Mycobacterium tuberculosis Beijing genotype, the Netherlands.

To determine whether the Beijing genotype of Mycobacterium tuberculosis is emerging in the Netherlands, we collected data on 6,829 patients during 1993 to 2000. Six percent had the Beijing genotype. This genotype was associated with diagnosis in recent years, young age, nationality, and multidrug resistance.

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The Beijing genotype is found frequently in Asia (1-3) but also in outbreaks of multidrug-resistant tuberculosis (MDRTB) in various parts of the world, including Cuba, Germany, Russia, and Estonia (4-7). The largest known epidemic of MDRTB in North America was caused by the W strain, a variant of the Beijing genotype (8 9). A recent study showed this strain's emergence and association with drug resistance in Vietnam (10). The Beijing genotype was also responsible for a recent outbreak of tuberculosis (TB) on the Canary Islands (11).

The relatively high degree of genetic conservation of Beijing genotype strains found in a widespread area (12) suggests a recent dissemination and, hence, selective advantages associated with this genotype of Mycobacterium tuberculosis. Recently, Beijing genotype bacteria were reported to carry mutations in putative mutator genes, which may explain a higher adaptability of these bacteria under stress conditions such as exposure to the intracellular environment or anti-TB drugs (13). These mutations may also be the basis of differential interaction between Beijing genotype bacteria and the host immune defense system suggested in a recent study in Indonesia (14).

In the Netherlands, MDRTB is affecting <1% of new TB patients, in particular immigrants (15-17). The incidence of the Beijing genotype has not yet been described. The goal of our study was to determine whether the Beijing genotype is emerging in the Netherlands and whether this genotype is associated with multidrug resistance.

The Study

Patient data were obtained from the Netherlands Tuberculosis Register, which is maintained by the Royal Netherlands Tuberculosis Association and has been in place since 1993. Municipal health services, which are responsible for the followup of all TB patients, send information using standardized, precoded forms on all reported TB cases to the register. The register includes information on demographic characteristics, case detection, risk groups, type of disease, treatment, and treatment outcome.

Isolates obtained from all 8,210 culture-positive patients underwent restriction fragment length polymorphism (RFLP)-typing with IS6110 as a probe at the National Institute of Public Health and the Environment. The Beijing genotype was defined on the basis of spoligotype (no spacers 1-34; at least 4 of spacers 35-43) and a specific region A insertion (18). Certain IS6110 RFLP-genotype families (clades 47 and 61) were consistently found to be Beijing genotype (18). Isolates were assigned to the Beijing genotype on the basis of the IS6110 RFLP pattern (18). If any doubt about clade membership existed, we used spoligotyping for the final allocation.

The RFLP results for the period 1993-2000 were matched to patient data on the basis of date of birth, postal area code, and sex, resulting in a perfect match for 5,994 (73%) patients and a near-perfect match for 835 (10%) patients; overall, 83% of the samples matched (6,829 patients). During this period, the Beijing genotype was observed in 516 cultures, representing 6% of the 8,210 patients. In the matched dataset, 415 (6%) of 6,829 were of the Beijing genotype. The number of TB cases attributable to the Beijing genotype tended to increase over time (Figure; p>0.05). This tendency was observed both among Dutch patients (r-0.66, 95% confidence interval [CI]-0.08 to 0.93) and non-Dutch patients (r=0.59, 95% CI-0.20 to 0.91) (Figure). While the increase in the number of cases among immigrants may be associated with an overall increase of immigrants to the Netherlands during the study period, the increasing numbers among Dutch patients likely reflect an increasing incidence rate.

The proportion of TB patients who had the Beijing genotype was significantly associated in univariate analysis with a later year of diagnosis, young age, nationality (increased among immigrants from Asia and decreased among those from Morocco, Turkey, Somalia, and other African countries, compared with Dutch citizens), and multidrug resistance (Table). These associations persisted in multivariate analysis (Table).

The association between the proportion of TB cases with the Beijing genotype and age was observed in particular in Dutch patients ([chi square.sub.trend] 15.5; p<0.0001), and was not clear among non-Dutch patients ([chi square.sub.trend] 0.8; p>0.2). Among Dutch patients, the Beijing genotype was more commonly found in new cases (6%, 133/2,130)

than among retreated cases (4%, 18/498; p<0.05). Among non-Dutch patients, the Beijing genotype was slightly more common in retreated cases (8%, 19/229) than in new cases (6%, 204/3,218); however, this occurrence was not significant (p>0.2). The incidence pattern by person and time appears consistent with transmission of the Beijing genotype from immigrants to the Dutch population (19).

The proportion of TB cases with the Beijing genotype was not substantially associated with HIV infection, pulmonary localization, RFLP clustering, or duration of stay in the Netherlands (Table). The lack of association with RFLP-clustering suggests that the Beijing genotype is not spreading more quickly than other M. tuberculosis strains.

Of the nine patients with the Beijing genotype and multidrug resistance, four were from central and eastern Europe, three from Asia, one from Africa, and one from the Netherlands. One of these nine patients was RFLP-clustered; the other patient in that RFLP cluster had been diagnosed previously and had isolated isoniazid resistance.

During 1993 to 2000 in the Netherlands, the Beijing genotype represented 6% of TB cases. The Beijing genotype was associated with recent diagnoses, young age (in particular among Dutch citizens), nationality (Eastern Europe and Asia), and multidrug resistance. Although the Beijing genotype was associated with multidrug resistance, the number of MDRTB cases was small. No secondary cases were observed with the Beijing genotype and MDRTB. Of the nine cases of MDRTB, only one case may have been caused by transmission within the Netherlands from a case-patient who had isoniazid resistance at the start of treatment and may have acquired rifampicin resistance during treatment. However, the spread of the Beijing genotype among young people suggests the possible emergence of multidrug resistance and emphasizes the need for continued surveillance.

Table. Risk factors for the Beijing genotype of Mycobacterium
tuberculosis, the Netherlands, 1993-2000

Risk factor                       Beijing genotype    Total    %

Y of diagnosis
1993                                     23             669     3
1994                                     41             941     4
1995                                     56             863     6
1996                                     50             946     5
1997                                     64             872     7
1998                                     58             804     7
1999                                     74             879     8
2000                                     49             855     6

Sex
Male                                    254           4,135     6
Female                                  161           2,694     6

Age group
<25                                     120           1,522     8
25-34                                   123           1,925     6
35-44                                    64           1,080     6
45-54                                    23             614     4
55-64                                    24             493     5
65-74                                    40             517     8
75+                                      21             678     3

Nationality
Netherlands                             160           2,825     6
Europe (central and eastern)             20             231     9
Turkey                                    3             339     1
Morocco                                  19             650     3
Somalia                                  21             957     2
Africa (other)                           28             594     5
Asia                                    140             786    18
Other                                    19             345     6
Unknown                                   5             102     5

RFLP clustering (a)
No                                      212           3,227     7
Yes, first case                          57           1,052     5
Yes, later case                         146           2,550     6

Localization
Pulmonary                               268           4,064     7
Extrapulmonary                          113           2,122     5
Pulmonary and extrapulmonary             34             643     5

Residing in Netherlands
<6 months                                46             641     7
6-11 mo                                  12             301     4
12 23 mo                                 28             358     8
2-4 y                                    53             767     7
[greater than or equal to] 5 y          122           1,710     7
Born in Netherlands                     117           2,437     5
No information                           37             615     6

Drug resistance
Susceptible                             332           5,910     6
H only                                   11             227     5
S only                                   34             358     9
Other patterns                           26             221    12
Multidrug-resistant                       9              53    17
Unknown                                   3              60     5

Multidrug resistance
Yes                                       9              53    17
No                                      406           6,776     6

HIV infection
Yes                                      14             294     5
No                                      401           6.535     6

                                                Odds ratio

                                             Adjusted (b) (95%
Risk factor                       Crude    confidence intervals)

Y of diagnosis
1993                              1.09     1.08 (1.03 to 1.13)
1994                                            (per year)
1995
1996
1997
1998
1999
2000

Sex
Male                              1
Female                            0.97

Age group
<25                               1                  1
25-34                             0.80     0.69 (0.53 to 0.91)
35-44                             0.74     0.60 (0.43 to 0.83)
45-54                             0.45     0.36 (0.23 to 0.57)
55-64                             0.60     0.55 (0.35 to 0.88)
65-74                             0.98     0.78 (0.52 to 1.15)
75+                               0.37     0.30 (0.18 to 0.50)

Nationality
Netherlands                       1                  1
Europe (central and eastern)      1.58     1.27 (0.77 to 2.09)
Turkey                            0.15     0.12 (0.04 to 0.38)
Morocco                           0.50     0.41 (0.25 to 0.68)
Somalia                           0.37     0.27 (0.17 to 0.44)
Africa (other)                    0.82     0.63 (0.41 to 0.96)
Asia                              3.61     3.01 (2.32 to 3.91)
Other                             0.97     0.86 (0.53 to 1.42)
Unknown                           0.86     0.73 (0.29 to 1.84)

RFLP clustering (a)
No                                1
Yes, first case                   0.81
Yes, later case                   0.86

Localization
Pulmonary                         1
Extrapulmonary                    0.80
Pulmonary and extrapulmonary      0.79

Residing in Netherlands
<6 months                         1
6-11 mo                           0.54
12 23 mo                          1.10
2-4 y                             0.96
[greater than or equal to] 5 y    0.99
Born in Netherlands               0.65
No information                    0.83

Drug resistance
Susceptible                       1
H only                            0.86
S only                            1.76
Other patterns                    2.24
Multidrug-resistant               3.44
Unknown                           0.88

Multidrug resistance
Yes                               3.21     2.64 (1.22 to 5.74)
No                                1                  1

HIV infection
Yes                               0.76
No                                1

(a) RFLP, restriction fragment length polymorphism.

(b) Adjusted for year of diagnosis, age, nationality, and multidrug
resistance.

Acknowledgments

We thank all municipal health services in the Netherlands for their contribution to the Netherlands Tuberculosis Register.

This study was performed within the framework of Concerted Action project QLK2-2000-00630 on Molecular Epidemiology of Tuberculosis.

References

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Martien W. Borgdorff, * Petra de Haas, ([double dagger]) Kristin Kremer, ([double dagger]) and Dick van Soolingen ([double dagger])

* Royal Netherlands Tuberculosis Association, the Hague, the Netherlands; ([dagger]) University of Amsterdam, Amsterdam, the Netherlands; and ([double dagger]) National Institute of Public Health and the Environment (RIVM), the Hague, the Netherlands

Dr. Borgdorff is the chief epidemiologist of the Royal Netherlands Tuberculosis Association (KNCV) and professor of international health with special reference to tuberculosis at the University of Amsterdam. His areas of interest include the molecular epidemiology of tuberculosis and other communicable diseases.

Address for correspondence: M.W. Borgdorff, Royal Netherlands Tuberculosis Association (KNCV), RO. Box 146, 2501 CC the Hague, the Netherlands; fax: [phone]; email: [e-mail]