Introduction

Resistance of Mycobacterium tuberculosis (MTB) to anti-tuberculosis (TB) drugs, particularly to isoniazid (INH) and rifampicin (RMP), which results in multidrug-resistant (MDR)-TB, presents a serious challenge in the control of TB worldwide [1,2]. The World Health Organization (WHO) estimates that the prevalence of MDR-TB varies from 0% to 65.1% across the world [1]. Despite progress in disease surveillance, more than 80% of MDR-TB patients are unaware of their disease status, indicating that the transmission status of MDR-TB is mostly unknown in high-TB burden countries [1].

Drug-resistant TB, including MDR-TB, develops as a result of inadequate treatment of an individual who was initially infected with a fully or partly sensitive strain or by direct transmission of a drug-resistant strain from one individual to another [3]. Although previous treatment is the strongest risk factor of MDR-TB, other risk factors such as younger age, male gender, and human immunodeficiency virus (HIV) coinfection have also been reported [4-6]. Further analysis may provide information on the dynamics of its transmission and better countermeasures against increasingly drug-resistant TB.

Viet Nam is one of the 22 countries with a high TB burden and is one of the 27 countries with a high MDR-TB burden [1]; the prevalence of any drug resistance and MDR-TB among newly diagnosed cases in a 2006 countrywide survey was 30.7% and 2.7%, respectively [7]. Although drug resistance, including MDR, and potential risk factors have been investigated in some areas [8-10], host-, pathogen-, and environment-related factors, such as patients’ HIV status; residential area; and genotypes of the MTB isolates, have not been comprehensively assessed in Viet Nam. We conducted this study to estimate the status of primary anti-TB drug resistance, including MDR, among newly diagnosed TB patients in Hanoi, the capital and second largest city of Viet Nam, and to investigate the role of the above risk factors in resistance to each of the first-line drugs.

Materials and Methods

Results

Study samples and patient characteristics

In total, 546 newly diagnosed smear-positive pulmonary TB patients were recruited. From 506 culture-positive cases, microbial isolates were collected from 495 patients (97.8%), of which six were infected with nontuberculous mycobacteria. As a result, 489 MTB isolates were tested for drug susceptibility. Because of insufficient quality of the extracted DNA samples, 467 MTB isolates further underwent spoligotyping and 465 underwent VNTR typing (Figure 1). The median age was 38.6 years (range = 16.6–85.4), the proportion of male patients was 78.9%, and HIV coinfection was observed in 9.0% of the patients (Table 1).

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Figure 1. Study flow.

TB: tuberculosis; MTB: Mycobacterium tuberculosis; NTM: nontuberculous mycobacterium; VNTR: variable numbers of tandem repeats; DNA: deoxyribonucleic acid.

https://doi.org/10.1371/journal.pone.0071867.g001

		Number	%
Age (median, range)		(38.6,	16.6–85.4)
Gender	Male	386	78.9
	Female	103	21.1
Body mass index	<16	70	14.3
	16–18.4	201	41.1
	18.5–24.9	213	43.6
	≥25	4	0.8
	Not available	1	0.2
Residential area	Suburban	100	20.4
	New urban	228	46.6
	Old urban	161	32.9
Smoking habit	Smoker	189	38.7
	Ex-smoker	134	27.4
	Nonsmoker	165	33.7
	No answer	1	0.2
HIV status	Positive	44	9.0
	Negative	443	90.6
	Not available	2	0.4

Table 1. Characteristics of the study population (n = 489).

HIV: human immunodeficiency virus

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Discussion

We found that the proportion of drug-resistant cases, including MDR, was considerably high among newly diagnosed smear-positive culture-positive pulmonary TB patients residing in Hanoi city. Depending on the type of drug resistance, the drug resistance-associated risk factors showed a pronounced variation and revealed complicated aspects in a large city. The majority of MDR-TB cases revealed that infection with Beijing strains was predominantly spread in this area, while non-Beijing MDR strains were also observed.

INH or SM resistance was not uncommon, and most RMP-resistant strains were also associated with SM and INH resistance, resulting in MDR. These findings were consistent with a previous report in Ho Chi Minh city in Viet Nam [9]. The high prevalence of primary resistance to INH and SM (28.2% and 28.2%, respectively) and moderate prevalence of RMP resistance and MDR (4.9% and 4.5%, respectively) shown in our study might be considered noteworthy, when comparing with those of South East Asian region (10.3%, 8.9%, 3.4%, and 2.8%) [7], and of China (16.0%, 27.7%, 6.7%, and 5.7%) [18]. In this situation, the use of a regimen with RMP for only 2 months of the intensive phase, which is still accepted in Viet Nam, may pose the risk for poor treatment outcome [19] and accumulation of further drug resistance [20].

The association between younger age and anti-TB drug resistance has been reported previously [9,21]. The results of univariate and multivariate analyses performed in our study indicate that primary drug resistance among the younger population may be confounded by the recent transmission of Beijing strains [9,22]. In the current study, living in an old urban area and infection with clustered strains were associated with INH, but not SM, resistance, suggesting that the transmission of INH-resistant strains is concentrated in areas with a high population density, whereas SM-resistant strains are spreading more diffusely throughout the city. Initially, SM was used for treatment of wound infections during the war in Viet Nam in the early 1950s, which may partly explain the widespread development of SM-resistant nonclustered strains, whereas INH was first circulated in 1960s, and RMP was introduced at around 1975 [23,24]. The Beijing genotype was significantly associated with resistance to any drug, INH, and SM, but it was not associated with either RMP resistance or MDR. A direct role of Beijing strains in drug resistance remains controversial [22,25-27].

The spoligotype and VNTR analyses demonstrated that any-drug resistant strains showed a higher tendency for clustering than fully-sensitive strains; and almost half of the MDR strains were clustered and presumably derived from common infection sources or infection with different sources sharing ancestors [16,28]. Three of the MDR strains (13.6%) belonged to the largest Beijing cluster, accounting for approximately 10% of the study population. Although the Beijing genotype was predominant among clustered MDR strains, three non-Beijing genotype strains were closely related to each other based on their VNTR patterns and showed unclassified spoligo patterns resembling EAI5 or EAI4_VNM, a possibly indigenous MTB subtype mainly observed in Viet Nam. Research into the origin and transmission dynamics of these variant MDR strains, as well as their molecular characteristics, may be important, because it is generally believed that the EAI lineage has conferred significantly less drug resistance compared with other genotypes in Asian countries [29,30].

HIV coinfection was significantly associated with only RMP resistance and MDR in multivariate analysis, although it showed significant associations with all types of drug resistance in univariate analysis. This independent association with RMP resistance and MDR has also been reported in other studies [31,32], including one in the northern area [10], but was not observed in a study of the southern area of Viet Nam [9]. The southern study was conducted between 1998 and 2000, when HIV prevalence was low in Viet Nam [33]. This may explain the lower percentage of HIV, compared with ours (2.8% vs. 9.0%), resulting in a low statistical power (20%) [9]. In Hanoi, approximately 25% of injecting drug users tested were HIV positive [31]. Drug use is a risk factor for nonadherent treatment, and it promotes development of drug resistance [34], thus increasing the chance of resistance transmission among the group. HIV coinfection is also associated with pharmacokinetic alteration of RMP, resulting in a 39% reduction of drug concentration [35]. The decreased bioavailability of RMP may contribute to the development of RMP resistance as well. In addition, HIV-coinfected TB patients receiving antiretroviral treatment often suffer from the adverse effects of RMP when an alternative drug is not available, which may cause poor treatment outcomes [36] and facilitate drug resistance. The negative effect of HIV coinfection on RMP resistance, together with the recent spread of Beijing strains associated with INH resistance, may pose a combined risk for the acquisition and transmission of MDR-TB in a large city like Hanoi.

SM resistance was independently associated with smoking, after adjusted for HIV coinfection. The reason for this association is unknown, although smoking is known to be associated with TB [37]. The proportion of PZA resistance tested using the pyrazinamidase assay was low among the total study population [38]. Nevertheless, the proportion of PZA resistance was significantly higher in the MDR group than that in the non-MDR group, indicating a need for evaluation of the susceptibility of MTB strains to this drug.

The clustering rate in Hanoi (43.4%) was high, presumably because our study was conducted in a capital city with high population density and enrolled only patients with smear-positive pulmonary TB. Others have reported relatively lower clustering rates (28.3% in China [39], 37.7% in Zambia [40], and 16.8% in Uganda [41]). However, these studies were conducted in peripheral areas (Zambia) or enrolled patients with smear-negative pulmonary TB (China, Uganda). In addition, it is known that the resolution of 15 MIRU-VNTR for Beijing strains is suboptimal and may overestimate the clustering rate. Addition of more loci to the standard VNTR loci may increase the resolution in a setting where Beijing-genotype strains prevail [42]. Nevertheless, the data can be analyzed using the standard 15 MIRU-VNTR typing method first, since it has been used internationally for a long time [39-41,43,44].

Our study has some limitations. First, we did not have enough information about direct epidemiologic links among clustered patients. In high TB burden countries, however, a TB outbreak is difficult to identify. In addition, we may not have analyzed all representative isolates in Hanoi city. However, the seven districts participating in this study cover old urban, new urban, and suburban areas in this city, and analysis of a relatively large number of isolates definitely provided information that would be useful in the management of drug-resistant TB. Despite the aforementioned limitations, we investigated a variety of host-, bacteria-, and environment-related factors and developed a multidimensional picture of the status of drug-resistance in the studied area.

In conclusion, the transmission status of drug-resistant TB in a large city with a high proportion of Beijing strains, particularly in HIV-prevalent areas, should be carefully monitored to avoid an increase in the incidence of MDR and generation of extensively drug-resistant TB. Drug susceptibility testing should be considered. On the basis of the results, an optimal treatment regimen, together with intensive monitoring of treatment adherence, is suggested to avoid further increases in drug resistance.

Acknowledgments

The authors would like to thank Dr. Bui Thi Nguyet, Ms. Vu Thi Xuan Thu, Dr. Pham Thu Anh (Hanoi Lung Hospital), Dr. Phan Thi Minh Ngoc, Ms. Nguyen Thi Ha (NCGM-BMH Medical Collaboration Center), and all the healthcare staff of relevant district TB centers for supporting site implementation.