Abstract
Mixed infection with Beijing and non-Beijing strains of Mycobacterium tuberculosis has been reported and has been suggested to mediate elevation of the reinfection rate in regions with a high incidence of tuberculosis (TB). To evaluate the prevalence of infection with both Beijing and non-Beijing strains of M. tuberculosis in eastern Taiwan, the region with the highest TB incidence in Taiwan, 185 active pulmonary TB patients were enrolled at Tzu Chi General Hospital from October 2007 to September 2008. A modified multiplex PCR method was developed to distinguish Beijing and non-Beijing strains directly using the sputum of patients. Of the 185 patients, 46.5% were infected with a Beijing strain, 42.2% were infected with a non-Beijing strain, and 11.3% were infected with both strain types. Notably, mixed infection with both strain types was not associated with TB treatment history or the high-incidence race group, aborigines. In addition, the incidence rate of mixed infection before treatment with anti-TB medication was as high as that in patients with a history of anti-TB treatment. Further analysis of antibiotic susceptibility revealed that Beijing strains alone had the highest multidrug resistance rate (17.5%), mixed infection had the highest rate of resistance to at least one drug (23.8%), and non-Beijing strains had the highest rate of sensitivity to all drugs (79.5%), implying that Beijing strains are predominant in the development of drug resistance in tuberculosis.
Tuberculosis (TB) remains a major public problem globally, and the prevalence of drug resistance in tuberculosis has been increasing (3). The disease is caused by infection with Mycobacterium tuberculosis and remains the single most important fatal infection in humans, causing around 1.3 million deaths and 9.27 million cases worldwide in 2007 (5, 28). The highest number of cases in 2007 was in Asia (55%), followed by Africa (33%) (28). A particular threat to the Asian community is the high prevalence of the Beijing strain (25); this strain is more prevalent in eastern Asian countries, including Taiwan, and is associated with higher frequencies of multidrug resistance (MDR) (8, 12, 16, 18, 22) and extensive drug resistance (XDR) (12). Notably, 46.2% of TB patients were found to be infected with a Beijing strain in eastern Taiwan in a study by Jou et al. (8).
The TB incidence rate in Hualien County, located in eastern Taiwan, in 2005 was 180.9 cases per 100,000 population, which was 2.86 times the average incidence rate in Taiwan as a whole (63.2 cases per 100,000 population) (11). In addition, the TB incidence rates in the three aboriginal townships (Sioulin, Wanrong, and Jhuosi) of Hualien County were 639.8, 249.4, and 330.1 cases per 100,000 population, respectively, all much higher than the rate in the whole of Hualien County (11). TB has been one of the major causes of death in Taiwan for decades, and the mortality rate due to TB is the highest in eastern Taiwan (6). However, the underlying factors mediating the persistent high TB incidence and mortality rates in eastern Taiwan are not conclusive.
Traditionally, infection and recurrence of TB have been believed to be caused by primary infection or reactivation from a single strain (21). However, infection with multiple M. tuberculosis strains, termed mixed infection, within a single individual has been increasingly documented and has recently attracted the attention of clinicians and TB control programs (2, 17, 19, 26). In general, mixed infection refers to infection with one strain that does not protect against infection with another strain (21). It has been documented that mixed infection can influence a clinician's judgment regarding treatment and the pharmaceutical development of a vaccine (1). In addition, anti-TB drug resistance may emerge in patients with mixed infection (19, 23, 26), due to the undetected strains (1). Thus, it is critical to accurately and rapidly discriminate mixed infection with different strains of M. tuberculosis simultaneously within a single individual.
Warren et al. developed a singleplex PCR method based on the comparative genomic data of M. tuberculosis to distinguish between Beijing and non-Beijing evolutionary lineages of M. tuberculosis in isolates from patients in an epidemiologic study in a city in South Africa (26). Their study showed that mixed infection with both Beijing and non-Beijing strains of M. tuberculosis was more frequent in retreatment cases (23%) than in new cases (17%). In the present study, we enrolled 185 active pulmonary TB patients in a high-incidence region of Taiwan to evaluate the prevalence of infection with Beijing and/or non-Beijing strains of M. tuberculosis. As no single-step, sensitive multiplex PCR method to rapidly differentiate evolutionary lineages of M. tuberculosis in active pulmonary TB patients was available, a modified multiplex PCR method was established in this study to distinguish Beijing and non-Beijing strains directly using the sputum of patients.
MATERIALS AND METHODS
Study population.
Patients at Tzu Chi General Hospital (TCGH) suspected of having active pulmonary TB from whom sputum specimens were collected from October 2007 to September 2008 were enrolled in this study. All patients were resident at an epidemiologic site in Hualien, eastern Taiwan. The final diagnosis of pulmonary TB was confirmed by sputum cultures. Charts were reviewed to obtain the following information: age, sex, race (aborigine or nonaborigine), history of TB treatment, results of sputum smear by acid-fast staining for M. tuberculosis, cavitations on chest radiograph, steroid use, and concurrent diseases such as diabetes mellitus, cancer, liver cirrhosis, uremia, pneumoconiosis, and AIDS. The study was approved by the ethics committee of TCGH.
Specimen preparation.
Sputum specimens were mechanically homogenized, liquefied with dithiothreitol in distilled water, and then split for two analyses, one for culture in BACTEC medium (BD, Franklin Lakes, NJ) and the other for DNA preparation. Sputum DNA preparation was done according to the manufacturer's instructions (RMtb-PCR; Roche PCR Diagnostics, Branchburg, N.J.). Briefly, a volume of 100 μl of each specimen was mixed with 500 μl of Sputum Wash Solution and centrifuged at 12,000 rpm for 10 min. The pellet was resuspended with 100 μl of Sputum Lysis Reagent, incubated at 60°C for 45 min, mixed with 100 μl of Sputum Neutralization Reagent, and stored for less than 1 week.
Beijing and non-Beijing differentiated-multiplex PCR.
A modified multiplex PCR method was developed in this study using primers described by Warren et al. (26). The multiplex PCR employed two sets of primers for the regions flanking the direct repeat (DR) of Beijing and non-Beijing strains. To identify M. tuberculosis with Beijing evolutionary lineage, primers complementary to the 3′ end of the IS6110 element and Rv2820 (5′-ACCGAGCTGATCAAACCCG-3′ and 5′-ATGGCACGGCCGACCTGAATGAACC-3′) were used and a positive amplification product of 239 bp was obtained. To determine the presence of M. tuberculosis strains of origins other than the Beijing evolutionary lineage, primers complementary to Rv2819 (5′-GATCGCTTGTTCTCAGTGCAG-3′ and 5′-CGAAGGAGTACCACGTGGAG-3′) were used and a positive amplification product of 569 bp was obtained. PCR was performed using a total volume of 25 μl in a DNA thermal cycler (Perkin-Elmer Corporation, Norwalk, CT) that had been programmed for 35 cycles of 1 min at 94°C, 1 min at 60°C, and 2 min at 72°C.
Spoligotyping validation.
Spoligotyping was performed according to our previous study (7). Only strains that hybridized to all of the last nine spacer oligonucleotides (spacers 35 to 43) were defined as Beijing strains, and M. tuberculosis strains of all other spoligotypes were defined as non-Beijing strains. We randomly selected spoligotyping-confirmed isolates of Beijing and non-Beijing strains to validate the results of the modified multiplex PCR method.
Drug susceptibility testing.
Drug susceptibility tests were performed by the indirect agar proportion method (Buddhist TCGH) as recommended by the WHO. The inoculums for susceptibility testing were grown from a primary culture or subculture, and tests were conducted using Middlebrook 7H10 agar medium impregnated with isoniazid (INH), rifampin (RIF), ethambutol (EMB), and streptomycin (SM). The antibiotic concentrations in the medium were 1.0 g/ml for INH, 1.0 g/ml for RIF, 10.0 g/ml for EMB, and 10.0 g/ml for SM. Bacteria in which growth on drug-containing media represented more than 1% of the number of colonies that developed on drug-free media were considered to be resistant to that agent. Primary resistance was defined as the presence of drug susceptibility in isolates from TB patients who had never received treatment previously, while acquired resistance was defined as the presence of drug susceptibility in isolates from TB patients who had a history of previous treatment. Isolates resistant to both INH and RIF were defined as MDR M. tuberculosis, while isolates resistant to at least one drug were designated non-MDR M. tuberculosis.
Statistical analysis.
Data were analyzed by SPSS 11.5 for Windows (SPSS Inc. Chicago, IL). Categorical variables were analyzed using the chi-square test or Fisher's exact test. A P value of <0.05 was considered statistically significant.
RESULTS
Description of patients.
During the study period, 185 TB patients whose sputum cultures were positive for M. tuberculosis were enrolled (Table 1), including 128 (69.2%) males and 57 (30.8%) females with an average age of 52.4 ± 19.6 years (range, 2 to 92 years). The numbers of aboriginal and nonaboriginal patients were 90 (48.6%) and 95 (51.4%), respectively. Chest radiographs showed cavitation in 61 patients (33.0%) and no cavitation in 124 patients (67.0%). Concurrent diseases were noted in 59 (31.9%) patients; 1 patient had been diagnosed with AIDS according to the chart record. Positive sputum smear reports were noted in 96 (51.9%) patients. Among the whole group of patients, 144 (77.8%) had never been treated with anti-TB medication.
TABLE 1.
Characteristics of patients infected with Beijing, non-Beijing, and both M. tuberculosis strain types
| Characteristic | Total | No. (%)a of patients infected with: |
P value | ||
|---|---|---|---|---|---|
| Beijing | Non-Beijing | Both | |||
| No. (%) of patients | 185 | 86 (46.5) | 78 (42.2) | 21 (11.3) | |
| Sex | |||||
| Male | 128 | 64 (50.0) | 47 (36.7) | 17 (13.3) | 0.068 |
| Female | 57 | 22 (38.6) | 31 (54.4) | 4 (7.0) | |
| Age (yr) | |||||
| ≤24 | 15 | 5 (33.3) | 8 (53.3) | 2 (13.3) | 0.101 |
| 25-44 | 58 | 22 (37.9) | 30 (51.7) | 6 (10.3) | |
| 45-64 | 55 | 23 (41.8) | 25 (45.5) | 7 (12.7) | |
| ≥65 | 57 | 36 (63.2) | 15 (26.3) | 6 (10.5) | |
| Aboriginal | |||||
| Yes | 90 | 36 (40.0) | 45 (50.0) | 9 (10.0) | 0.110 |
| No | 95 | 50 (52.6) | 33 (34.7) | 12 (12.6) | |
| Concurrent disease | |||||
| Yes | 59 | 25 (42.4) | 26 (44.1) | 8 (13.6) | 0.683 |
| No | 126 | 61 (48.4) | 52 (41.3) | 13 (10.3) | |
| History of TB treatmentb | |||||
| Yes | 41 | 21 (51.2) | 14 (34.1) | 6 (14.6) | 0.459 |
| No | 144 | 65 (45.1) | 64 (44.4) | 15 (10.4) | |
| Sputum smear | |||||
| Positive | 96 | 41 (42.7) | 42 (43.8) | 13 (13.5) | 0.455 |
| Negative | 89 | 45 (50.6) | 36 (40.4) | 8 (9.0) | |
| Cavitation on chest X-ray | |||||
| Positive | 61 | 33 (54.1) | 20 (32.8) | 8 (13.1) | 0.194 |
| Negative | 124 | 53 (42.7) | 58 (46.8) | 13 (10.5) | |
The percentage in parentheses represents the number of patients divided by the total number of patients within each category.
A history of TB treatment was defined as treatment for 1 month or more.
Beijing and non-Beijing differentiated-multiplex PCR.
To differentiate the Beijing and non-Beijing strains directly using sputum from the enrolled patients, we established a modified multiplex PCR method based on that described by Warren et al. (26). In order to perform a Beijing and non-Beijing differentiated-multiplex PCR in a single reaction mixture, we first applied a combination of two sets of primers to the DNA extracted from known Beijing and non-Beijing strains of cultured M. tuberculosis, validated by spoligotyping in our previous study (7). An amplified product of 239 bp was identified in the DNA from the Beijing strain alone (Fig. 1A, line 1), whereas an amplified product of 569 bp was identified in that from the non-Beijing strain alone (Fig. 1A, line 2). We then mixed DNA from the two strains and subjected the mixture to amplification with the two sets of primers, which resulted in two PCR products of 239 bp and 569 bp (Fig. 1A, line 3). Further validation by spoligotyping was performed on 10 randomly selected Beijing strain isolates (no. 1 to 10) and nine randomly selected non-Beijing strain isolates (no. 11 to 19) (Fig. 1B). Beijing strain isolates (no. 1 to 10) showed an amplified product of 239 bp, whereas non-Beijing strain isolates (no. 11 to 19) showed an amplified product of 569 bp (Fig. 1C). Thus, the strain discrimination by PCR is in accord with that by spoligotyping. These results confirmed the sensitivity and specificity of the multiplex PCR detection system in distinguishing between Beijing and non-Beijing strains of M. tuberculosis.
FIG. 1.
Beijing and non-Beijing differentiated-multiplex PCR. (A) Multiplex PCR of cultured Beijing and non-Beijing strains. PCR-amplified products were obtained from DNA extracted from a Beijing strain alone (lane 1), a non-Beijing strain alone (lane 2), and both strain types (lane 3). Lane M, 100-bp ladder for molecular size comparison. (B) Spoligotyping of 10 randomly selected clinical Beijing strain isolates (no. 1 to 10) and nine randomly selected clinical non-Beijing strain isolates (no. 11 to 19). (C) Validation of the spoligotyping results by multiplex PCR. Isolates 1 to 10 showed an amplified product of 239 bp, and isolates 11 to 19 showed an amplified product of 569 bp.
Detection of infectious strains of M. tuberculosis.
The Beijing and non-Beijing differentiated-multiplex PCR was then applied to determine the presence of Beijing and/or non-Beijing strains directly using sputum specimens. A representative PCR assay of 21 randomly selected sputum specimens is shown in Fig. 2. A PCR-amplified product of 239 bp from the sputum specimen indicated the presence of a Beijing strain in the sputum of patients 1, 3, 5, 8 to 9, 11, 13, and 16 to 21. A positive amplification product of 569 bp from the sputum specimen indicated the presence of a non-Beijing strain in the sputum of patients 2, 6, 7, 10, 12, and 15. Two PCR-amplified products of 239 bp and 569 bp from the sputum specimen indicated the presence of both Beijing and non-Beijing strains in the sputum of patients 4 and 14. On the basis of the 185 clinical samples, we identified 86 patients (46.5%) as being infected with a Beijing strain, 78 patients (42.2%) with a non-Beijing strain, and 21 patients (11.3%) with both Beijing and non-Beijing strains (Table 1). Among the patients infected with Beijing, non-Beijing, and both types of strains, no significant differences were present with regard to sex, age, race, concurrent diseases, TB treatment history, results of sputum smear, or cavitation on the chest radiograph (Table 1).
FIG. 2.
Multiplex PCR amplification of DNA directly using sputum of patients. Shown is a representative PCR assay of 21 randomly selected sputum specimens in this study. The sputum specimens from patients 1, 3, 5, 8, 9, 11, 13, and 16 to 21 showed an amplified product of 239 bp; the sputum specimens from patients 2, 6, 7, 10, 12, and 15 showed an amplified product of 569 bp; and the sputum specimens from patients 4 and 14 showed two PCR products of 239 bp and 569 bp. Lane M, 100-bp ladder for size comparison.
Drug susceptibility analysis.
In accordance with the recommendations of the TB control program (11), drug susceptibility testing by the proportion method is routinely performed for all culture-confirmed cases of TB. The results of the drug susceptibility tests of the 185 M. tuberculosis isolates collected in this study were analyzed and are summarized in Table 2 . The overall drug sensitivity rates of pansusceptible M. tuberculosis (sensitive to all drugs), non-MDR M. tuberculosis (resistant to at least one drug but not MDR M. tuberculosis), and MDR M. tuberculosis (resistant to INH and RIF at least) were 74.6%, 15.1%, and 10.3%, respectively. The individual drug resistance rates were as follows: 7.0% were resistant to INH, 7.6% to SM, 1.1% to RIF, and 1.1% to EMB. The primary drug susceptibility rates of pansusceptible M. tuberculosis, non-MDR M. tuberculosis, and MDR M. tuberculosis were 79.2%, 15.3%, and 5.6%, respectively, and the acquired resistance rates were 58.5%, 14.6%, and 26.8%, respectively. Statistical analysis revealed significant differences in drug susceptibility between patients with a history of TB treatment (acquired resistance) and those without a history of TB treatment (primary resistance) (P < 0.001 by chi-square test). In particular, patients with a history of TB treatment had a lower rate of infection with pansusceptible M. tuberculosis and a higher rate of infection with MDR M. tuberculosis than did those without a history of TB treatment.
TABLE 2.
Prevalence of drug susceptibility to first-line anti-TB agents
| Drug susceptibility | Total (n = 185) | No. (%)a of isolates grouped by history of TB treatment |
|
|---|---|---|---|
| Primary resistance (n = 144)b | Acquired resistance (n = 41)c | ||
| Pansusceptibled | 138 (74.6) | 114 (79.2) | 24 (58.5) |
| Non-MDRe | 28 (15.1) | 22 (15.3) | 6 (14.6) |
| INH | 13 (7.0) | 9 (6.3) | 4 (9.8) |
| SM | 14 (7.6) | 11 (7.6) | 3 (7.3) |
| RIF | 2 (1.1) | 2 (1.4) | 0 (0.0) |
| EMB | 2 (1.1) | 2 (1.4) | 0 (0.0) |
| MDRf | 19 (10.3) | 8 (5.6) | 11 (26.8) |
The percentage in parentheses represents the number of isolates divided by the total number of isolates within each group.
Primary resistance was defined as the presence of drug susceptibility in isolates from TB patients who had never received any treatment for TB.
Acquired resistance was defined as the presence of drug susceptibility in isolates from TB patients who had a history of previous TB treatment.
Pansusceptibility was defined as sensitivity of isolates to all antibiotics.
Non-MDR TB was defined as resistance of isolates to at least one drug.
MDR TB was defined as resistance of isolates to both INH and RIF.
Drug susceptibilities of different strains of M. tuberculosis.
Further analysis of the distribution of drug susceptibility in groups infected with Beijing, non-Beijing, and both strain types revealed that the resistance rates of pansusceptible M. tuberculosis were 70.9%, 79.5%, and 71.4%, respectively (Table 3); the resistance rates of non-MDR M. tuberculosis were 11.6%, 16.7%, and 23.8%, respectively; and the resistance rates of MDR M. tuberculosis were 17.4%, 3.8%, and 4.8%, respectively. Statistical analysis identified significant differences in the drug resistance rate among the groups infected with a Beijing strain alone, a non-Beijing strain alone, and a mixture of both strain types (P = 0.035 by Fisher's exact test). Among these three groups, the group infected with a Beijing strain alone had the highest MDR M. tuberculosis infection rate, 17.4%. In addition, the group infected with both strain types had the highest non-MDR M. tuberculosis infection rate, 23.8%, whereas the group infected with a non-Beijing strain alone had the highest pansusceptible M. tuberculosis infection rate, 79.5%.
TABLE 3.
Distribution of drug susceptibility in groups of patients infected with strains of different evolutionary lineages
| Drug susceptibility | No. (%)a of isolates grouped by evolutionary lineage |
||
|---|---|---|---|
| Beijing (n = 86) | Non-Beijing (n = 78) | Both (n = 21) | |
| Pansusceptible | 61 (70.9) | 62 (79.5) | 15 (71.4) |
| Non-MDR | 10 (11.6) | 13 (16.7) | 5 (23.8) |
| MDR | 15 (17.4) | 3 (3.8) | 1 (4.8) |
The percentage in parentheses represents the number of isolates divided by the total number of isolates within each group.
Drug susceptibilities of different strains within the subgroup of TB treatment history.
We further analyzed the distribution of drug susceptibilities in patients infected with Beijing and/or non-Beijing strains within the subgroup of TB treatment history (Table 4). The distribution of drug susceptibilities in patients infected with Beijing, non-Beijing, and both strain types within the subgroup of primary resistance revealed that the resistance rates of pansusceptible M. tuberculosis were 46.5%, 44.7%, and 8.8%, respectively; the resistance rates of non-MDR M. tuberculosis were 27.3%, 50.0%, and 22.7%, respectively; and the resistance rates of MDR M. tuberculosis were 75.0%, 25.0%, and 0%, respectively. Within the subgroup of acquired resistance, the resistance rates in patients infected with Beijing, non-Beijing, and both strain types of pansusceptible M. tuberculosis were 33.3%, 45.8%, and 20.8%, respectively; the resistance rates of non-MDR M. tuberculosis were 66.7%, 33.3%, and 0%, respectively; and the resistance rates of MDR M. tuberculosis were 81.8%, 9.1%, and 9.1%, respectively. While no significant difference in drug resistance between groups infected with a Beijing strain alone, a non-Beijing strain alone, and a mixture of both strain types was identified within each subgroup of TB treatment history, the group infected with a Beijing strain alone had the highest MDR rate in both the primary and acquired resistance subgroups.
TABLE 4.
Distribution of drug susceptibilities in patients infected with Beijing and/or non-Beijing strains within the subgroup of TB treatment history
| Drug susceptibility | No. (%)a of isolates grouped by history of TB treatment |
|||||||
|---|---|---|---|---|---|---|---|---|
| Primary resistance (n = 144) |
Acquired resistance (n = 41) |
|||||||
| Total | Beijing | Non-Beijing | Both | Total | Beijing | Non-Beijing | Both | |
| Pansusceptible | 114 | 53 (46.5) | 51 (44.7) | 10 (8.8) | 24 | 8 (33.3) | 11 (45.8) | 5 (20.8) |
| Non-MDR | 22 | 6 (27.3) | 11 (50.0) | 5 (22.7) | 6 | 4 (66.7) | 2 (33.3) | 0 (0.0) |
| MDR | 8 | 6 (75.0) | 2 (25.0) | 0 (0.0) | 11 | 9 (81.8) | 1 (9.1) | 1 (9.1) |
The percentage in parentheses represents the number of isolates divided by the total number of isolates within each group.
DISCUSSION
Laboratory diagnosis of M. tuberculosis by acid-fast smear and culture of processed sputum specimens has been utilized for decades; however, there are several drawbacks to these diagnostic methods for M. tuberculosis regarding diagnostic sensitivity, labor intensiveness, and time required. Early and reliable diagnosis of M. tuberculosis is crucial for accurate detection and treatment of suspected TB cases. PCR has recently been utilized to detect M. tuberculosis directly using sputum or sputum cultured isolates in clinical settings (9, 20, 27). In this study, we performed a modified multiplex PCR to differentiate between Beijing and non-Beijing strains of M. tuberculosis directly using the sputum of active pulmonary TB patients. The sensitivity and specificity of the multiplex PCR results were validated by spoligotyping of cultured isolates. According to the study by Jou et al., which involved analysis by the spoligotyping method, the proportion of TB patients infected with Beijing strains in eastern Taiwan was 46.2% in 2003 (8). A similar proportion (46.5%) of TB patients was found to be infected with Beijing strains in our study, unequivocally confirming the accuracy and reliability of the detection method used in this study.
The World Health Organization estimated that the incidence rate in sub-Saharan Africa was nearly 363 cases per 100,000 population in 2007 (28), which is close to that in our aboriginal population but much higher than that in eastern Taiwan as a whole (180.9 cases per 100,000 population) in 2005 (11). The relatively low incidence rate in eastern Taiwan may partly explain the relatively low proportion of mixed infection identified in our study (11.3% [21/185] in this study versus 18.8% [35/186] in the study of Warren et al.). Notably, the TB incidence rate in the aborigine population is much higher than that in the nonaborigine population of eastern Taiwan (11). However, we found that mixed infection was not significantly associated with the high-incidence race group (10.0% for aborigines versus 12.6% for nonaborigines) or history of TB treatment (14.6% for retreatment versus 10.4% for new treatment) (Table 1). These findings differed from those of previous studies, in which it was demonstrated that mixed infection is associated with a high reinfection rate in high-incidence communities (19, 24, 26). Instead, our results revealed that the incidence rate of mixed infection in patients who had not previously taken anti-TB medication was as high as in those with a history of TB treatment, which implied that before any treatment with anti-TB medication, infection with one M. tuberculosis strain does not protect against infection with another M. tuberculosis strain. Therefore, our findings will have a significant effect on treatment strategy and the development of vaccines; i.e., a vaccine against one single strain may not confer protection against other strains.
Our findings that, of the overall resistance rate, 15.1% was due to resistance to at least one drug and 10.3% was due to MDR (Table 2) are similar to the results of our previous study in 2001 (28.6% and 12.7%, respectively) (13). The results demonstrated that the incidence rate of pansusceptible M. tuberculosis was lower in patients with a history of TB treatment (Table 2), which was consistent with the conclusion of the study of Chiang et al. (3). In addition, the finding of a higher MDR rate in Beijing strains was similar to the results of other studies (4, 10, 14, 15, 22). While our study did not reveal a significant association between infection with a Beijing strain and a history of TB treatment (Table 1), isolates from these patients exhibited the highest MDR rate (Table 3), which suggests that anti-TB medication may not contribute to the prevalence of MDR in Beijing strains. These results implied that Beijing strains are predominant in the development of drug resistance in tuberculosis.
Among the groups infected with different M. tuberculosis strains, the mixed-infection group had the highest rate of resistance to at least one drug (non-MDR M. tuberculosis; 23.8%) (Table 3), and the rate of sensitivity to all drugs (pansusceptible M. tuberculosis; 71.4%) in the mixed-infection group was as low as that in the group infected with a Beijing strain alone (70.9%). Although it is not clear whether the coexistence of mixed strains of M. tuberculosis in the same host would increase the probability of genetic mutation, undetected multidrug-resistant strains in mixed infection may outcompete drug-susceptible strains during antibiotic treatment (1). In support of this notion, mixed infection has been reported to be an important factor underlying changes in the drug susceptibility pattern in a high-incidence region (23). Our results indicating a lower proportion of strains sensitive to all drugs and the highest proportion of strains resistant to at least one drug in the mixed-infection group unequivocally suggest that the mixed-infection group represents an intermediate risk group, and this may contribute to an increased risk of MDR. Therefore, earlier identification of mixed infection would enable prompt treatment with a more appropriate drug regimen, resulting in a more successful outcome.
There are some limitations of this study. First, the number of cases of mixed infection was relatively small compared with that in the study by Warren et al. (21 versus 35), which might reduce the statistical power and affect the conclusions regarding the significance of mixed infection made in our study. Second, accurate discrimination of M. tuberculosis strains originating from exogenous reinfection or endogenous reactivation remains limited in this study. Third, separation of all M. tuberculosis strains into Beijing and non-Beijing strains in our study might have been oversimplified, and the proportion of mixed-infection cases may therefore have been underestimated. Finally, owing to the pattern of high drug resistance identified in this study, particularly in Beijing strains, further studies of the epidemiology and XDR patterns of Beijing and/or non-Beijing strains are necessary.
In conclusion, we developed a Beijing and non-Beijing differentiated-multiplex PCR method in this study to identify Beijing and/or non-Beijing strains of M. tuberculosis directly using patient sputum in a single step, and the results demonstrated that mixed infection is present in patients with active pulmonary TB in eastern Taiwan. Comparison of the clinical parameters of patients infected with a single strain and those infected with mixed strains did not reveal any significant differences. In addition, mixed infection was not found to be associated with a history of TB treatment or the high-incidence race group, aborigines. Finally, the results of this study, which was performed in a region of Taiwan where TB is endemic, indicate that mixed infection, i.e., the simultaneous presence of Beijing and non-Beijing M. tuberculosis strains in a single individual, was high in patients who had not received any anti-TB medication. Thus, improper prescription of treatment regimens and the inability to accurately discriminate M. tuberculosis strains and resistance patterns in cases of mixed infection may facilitate the emergence of resistant bacteria and the increase in multidrug-resistant strains.
Acknowledgments
We thank the TB laboratory of TCGH for providing access to specimens and Fang-Hao Yeh for excellent technical support and preparation of specimens.
This work was supported by grants from the National Science Council of Taiwan (98-2627-M-006-015 and 99-2320-B-006-014-MY3).
Footnotes
Published ahead of print on 27 October 2010.
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