Mycobacterium tuberculosis Beijing genotype and risk for treatment failure and relapse, Vietnam.

Among 2,901 new smear-positive tuberculosis cases in Ho Chi Minh City, Vietnam, 40 cases of treatment failure and 39 relapsing cases were diagnosed. All initial and follow-up Mycobacterium tuberculosis isolates of these case-patients had (nearly) identical restriction fragment length polymorphism patterns, and the Beijing genotype was a significant risk factor for treatment failure and relapse (odds ratio 2.8, 95% confidence interval 1.5 to 5.2).

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The Beijing genotype is widespread in Asia (1-3), and has been involved in outbreaks of multidrug-resistant tuberculosis in various parts of the world, including Cuba, Germany, Russia, and Estonia (4-7). The W strain, which caused a large outbreak of multidrug-resistant tuberculosis in the United States, is a variant of the Beijing genotype (8-10). The Beijing genotype is emerging in Vietnam in association with drug resistance in this region (11).

In a recent study on acquired drug resistance in Ho Chi Minh City, Vietnam, drug resistance at time of enrollment in the study was shown to be an important risk factor for treatment failure and for relapse of tuberculosis after treatment was completed successfully (12). We used the materials collected for this study to determine the extent to which the Beijing genotype is a risk factor for treatment failure or relapse.

The methods of this study have been described previously (12). In brief, 2,901 new case-patients with smear results positive for Mycobacterium tuberculosis were enrolled in Ho Chi Minh City, Vietnam, from August 1996 through July 1998. After a case was diagnosed at the district tuberculosis center, a sputum sample from the case-patient was sent to the reference laboratory, for a repeat microscopy examination of the sputum smear to confirm the diagnosis and to be stored at -20[degrees]C. All patients received the standard regimen of the National Tuberculosis Program, i.e., 2 mouths of streptomycin, isoniazid, rifampicin, and pyrazinamide, followed by 6 months of isoniazid and ethambutol (2SHRZ/6HE). When treatment failure (defined as a positive sputum smear 5 or 8 months after the onset of treatment) or relapse (defined as a positive sputum smear within 2 years alter scheduled treatment cessation) was noted, another sputum sample was collected, and both samples were cultured and tested for drug susceptibility with the proportion method. Restriction fragment length polymorphism (RFLP) typing was performed by using insertion element IS6110 as a probe (13,14) to exclude reinfection and laboratory cross-contamination.

A random sample of sputum samples was collected at enrollment for culture and sensitivity testing from 10% of patients who had nut experienced treatment failure or relapse (controls). This sample size would allow approximately two controls per case-patient. We performed spoligotyping on the sputum samples of case-patients who had experienced treatment failure or relapse and controls to identify the samples that belonged to the Beijing genotype (15). The Beijing genotype was defined as strains without spacers 1-34 and the presence of (at least 3) the spacers 35-43 (16).

Over the enrollment period, 6,113 new smear-positive tuberculosis patients began a treatment regimen, 2,901 of whom were included in the study. Slightly more men were enrolled than women (age-adjusted odds ratio [OR] 1.2, 95% confidence interval [CI] 1.0 to 1.3), and enrollment was particularly low in those [greater than or equal to] 65 years of age (sex-adjusted OR 0.3, 95% CI 0.2 to 0.4). Of the 2,901 enrolled patients, 2,568 (88%) recovered, and 12 (0.4%) completed treatment; in 125 (4.3%), treatment failed; 63 (2.2%) died; 53 (1.8%) were transferred out; and 80 (2.8%) did not complete the study. Through December 1999, a total of 168 case-patients who experienced a relapse (6.5% of those cured or with treatment completed) were identified. Forty of 125 case-patients whose treatment failed and 39 of 168 case-patients who had a relapse had two positive cultures with nearly identical RFLP patterns (12). Spoligotyping results were available for 136 controls.

Case-patients were somewhat less likely than controls to be female and tended to be somewhat older than controls. However, these differences were not significant. Primary drug resistance (in comparison with full susceptibility) was a strong risk factor for treatment failure or relapse with combined ORs of 3.4 for streptomycin monoresistance, 4.2 for isoniazid monoresistance, and 23 for other susceptibility patterns (Table). The Beijing genotype was associated with treatment failure (OR 3.3 95% CI 1.3 to 8.3; p < 0.01) and relapse (OR 2.4 95% CI 1.0 to 5.7; p < 0.05). In view of the small numbers and similar odds ratios, these two groups were combined (OR 2.8, 95% CI 1.5 to 5.2) (Table). The association between the Beijing genotype and treatment failure or relapse hardly changed when taking into account primary drug resistance, age, and sex (OR 3.2, 95% CI 1.4 to 7.1). We conclude that the Beijing genotype is a risk factor for treatment failure and relapse in Vietnam, irrespective of primary drug resistance. This finding suggests that infections with Beijing genotype strains are more persistent than infections with other M. tuberculosis strains, which may explain the emergence of Beijing genotype strains in this region (11).

This study had limited power to detect risk factors for relapse and treatment failure, mainly because of the relatively small numbers of case-patients in those categories. Recruiting a larger number of controls could not change this, since the selection of more than two controls per case, while increasing workload, has relatively little impact on the statistical power of the analysis. However, since the association between the Beijing genotype and treatment failure or relapse was strong, the association was significant despite limited power.

Beijing genotype strains may have several selective advantages over other genotypes of M. tuberculosis. In many, but not all, areas where Beijing genotype strains are prevalent, this genotype is associated with resistance to antitubereulosis drugs (17). The basis for this correlation has so far not been disclosed. However, recent findings indicated that exclusively in Beijing genotype strains, mutations are present in putative mutator genes (18). This finding may indicate that Beijing genotype strains have a higher ability than other strains to allow particular critical mutations in resistance genes, which enables them to acquire resistance to the drugs used in a standard treatment regimen.

This enhanced flexibility due to alterations in the DNA repair mechanism of Beijing genotype bacteria may also play a role in the interaction with the host immune defense system to deal with the less favorable conditions like exposure to oxygen and nitrogen radicals in intracellular environment. Extended research on the immunopathology caused by M. tuberculosis strains of different genotypes in a BALB/c mouse model has shown that most, but not all, Beijing genotype strains cause a more severe pathology, but a reduced immune response in comparison to other genotypes of M. tuberculosis (19).

If Beijing genotype strains have a selective advantage over other genotypes of M. tuberculosis, this may have important implications for future tuberculosis control. The enhanced capability to develop resistance and to interact with the host immune defense system may facilitate the spread of tuberculosis in Asia and in other areas. Currently, a worldwide survey is being conducted to measure the global spread of this genetically conserved group of M. tuberculosis strains and its association with resistance, active transmission (young age), and other factors. Although the conservation of Beijing genotype strains in Asia is highly pronounced, the conserved population structure of M. tuberculosis in other high-prevalence areas such as Africa also merits further research on the possible development of selective advantages.

Table. Characteristics at enrollment of case-patients who
experienced treatment failure and relapse and of controls
who did not experience treatment failure, relapse, or dies (a)

                                             All
                                            case-
Characteristic       Failure    Relapse    patients   Controls
Sex
  Male                  28         36         64        104
  Female                12         3          15         39
Age group (y)
  15-34                 15         9          24         60
  35-54                 22         25         47         72
  [greater than
   or equal to] 35      3          5          8          11
Genotype
  Beijing               32         29         61         75
  Other                 8          10         18         61
  Unknown                                                7
Resistance pattern
  Fully suss.           4          13         17        101
  S only                3          9          12         21
  H only                3          4          7          10
  Other               30 (d)     13 (e)       43         11
TOTAL                   40         39         79        143

                         Crude               Adjusted (b)

Characteristic        OR       95% CI       OR       95% CI
Sex
  Male                1
  Female             0.63   0.32 to 1.22
Age group (y)
  15-34               1
  35-54              1.63   0.90 to 3.0
  [greater than
   or equal to] 35   1.82   0.65 to 5.1
Genotype
  Beijing            2.8    1.48 to 5.15   2.5     1.2 to 5.2
  Other               1                     1
  Unknown
Resistance pattern
  Fully suss.         1                     1
  S only             3.4    1.41 to 8.2    4.0     1.6 to 9.9
  H only             4.2     1.39 to 12    4.7     1.5 to 15
  Other               23     10.0 to 54     23     9.7 to 55
TOTAL

                         Adjusted (c)

Characteristic        OR       95% CI
Sex
  Male                1
  Female             0.55   0.22 to 1.38
Age group (y)
  15-34               1
  35-54              2.0    0.90 to 4.6
  [greater than
   or equal to] 35   2.4    0.60 to 9.9
Genotype
  Beijing            3.2     1.4 to 7.1
  Other               1
  Unknown
Resistance pattern
  Fully suss.         1
  S only             3.9    1.54 to 9.9
  H only             5.0     1.54 to 16
  Other               26     10.3 to 64
TOTAL

(a) OR, odds ratio; CI, confidence interval; sucs., susceptible.

(b) Adjusted for genotype and resistance pattern.

(c) Adjusted for age, sex, genotype, and resistance pattern.

(d) Of These 30, 12 had resistance to H and S, 1 to H, S, and E,
10 to H, R, and S, and 7 to H, R, S, E.

(e) Of these 13, 11 had resistance to H and S, and 2 to H, S, and E.

This study was financially supported by the European Union project "New generation genetic markers and techniques for the epidemiology and control of tuberculosis" (grant QLK2-CT-2000-00630).

References

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Nguyen Thi Ngoc Lan, * Hoang Thi Kim Lien, * Le B. Tung, * Martien W. Borgdorff, ([dagger]) ([double dagger]) Kristin Kremer ([section]), and Dick van Soolingen ([section])

* Pham Ngoe Thach Tuberculosis and Lung Diseases Centre, Ho Chi Minh City, Vietnam; ([dagger]) Royal Netherlands Tuberculosis Association, The Hague, the Netherlands; ([double dagger]) University of Amsterdam, the Netherlands; and ([section]) National Institute of Public Health and the Environment, Bilthoven, the Netherlands

Dr. Lan is the director of the National Tuberculous Rreference Laboratory in Ho Chi Minh City, Vietnam, and is responsible for science and technology at the Pham Ngoc Thach Hospital (Tuberculosis and Lung Diseases Hospital for the South of Vietnam). Her research interests include the microbiology, epidemiology, and molecular biology of tuberculous.

Address for correspondence: Dick van Soolingen, National institute of Public Health and the Environment, P.O. Box 1, 3720 BA Bilthoven, the Netherlands; fax: [phone]; email: [e-mail]