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 Table of Contents  
ORIGINAL ARTICLE
Year : 2018  |  Volume : 7  |  Issue : 4  |  Page : 375-379

Infection of multiple Mycobacterium tuberculosis strains among tuberculosis/human immunodeficiency virus co-infected patients: A molecular study in Myanmar


1 Department of Regional Public Health, Nay Pyi Taw Union Territory, Ministry of Health and Sports, Myanmar; Department of Epidemiology Unit, Faculty of Medicine, Prince of Songkla University, Hat Yai, Songkhla, Thailand
2 Department of Microbiology, Faculty of Science, Mahidol University, Bangkok, Thailand
3 Department of Research and Development Affairs, Faculty of Medicine, Siriraj Hospital, Mahidol University, Bangkok, Thailand
4 Department of Epidemiology Unit, Faculty of Medicine, Prince of Songkla University, Hat Yai, Songkhla, Thailand

Date of Web Publication5-Dec-2018

Correspondence Address:
Virasakdi Chongsuvivatwong
Epidemiology Unit, Faculty of Medicine, Prince of Songkla University, Hat Yai, Songkhla
Thailand
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijmy.ijmy_108_18

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  Abstract 


Background: Appearance of Mycobacterium tuberculosis (MTB) in the sputum of a tuberculosis (TB)/human immunodeficiency virus (HIV) co-infected patient under treatment may indicate either failure or new infection. This study aims to evaluate whether TB treatment failure among TB/HIV co-infected patients is a real failure. Methods: A prospective cohort study was conducted among 566 TB/HIV co-infected patients who started TB treatment in 12 townships in the upper Myanmar. Among the 566 participants, 16 (2.8%) resulted in treatment failure. We performed a molecular study using mycobacterial interspersed repetitive-unit-variable number of tandem repeat (MIRU-VNTR) genotyping for them. The MIRU-VNTR profiles were analyzed using the web server, MIRU-VNTRplus. All data were entered into EpiData version 3.1 and analyzed using R version 3.4.3. Results: Among 16 failure patients, seven had incomplete laboratory results. Of the nine remaining patients, nobody had exactly the same MIRU-VNTR pattern between the initial and final isolates. Four patients had persistent East-African Indian (EAI) lineages and one each had persistent Beijing lineage, changing from EAI to Beijing, from Beijing to EAI, NEW-1 to Beijing, and NEW-1 to X strains. Female patients have significantly larger genetic difference between MTB of the paired isolates than male patients (t-test, P = 0.04). Conclusion: Thus, in our study patients, infection of multiple MTB strains is a possible cause of TB treatment failure. Explanation for the association between gender and distance of genotypes from the initial to subsequent MTB infection needs further studies.

Keywords: Infection of multiple Mycobacterium tuberculosis strains, tuberculosis treatment failure, tuberculosis/human immunodeficiency virus co-infection


How to cite this article:
Kyi MS, Palittapongarnpim P, Chaiprasert A, Ajawatanawong P, García HG, Chongsuvivatwong V. Infection of multiple Mycobacterium tuberculosis strains among tuberculosis/human immunodeficiency virus co-infected patients: A molecular study in Myanmar. Int J Mycobacteriol 2018;7:375-9

How to cite this URL:
Kyi MS, Palittapongarnpim P, Chaiprasert A, Ajawatanawong P, García HG, Chongsuvivatwong V. Infection of multiple Mycobacterium tuberculosis strains among tuberculosis/human immunodeficiency virus co-infected patients: A molecular study in Myanmar. Int J Mycobacteriol [serial online] 2018 [cited 2018 Dec 11];7:375-9. Available from: http://www.ijmyco.org/text.asp?2018/7/4/375/246908




  Introduction Top


Tuberculosis (TB) treatment outcome is poor among human immunodeficiency virus (HIV)-positive TB patients compared with HIV-negative TB patients.[1],[2],[3],[4],[5],[6],[7],[8] Moreover, people living with HIV (PLHIV) are at higher risk of recurrence after successful previous TB treatment.[9],[10],[11] Many molecular studies reported that recurrent TB among PLHIV was mostly due to exogenous reinfection of Mycobacterium tuberculosis (MTB),[12],[13],[14],[15],[16],[17] especially those who reside in TB-endemic countries.[18],[19],[20] However, the evidence to support this idea among TB/HIV co-infected patients who have treatment failure is not solid.

There is a need to conduct such assessment in Myanmar where TB/HIV is a high burden. The objectives of this study therefore were to evaluate whether TB treatment failure among TB/HIV co-infected patients was a real failure and to assess the associated factors.


  Methods Top


Study design, setting, and participants

A prospective cohort study was conducted in 12 townships in the upper Myanmar. TB/HIV co-infected patients aged ≥15 years were diagnosed according to the WHO and National guidelines.[21],[22] An individual with rifampicin resistance by Xpert MTB/RIF assay was excluded due to the need of long period of treatment. Five hundred and sixty-six newly registered TB/HIV co-infected patients at the TB clinics during the study period were included. They were followed up bacteriologically until the end of TB treatment.

Sample collection and molecular typing

Two sputum samples from each patient were collected before starting TB treatment and sent to the Upper Myanmar TB Reference Laboratory in Mandalay. Those samples were inoculated onto Mycobacteria Growth Indicator Tube (MGIT) by standard procedures.[23] The isolates were frozen in 7H9 broth plus glycerol to preserve for further analysis. After getting TB treatment outcomes, sputum samples were collected and inoculated into MGIT again if the sputum smear result was positive. The initial and final isolates of the same patient were confirmed by having the same identification number, name, gender, and address. The DNA of eleven paired (initial and final) culture isolates were extracted as described by van Helden et al.[24] Those extracted DNAs were sent to perform mycobacterial interspersed repetitive-unit-variable number of tandem repeat (MIRU-VNTR) genotyping at the Department of Microbiology, Faculty of Science, Mahidol University, Bangkok, Thailand. The copy numbers of DNA tandem repeat of 24 MIRU-VNTR loci were identified using 24 sets of standard polymerase chain reaction (PCR) primers.[25],[26] Determination of the PCR product sizes was done by agarose gel electrophoresis. Any paired samples with more than two MIRU-VNTR loci failed in genotyping were not included in the statistical analysis.

Data analysis

Analysis of the MIRU-VNTR profiles using the web server, MIRU-VNTRplus (http://www. miru-vntrplus.org/), enabled a comparison of profiles by generating Neighbour Joining (NJ) tree and identification of clusters of strains of MTB.[27] Isolates that had identical type or a single mismatch among 24 MIRU-VNTR loci were considered clonally related. Under the constraints of small sample size, statistical analysis was carried out to explore possible factors associated with the genotype changes. All data were entered into Epidata version 3.1 (http://www.epidata.dk/) and analyzed using R version 3.4.2 (https://cran.r-project.org/). Statistical significance was tested by t-test because of using continuous variable. P < 0.05 with 95% confidence interval was set as significance. The results would be a foundation for further in-depth studies.

The study was approved by the Ethical Review Committee of the Prince of Songkla University, Thailand, and Department of Medical Research, Myanmar.


  Results Top


Patient characteristics

Data were collected from October 2016 to February 2018. A total of 566 TB/HIV patients were identified. At the end of the 5th month of TB treatment, 16 patients (2.8%) were notified as treatment failure according to the WHO's definition.[28] Five of them had no complete laboratory results. Two other had at least more than two MIRU-VNTR loci without genotyping results. Finally, nine patients had data available for analysis. These five male and four female patients had an average age of 37.1 ± 4.9 years, which was not significantly different from the five male and two female excluded patients. Among the included nine patients, five were jobless and among four who had job, one female patient had experience in sex work.

Genotyping results and associated factors

[Table 1] shows the MIRU-VNTR genotyping results of the initial and final isolates of nine patients. All patients had discordant fingerprints indicating that they have a new strain of MTB. Their paired isolates were different in 2–17 loci (median = 8) as mentioned in the last column of [Table 1].
Table 1: Mycobacterial interspersed repetitive-unit-variable number of tandem repeat analysis of 18 isolates from nine patients

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[Table 2] describes the mean and standard deviation of the number of un-identical loci by various independent variables of the patients. The only statistically significant variable associated with the number of un-identical loci was gender. In male patients, the initial and final isolates had an average of 5.6 un-identical loci compared to the average of 12.5 loci in the female (t-test, P = 0.04).
Table 2: Mean and standard deviation of number of un-identical loci by patient characteristics

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Determination of Mycobacterium tuberculosis strain diversity

The MIRU-VNTR profiles were determined for 18 isolates and compared with profiles present in the MIRU-VNTRplus database by generating trees using the NJ algorithm. Among 18 strains from nine patients, ten formed a cluster with the most closely related reference strain belonging to the East-African Indian (EAI), five to the Beijing, two to the NEW-1, and one to the X lineage. Both X and NEW-1 belong to the Euro-American lineage. [Figure 1] links isolates from the same patients with green solid lines for male patients and red dotted lines for female patients. Male patients were more likely to have the isolate pairs closer genetically than female patients did.
Figure 1: Neighbour-Joining tree (displayed as radial tree) showing the extent of difference between initial and final strains of the same patient (five males and four females). The strains of male patients are linked in green straight lines and those of female patients in red dotted lines

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[Table 3] describes the distribution of nine patients by their initial and final strains according to gender. The initial and final strains of all the five male patients (M1, M3, M4, M5, and M6) belonged to the same lineages, mostly EAI. Even though the initial and final strains of a patient (M6) converted from New-1 to X subfamily, both were Euro-American strains. In contrast, only one (F2) of the four female patients had the same Beijing genotyping of both strains. Other female patients (F7, F8, and F9) had different strains.
Table 3: Distribution of nine patients by their initial and final strains according to gender

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  Discussion Top


None of our patients with MTB in their initial and final sputum had genetically unchanged MTB strains. Female patients were more likely to have greater genetic distances between the initial and final strains of MTB.

Exogenous reinfection of TB has also been observed in many other studies in TB-endemic areas.[9],[12],[13],[15],[16],[17],[29] This could have a few possible explanations. First, the patient could initially simultaneously harbor multiple strains of MTB before starting the treatment.[30],[31] Bacterial isolation may pick up only one but different strain at a time. Otherwise, the patients could have been re-infected by a new strain of MTB during the treatment course as PLHIV are highly susceptible to TB infection.[1]

Female patients were more likely to have two different MTB strains with greater genetic distances than male patients. This concurred with a study in Sydney where two re-infected patients were both female.[18] In our study, a female patient was a sex worker. Having high number of sex partners may have the chance to get infection of different MTB strains. However, with limited information on sexual behaviors of the participants and the smallness of our sample size, this conjecture needs to be proved by further studies.

The Beijing lineage is the most predominant MTB strain in many Asian countries.[32] Some studies reported that the EAI, one of the ancient MTB lineages, is prevalent in South East Asian countries.[33],[34],[35] The genetic diversity of MTB in Myanmar is mainly driven by both the EAI and the Beijing subfamilies.[36],[37] In this study, most patients have the EAI lineage followed by the Beijing, which concurred with those results.


  Conclusion Top


Infection of multiple MTB strains could be a cause of apparent treatment failure in our study population. The association between gender and distance of genotypes from the initial to subsequent MTB infection was a result of our preliminary study. More data are needed to get a comprehensive conclusion.

Acknowledgments

This study was a part of the PhD thesis of the first author to fulfill the requirement for the TB/MDR-TB research training program at Epidemiology Unit, Prince of Songkla University, under the support of Fogarty International Center, National Institute of Health (Grant number D43TW009522). We are grateful to Dr. Si Thu Aung, Director of Disease control session, Dr. Kyi Kyi Swe, microbiologist and laboratory technicians from the Upper Myanmar TB Reference Laboratory of NTP and the respective township health departments in Myanmar and the Microbiology Department, Faculty of Science at the Mahidol University in Bangkok, Thailand, for their support. We also thank the patients who participated in our study.

Financial support and sponsorship

This study was financially supported by Fogarty International Center, National Institute of Health (Grant number D43TW009522).

Conflicts of interest

There are no conflicts of interest.



 
  References Top

1.
World Health Organization. Global Tuberculosis Report 2017. World Health Organization; 2017.  Back to cited text no. 1
    
2.
Tweya H, Feldacker C, Phiri S, Ben-Smith A, Fenner L, Jahn A, et al. Comparison of treatment outcomes of new smear-positive pulmonary tuberculosis patients by HIV and antiretroviral status in a TB/HIV clinic, Malawi. PLoS One 2013;8:e56248.  Back to cited text no. 2
    
3.
Agbor AA, Bigna JJ, Billong SC, Tejiokem MC, Ekali GL, Plottel CS, et al. Factors associated with death during tuberculosis treatment of patients co-infected with HIV at the Yaoundé central hospital, Cameroon: An 8-year hospital-based retrospective cohort study (2006-2013). PLoS One 2014;9:e115211.  Back to cited text no. 3
    
4.
Adejumo OA, Daniel OJ, Otesanya AF, Adegbola AA, Femi-Adebayo T, Bowale A, et al. Factors associated with TB/HIV co-infection among drug sensitive tuberculosis patients managed in a secondary health facility in Lagos, Nigeria. Afr J Infect Dis 2017;11:75-82.  Back to cited text no. 4
    
5.
Tabarsi P, Chitsaz E, Moradi A, Baghaei P, Marjani M, Mansouri D, et al. Treatment outcome and mortality: Their predictors among HIV/TB co-infected patients from Iran. Int J Mycobacteriol 2012;1:82-6.  Back to cited text no. 5
  [Full text]  
6.
Mekonnen D, Derbie A, Mekonnen H, Zenebe Y. Profile and treatment outcomes of patients with tuberculosis in Northeastern Ethiopia: A cross sectional study. Afr Health Sci 2016;16:663-70.  Back to cited text no. 6
    
7.
Teshome Kefale A, Anagaw YK. Outcome of tuberculosis treatment and its predictors among HIV infected patients in Southwest Ethiopia. Int J Gen Med 2017;10:161-9.  Back to cited text no. 7
    
8.
Karo B, Krause G, Hollo V, van der Werf MJ, Castell S, Hamouda O, et al. Impact of HIV infection on treatment outcome of tuberculosis in Europe. Acquired Immune Deficiency Syndrome 2016;30:1089-98.  Back to cited text no. 8
    
9.
Charalambous S, Grant AD, Moloi V, Warren R, Day JH, van Helden P, et al. Contribution of reinfection to recurrent tuberculosis in South African gold miners. Int J Tuberc Lung Dis 2008;12:942-8.  Back to cited text no. 9
    
10.
Unis G, Ribeiro AW, Esteves LS, Spies FS, Picon PD, Dalla Costa ER, et al. Tuberculosis recurrence in a high incidence setting for HIV and tuberculosis in Brazil. BMC Infect Dis 2014;14:548.  Back to cited text no. 10
    
11.
Millet JP, Shaw E, Orcau A, Casals M, Miró JM, Caylà JA, et al. Tuberculosis recurrence after completion treatment in a European city: Reinfection or relapse? PLoS One 2013;8:e64898.  Back to cited text no. 11
    
12.
Crampin AC, Mwaungulu JN, Mwaungulu FD, Mwafulirwa DT, Munthali K, Floyd S, et al. Recurrent TB: Relapse or reinfection? The effect of HIV in a general population cohort in Malawi. Acquired Immune Deficiency Syndrome 2010;24:417-26.s  Back to cited text no. 12
    
13.
Narayanan S, Swaminathan S, Supply P, Shanmugam S, Narendran G, Hari L, et al. Impact of HIV infection on the recurrence of tuberculosis in South India. J Infect Dis 2010;201:691-703.  Back to cited text no. 13
    
14.
Houben RM, Crampin AC, Ndhlovu R, Sonnenberg P, Godfrey-Faussett P, Haas WH, et al. Human immunodeficiency virus associated tuberculosis more often due to recent infection than reactivation of latent infection. Int J Tuberc Lung Dis 2011;15:24-31.  Back to cited text no. 14
    
15.
Sonnenberg P, Murray J, Glynn JR, Shearer S, Kambashi B, Godfrey-Faussett P, et al. HIV-1 and recurrence, relapse, and reinfection of tuberculosis after cure: A cohort study in South African mineworkers. Lancet 2001;358:1687-93.  Back to cited text no. 15
    
16.
Houben RM, Glynn JR, Mallard K, Sichali L, Malema S, Fine PE, et al. Human immunodeficiency virus increases the risk of tuberculosis due to recent re-infection in individuals with latent infection. Int J Tuberc Lung Dis 2010;14:909-15.  Back to cited text no. 16
    
17.
Fitzpatrick LK, Okwera A, Mugerwa R, Ridzon R, Ehiner J, Onorato I, et al. An investigation of suspected exogenous reinfection in tuberculosis patients in Kampala, Uganda. Int J Tuberc Lung Dis 2002;6:550-2.  Back to cited text no. 17
    
18.
Dobler CC, Marks GB, Simpson SE, Crawford AB. Recurrence of tuberculosis at a Sydney chest clinic between 1994 and 2006: Reactivation or reinfection? Public Health 2008;188:3.  Back to cited text no. 18
    
19.
Wang JY, Lee LN, Lai HC, Hsu HL, Liaw YS, Hsueh PR, et al. Prediction of the tuberculosis reinfection proportion from the local incidence. J Infect Dis 2007;196:281-8.  Back to cited text no. 19
    
20.
Interrante JD, Haddad MB, Kim L, Gandhi NR. Exogenous reinfection as a cause of late recurrent tuberculosis in the United States. Ann Am Thorac Soc 2015;12:1619-26.  Back to cited text no. 20
    
21.
World Health Organization. WHO Policy on Collaborative TB/HIV activities: Guidelines For National Programmes and Other Stakeholders. 2012.  Back to cited text no. 21
    
22.
National AIDS Programme. Guidelines for the Clinical Management of HIV Infection in Myanmar. 5th ed. Myanmar: National AIDS Programme; 2017. p. 112.  Back to cited text no. 22
    
23.
The Global Laboratory Initiative. Mycobacteriology Laboratory Manual. 2014. p. 154.  Back to cited text no. 23
    
24.
van Helden PD, Victor TC, Warren RM, van Helden EG. Isolation of DNA from Mycobacterium tuberculosis. Methods Mol Med 2001;54:19-30.  Back to cited text no. 24
    
25.
Supply P, Allix C, Lesjean S, Cardoso-Oelemann M, Rüsch-Gerdes S, Willery E, et al. Proposal for standardization of optimized mycobacterial interspersed repetitive unit-variable-number tandem repeat typing of Mycobacterium tuberculosis. J Clin Microbiol 2006;44:4498-510.  Back to cited text no. 25
    
26.
Supply P, Mazars E, Lesjean S, Vincent V, Gicquel B, Locht C, et al. Variable human minisatellite-like regions in the Mycobacterium tuberculosis genome. Mol Microbiol 2000;36:762-71.  Back to cited text no. 26
    
27.
Weniger T, Krawczyk J, Supply P, Niemann S, Harmsen D. MIRU-VNTRplus: A web tool for polyphasic genotyping of Mycobacterium tuberculosis complex bacteria. Nucleic Acids Res 2010;38:W326-31.  Back to cited text no. 27
    
28.
World Health Organization. Definitions and Reporting Framework for Tuberculosis-2013 Revision. World Health Organization; 2014.  Back to cited text no. 28
    
29.
Glynn JR, Murray J, Bester A, Nelson G, Shearer S, Sonnenberg P, et al. High rates of recurrence in HIV-infected and HIV-uninfected patients with tuberculosis. J Infect Dis 2010;201:704-11.  Back to cited text no. 29
    
30.
Niemann S, Richter E, Rüsch-Gerdes S, Schlaak M, Greinert U. Double infection with a resistant and a multidrug-resistant strain of Mycobacterium tuberculosis. Emerg Infect Dis 2000;6:548-51.  Back to cited text no. 30
    
31.
Braden CR, Morlock GP, Woodley CL, Johnson KR, Colombel AC, Cave MD, et al. Simultaneous infection with multiple strains of Mycobacterium tuberculosis. Clin Infect Dis 2001;33:e42-7.  Back to cited text no. 31
    
32.
Chen YY, Chang JR, Huang WF, Kuo SC, Su IJ, Sun JR, et al. Genetic diversity of the Mycobacterium tuberculosis Beijing family based on SNP and VNTR typing profiles in Asian countries. PLoS One 2012;7:e39792.  Back to cited text no. 32
    
33.
Chen YY, Chang JR, Huang WF, Hsu CH, Cheng HY, Sun JR, et al. Genetic diversity of the Mycobacterium tuberculosis east African-Indian family in three tropical Asian countries. J Microbiol Immunol Infect 2017;50:886-92.  Back to cited text no. 33
    
34.
Zhang J, Heng S, Le Moullec S, Refregier G, Gicquel B, Sola C, et al. A first assessment of the genetic diversity of Mycobacterium tuberculosis complex in Cambodia. BMC Infect Dis 2011;11:42.  Back to cited text no. 34
    
35.
Chang JR, Chen YY, Huang TS, Huang WF, Kuo SC, Tseng FC, et al. Clonal expansion of both modern and ancient genotypes of Mycobacterium tuberculosis in Southern Taiwan. PLoS One 2012;7:e43018.  Back to cited text no. 35
    
36.
Tun T, Aye KS, Nyunt WW, Crump JA, Nakajima C, Suzuki Y, et al. Genotypic diversity of Mycobacterium tuberculosis strains in Myanmar. Infect Dis (Lond) 2017;49:237-9.  Back to cited text no. 36
    
37.
Phyu S, Stavrum R, Lwin T, Svendsen ØS, Ti T, Grewal HM, et al. Predominance of Mycobacterium tuberculosis EAI and Beijing lineages in Yangon, Myanmar. J Clin Microbiol 2009;47:335-44.  Back to cited text no. 37
    


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