures and DST. Van der Walt discussed South Africa’s experience when the country investigated requirements for implementation of the line probe assay for routine diagnosis of MDR TB in 2008. An evaluation revealed that more than mere implementation of a new test in the laboratory was necessary; the approach to case finding—the systematic surveying of the population to identify all cases of infection—also needed to be changed to derive the full benefit of the new technology. Because of the issues identified above, South Africa decided to pursue a revised strategy for case finding of drug-resistant TB, in which screening for drug resistance takes place simultaneously with case finding. With this strategy, all specimens that are smear positive proceed immediately to a drug resistance screening with a 1 For more information on line probe assays, refer to WHO’s policy statement on Molecular Line Probe Assays for Rapid Screening of Patients at Risk of MDR TB, issued in 2008, avail- able at http://www.who.int/tb/dots/laboratory/lpa_policy.pdf.

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 THREAT OF DRug-RESISTANT TubERCulOSIS line probe assay. If there is any indication of resistance to rifampicin or isoniazid, a specimen proceeds to a full range of resistance tests while the patient is also referred for initiation of appropriate treatment. This strategy will be effective for early case finding of patients with resistant disease, and it has major implications for laboratories, as well as for patient referral processes. Currently, molecular assays are available in South Africa only at two provincial laboratories and the one national reference laboratory. The new strategy requires that (molecular) screening for drug resistance occur at the much lower-level primary health care facilities; this in turn means that far more molecular tests will be performed, but the number of unnecessary culture-based tests will be reduced. Salmaan Keshavjee of Partners In Health discussed current strategies used by WHO and others to address issues of diagnostic capacity. In 2005, the World Health Assembly passed a resolution requesting the Director Gen- eral to implement and strengthen strategies for the effective control and management of patients with drug-resistant TB. In 2006, the Foundation for Innovative New Diagnostics (FIND), WHO, and Partners In Health collaborated to build a laboratory in Lesotho, a country with very rudimentary laboratory facilities. FIND’s strategy was to provide the laboratory with a full-time technical assistance consultant and the right equipment, and to work with the laboratory to develop capacity, conduct training, and help establish the laboratory network. This strategy was effective in converting the laboratory to an effective state-of-the-art facility capable of performing both cell culture and DST. Since this pilot effort, the initiative has grown. In 2007, WHO and the Stop TB Partnership created the GLI, which will be active in 16 additional countries in Africa, South Asia, the Western Pacific, and Eastern Europe. The GLI’s goal is to diagnose 74,000 new MDR TB patients by 2011. A critical component of such an effort is securing sustainable funding from bilateral and multilateral donors to support the construction of in- country DST and rapid testing laboratories, as well as ongoing external quality assessments by supranational reference laboratories. The Lesotho example demonstrates that instituting ongoing quality assurance is a critical component of establishing new laboratories. Until these new laboratories are built, however, there is an immediate need for laboratory support to diagnose and treat MDR TB. Keshavjee suggested that excess laboratory capacity in the developed world should be used for this purpose. POINT-OF-CARE (POC) DIAGNOSTICS In addition to building comprehensive laboratories to conduct extensive diagnosis and analysis, there is a great need for cost-effective POC testing, such as a TB dipstick test or blood test, so that diagnosis can take place

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 DIAgNOSIS during the patient’s visit, and appropriate treatment can begin immediately. Two POC diagnostic systems that have been developed were discussed during the workshop: the GeneXpert cartridge system from Cepheid and another model from Akonni Biosystems Inc. The Geneva-based FIND, with support from the Bill and Melinda Gates Foundation and NIAID, worked with Cepheid to develop a cartridge system specifically geared to the detection of TB and the simultaneous pre- diction of rifampicin resistance directly from the sputum of suspected TB cases. David Persing of Cepheid described the GeneXpert cartridge system as having the following characteristics: • It is able to process both macrofluidics—specimens such as sputum, blood, stool, and pus (up to 4.5 milliliters)—and microfluidics. • It has the ability to concentrate and purify samples from large volumes before they are processed for polymerase chain reaction (PCR) detection, so it does not require preprocessing of sputum specimens by centrifugation. • It is a closed system, with no opportunity for contamination. • The analysis can be performed in an on-demand, stat basis as soon as specimens have been collected. Cepheid already has GeneXpert cartridge systems on the market for such pathogens as Group B streptococcus, enterovirus, methicillin-resistant Staphylococcus aureus (MRSA), and MRSA/SA (i.e., Staphylococcus aureus that is methicillin-sensitive). In addition, the system has been deployed for 3 years in 265 U.S. Postal Service mail sorting centers to detect the pres- ence of bacillus anthracis—the largest biothreat detection program in the country. To date, more than 7.5 million GeneXpert cartridges have been used to test 100 billion letters, with no false positives. To customize the kit for use in detecting TB, scientists needed to develop an efficient method for working with sputum samples. Using a combination of sodium hydroxide, alcohol, and detergents, Cepheid developed a method for filtering and concentrating TB organisms from sputum without the use of a centrifuge. Once the TB organisms have been processed, sonic lysis, in the presence of glass beads, is used to blast them open inside the cartridge and release the DNA. This is followed by a nested PCR protocol, which is risky if performed in a laboratory because of contamination; as noted, however, the cartridge is completely contained, and the nested amplification yields about a 10,000-fold increase in sensitivity compared with conventional single-stage amplification of the same target. The PCR process used can detect a single genome of TB about 30–40 percent of the time and can consistently detect five genomes about 95 percent of the time. The PCR process amplifies the rifampin resistance locus, which carries the mutations that specify rifampin

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 THREAT OF DRug-RESISTANT TubERCulOSIS resistance, and this is used as a surrogate marker for MDR TB because all the circulating MDR and XDR strains are rifampin resistant. FIND is currently managing a clinical evaluation of the GeneXpert TB cartridge in five countries—Peru, Germany, Azerbaijan, India, and three sites in South Africa—which will be completed in December 2008. Those data will then be correlated with molecular data. Preliminary results for TB detection from Peru and Latvia indicate about 99 percent sensitivity for smear-positive, culture-positive cases. Sensitivity for smear negatives was approximately 87.5 percent, with a specificity of 97.3. Although the number of samples was small, preliminary findings indicate that 100 per- cent of all rifampin-resistant and rifampin-sensitive strains were correctly identified. Charles Daitch of Akonni Biosystems Inc. described an alternative tech- nology developed by his company in the early 1990s through a collaboration involving the U.S. Department of Energy, the Defense Advanced Research Projects Agency, and the Russian government. This system performs both genetic and immuno-based assays using a technology that is scalable and costs as little as US$8.00 per test. The technology, developed by the Engelhardt Institute of Molecular Biology in Moscow, is called three-dimensional gel drop arrays, and provides a hydrogel environment. A nano test tube can be loaded with a nucleic acid or an antibody for sample screening and detection. The assay can detect TB to very low limits, can hold hundreds of nano test tubes, and is adaptable to the addition of future sequences as they become available for MDR and XDR TB. The system has the capability to process large samples (milliliters), avoid- ing the risk of not capturing and detecting the TB in a smaller sample. There are microarray screens for 75 different probes for profiling drug resistance, including two probes for TB complex—IS-6110 and IS-1245—and a probe for Mycobacterium aium, an organism that is resistant to most anti-TB drugs and can be mistaken for TB. Quality controls have been built into the assay. For example, everything on the array is replicated in quadruplicate, and the analysis automates interpretation to eliminate technician error. The system looks at the difference between the wild-type signal and the mutation signal and can detect resistance, but only if the mutations are screened for in the assay. The assay’s sensitivity depends on having the ability to screen for a wide array of mutations from throughout the world; sensitiv- ity could be enhanced by creating a repository of markers or sequences that could be used to develop the test profile. Akonni Biosystems is currently working with collaborators to expand the chip coverage for sequences related to XDR TB. Harrington offered some additional observations on the need for POC diagnostics and a commitment to research addressing the problem of diag- nostic capacity. While the recent development of complex and expensive

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 DIAgNOSIS technology is an important breakthrough, this technology is ill suited to resource-poor settings and is likely to remain unattainable for those most in need. Harrington advocated for the development of a TB dipstick test similar to the HIV pinprick test. That test, which costs US$1.00 and is 99 percent sensitive and specific, revolutionized HIV testing and was a key element in the scale-up of antiretrovirals worldwide. A TB dipstick would revolution- ize TB care by providing a POC test that would reduce or eliminate delay. Harrington asserted, “In some ways it is even more important than a new drug or a new vaccine. There is a cure for most cases of TB, and there is rea- sonable treatment for MDR. But if it can’t be diagnosed, millions of people will die of a treatable and curable disease.” Harrington outlined what needs to be done to achieve the goal of an effective dipstick test. First, there must be support from large organiza- tions such as NIH and the Bill and Melinda Gates Foundation, as well as small-scale innovative efforts supported by smaller donors. Funding for TB diagnostics research currently represents only 6 percent of NIH’s TB research investment and only 11 percent of its global TB research and development investment. Development of a TB dipstick test will also require a larger effort to identify biomarkers for active disease; platform development; and large, well-characterized clinical specimen banks more widely available than those in use today. Harrington suggested that meeting these needs will depend to some degree on effective advocacy.

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