Tuberculosis remains a major public health problem worldwide. Almost two million people die of TB annually and an estimated one-third of the world's population has latent infection. The situation is worse in developing countries in South-East Asia and Africa where MDR is on the increase. In six Asian countries, drug resistance is reportedly present in an estimated 2.8% of new and 18.8% of previously treated TB patients . Thailand, which is ranked 18th on a list of the 22 countries with the largest TB burdens, had a prevalence of approximately 192/100,000 people for all forms and an incidence rate of 62 new smear-positive cases per 100,000 in 2007 . The first national drug-resistance survey conducted in 2002 reported 1% of MDR-TB in new TB cases and 20% in previously treated cases . The rates had increased to 1.7% and 34.5%, respectively, by the second survey in 2006 . Moreover, TB surveillance during 1996–1998 showed that a high proportion of TB cases with drug resistance occurred in Chiang Rai province in northern Thailand.
In general, patients who have undergone successful treatment with anti-TB drugs can develop active disease again subsequently, referred to as recurrent TB. However, the pathogenesis of recurrence and classification of recurrent cases are still unclear . Recurrence of TB may be a result of regrowth of the treated bacterial strain in patients otherwise previously treated successfully, or of re-infection . Relapse refers to a patient becoming culture-positive again, or evidencing clinical or radiographic deterioration consistent with active TB, sometime after completion of apparently successful anti-TB drug therapy that had resulted in culture-negativity . The term retreatment refers to patients with recurrent TB who defaulted before completing their previous therapy or in whom initial treatments failed . Whether recurrent TB represents exogenous re-infection by a new strain of M. tuberculosis or endogenous reactivation of the original strain is controversial and has been debated for decades , . The importance of each of these possibilities likely varies according to the epidemiological context, the spread of MDR-TB, HIV infection , and the immigration of people from developing countries, which could modify disease transmission in areas at low risk of TB . In patients infected with HIV and MDR-TB there is evidence for a greater risk of reactivation than of re-infection , .
The most-widely used method for typing M. tuberculosis to determine whether recurrent TB represents endogenous reactivation or exogenous re-infection has been a molecular method involving Southern blotting of PvuII-digested chromosomal DNA and hybridization with the insertion sequence (IS) 6110 . In practice, it is generally accepted that two or more isolates with identical or near-identical (± one band) IS6110 fingerprints (known as clusters) represent a recent transmission event . This technique is thus useful in distinguishing between recent epidemiological events (transmission) and distant epidemiological events (reactivation) . In a single TB patient with a TB-free interval, it is assumed that isolates with identical IS6110- RFLP patterns denote endogenous reactivation of the previously infecting bacteria .
Apart from differentiating between endogenous reactivation and exogenous re-infections, IS6110-RFLP has allowed identification of different M. tuberculosis strains with varying degrees of virulence and drug resistance in different geographical areas . Up to now, the largest family of M. tuberculosis strain has been the Beijing family. The highest prevalence of this family reportedly occurs in Asian patients  and it is associated with various phenotypes such as drug-resistance , treatment failure, relapse and febrile response to TB treatment . In several Asian studies, the proportion of TB due to Beijing strains has been > 50% . However, because the IS6110-RFLP patterns vary between different geographical areas, there is so far limited available data regarding recurrent TB in Thailand. Although there was a national anti-TB drug resistance survey during 1997–1998 as part of a global project to evaluate IS6110-RFLP patterns and the extent of clustering, this study did not assess linkage to TB treatment history and development of drug resistance. Other subsequent study in Chiang Rai assessed acquired drug resistance in patients who had become positive again after completion, default from or failure of a standardized treatment regimen . These researchers commonly found non-identical IS6110-RFLPs in the first and subsequent episodes in TB-HIV patients. Successful treatment of TB depends upon selection of an effective drug regimen; however, drug resistance can evolve in originally drug-susceptible strains during anti-TB treatment. Therefore, this study aimed to evaluate the relationship between the quality of treatment and development of resistance by assessing drug resistant M. tuberculosis in relation to the molecular patterns in recurrent TB patients with either relapse or retreatment TB in Chiang Rai province in northern Thailand.
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In this study, we analyzed anti-TB drug resistance and IS6110-RFLP patterns of M. tuberculosis strains from recurrent patients in their first, second and third TB registrations. Resistance to INH, RMP, EMB and SM occurred more frequently in M. tuberculosis strains from the second registrations than in those from the first. The MDR rate was 12.2% among new cases at first registration, 22.5% among recurrent cases with previously treated TB at the second registration and 12.5% at third registration, indicating higher rate of drugs resistance in recurrent TB. In the present study, we found resistance to INH or RMP, with or without resistance to any other drugs (MDR-TB), more commonly in patients with failure and default treatment. Moreover, two acquired MDR-TB isolates from retreatment patients after treatment failure developed resistance to all anti-TB drugs tested (RMP, INH, Sm and EMB). These strains could well develop XDR-TB eventually. Recently, it has been recognized that MDR-TB and XDR-TB are serious problems for TB control program because XDR-TB strains are virtually untreatable . There is evidence that inadequate treatment is the main cause of endogenous reactivation, which is likely to occur soon after completion of treatment for the first episode . However, apparently successful treatment sometimes fails to totally eradicate the bacteria from patients; they can then re-activate later. TB patients who fail primary treatment have significantly greater risks of any drug resistance or MDR-TB than do those with successfully complete treatment (treatment completion or cure) . There is evidence that irregular drug administration causes development of drug resistance during short-course therapy with multiple drugs, because the drugs are taken for only a few killing cycles and regrowth occurs when the drugs stop. During each cycle, it is possible that selection of mutants that are relatively resistant occurs . We found significant differences in monodrug resistance to isoniazid or rifampin and MDR among M. tuberculosis strains from primary and secondary registrations, which could be attributable primarily due to poorly administered TB treatment . Recurrent TB is common in patients who have failed to respond to first and second line drugs . In line with this, our findings that MDR-TB occurs most commonly in patients with failure and default treatment and rarely in those with previously successful treatment (complete and cure) imply that failure of previous treatment is associated with drug resistance. Therefore, in areas with a high prevalence of drug resistance, we recommend use of alternative regimens, especially during the continuation phase. Drug resistant TB is a man-made phenomenon; inadequate or poorly administered treatment regimens can allow drug-resistant strains to become dominant in patients with TB .
In general, there is a very high rate of unexplained recurrent TB in areas with a high incidence of TB . Endogenous reactivation is possibly the main cause of relapses after a period without TB and recurrent TB requiring retreatment in Chiang Rai province, an area with high prevalence of TB and a high proportion of drug resistance . In this study, endogenous reactivation (30/42, 71.4%) was the major cause of recurrent TB either from relapse or retreatment as evidenced by identical IS6110-RFLP of Beijing (21/42, 50%), Nonthaburi (3/42, 7.1%) and heterogenous (6/42, 14.3%) patterns in a large proportion of isolated strains. The classical IS6110-RFLP method fails to adequately differentiate M. tuberculosis strains with identical patterns of low copy numbers of IS6110 with 2–5 band (5/42, 11.9%) and single band patterns (5/42, 11.9%) . Other strain typing methods such as VNTR typing are required to infer epidemiological linkage between low-copy number isolates . However, VNTR typing systems cannot define all unique isolates. If the primary genotype is IS6110-RFLP, VNTR typing is certainly useful as a secondary means of typing M. tuberculosis with small copy numbers of IS6110. . It is possible that the identical IS6110-RFLP patterns that we found in first and subsequent TB registrations did not truly represent endogenous reactivation because these patterns appearances may have reflected the duration of the study, incidence of M. tuberculosis strains in the population and prevalence of dominant strains. Therefore, we recommend further comprehensive investigation of the prevalence of IS6110-RFLP patterns among M. tuberculosis strains from this set of patients during 1997 through 2006 to determine the proportion of these strains in recurrent TB.
The non-identical IS6110-RFLP patterns observed in two retreatment patients after failure could be caused by either exogenous re-infection (2/42, 5%) with new M. tuberculosis strains or mixed infection, which reportedly occurs after successful treatment ,  and even during treatment . Exogenous re-infection plays a dominant role in the pathogenesis of post-primary TB in areas with a high incidence of the disease such as in South Africa and China , . Simultaneous infection with multiple strains of M. tuberculosis can cause exogenous reinfection of patients: this provides further evidence for occurrence of reinfection .
We found that the Beijing family IS6110-RFLP pattern predominated among strains causing recurrent TB (50%). The rest of the isolates belonged to the Nonthaburi family, were heterogeneous, or had 2–5 band or single band patterns. These findings are quite different from those of a previous study in which the Beijing family was not the predominant pattern in northern Thailand, comprising only 17.7% of isolates) . The prevalence rates of Beijing strain in other geographical areas is diverse, for example being 42% in Bangkok, 27.9% in central and 31.3% in western Thailand . However, such discrepancies may be attributable to previous studies including all forms of pulmonary TB without differentiating between different histories of anti-TB treatment. In this study, the major patterns in MDR-TB belonged to the Beijing family, the rest being heterogeneous and having 2–5 bands. Additionally, isolates with acquired MDR evidenced identical IS6110-RFLP patterns of the Beijing family, suggesting its strong association with response to treatment , . In Germany, where the incidence of TB is steadily decreasing and the estimated overall percentage of MDR-TB is less than 3% of all TB cases, researchers have found similar evidence for Beijing genotypes among MDR-TB strains . A study in Thailand between 1996 and 2007 proposed a classification of ancestral and modern Beijing sublineages based on the VNTR in which they identified 78.8% as modern Beijing strains and the remaining 21.2% as ancestral Beijing isolates . Although researchers have further analyzed such data and combined it with those of previous studies to construct a comprehensive phylogenetic tree, they have not assessed linkage between TB treatment history and development of drug resistance . Therefore, we recommend further comprehensive investigation of the genetic diversity and evolution of the Beijing genotype in M. tuberculosis isolates from patients with recurrent TB.
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We thank the participating patients for their kind participation in this study and staff of the Chiang Rai provincial hospital and the National TB Reference Laboratory, Bureau of Tuberculosis, Bangkok for their technical support. This work was supported by Health and Labor Science Research Grants for Research on Emerging and Re-emerging Infectious Diseases (H17-shinko-021 and H20-shinko-014), Ministry of Health, Labor and Welfare, Japan; the TB/HIV Research Project, Thailand, a collaborative research project between the Research Institute of Tuberculosis and the Japan Anti-tuberculosis Association; and the Faculty of Tropical Medicine, Mahidol University.