- Email: [email protected]
HIV co-infected patients in Ho Chi Minh city, Vietnam
Accepted Manuscript Title: Drug resistance and Mycobacterium tuberculosis strain diversity in TB/HIV co-infected patients in Ho Chi Minh city, Vietnam Authors: Trinh Quynh Mai, Nguyen Thi Van Anh, Nguyen Tran Hien, Nguyen Huu Lan, Do Chau Giang, Pham Thi Thu Hang, Nguyen Thi Ngoc Lan, Ben J. Marais, Vitali Sintchenko PII: DOI: Reference:
S2213-7165(17)30126-1 http://dx.doi.org/doi:10.1016/j.jgar.2017.07.003 JGAR 450
To appear in: Received date: Revised date: Accepted date:
8-3-2017 22-6-2017 12-7-2017
Please cite this article as: Trinh Quynh Mai, Nguyen Thi Van Anh, Nguyen Tran Hien, Nguyen Huu Lan, Do Chau Giang, Pham Thi Thu Hang, Nguyen Thi Ngoc Lan, Ben J.Marais, Vitali Sintchenko, Drug resistance and Mycobacterium tuberculosis strain diversity in TB/HIV co-infected patients in Ho Chi Minh city, Vietnam (2010), http://dx.doi.org/10.1016/j.jgar.2017.07.003 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Drug resistance and Mycobacterium tuberculosis strain diversity in TB/HIV coinfected patients in Ho Chi Minh city, Vietnam Trinh Quynh Mai1,2,3, Nguyen Thi Van Anh1, Nguyen Tran Hien1, Nguyen Huu Lan4, Do Chau Giang4, Pham Thi Thu Hang4, Nguyen Thi Ngoc Lan4, Ben J Marais2, Vitali Sintchenko2,3
1
National Institute of Hygiene and Epidemiology, Hanoi, Vietnam; 2Sydney Medical
School and Marie Bashir Institute for Infectious Diseases and Biosecurity, The University of Sydney, Australia; 3Centre for Infectious Disease and Microbiology – Public Health, ICPMR, Westmead Hospital, Sydney, Australia; 4Pham Ngoc Thach TB and Lung Disease Hospital, Ho Chi Minh City, Vietnam.
Corresponding author Trinh Quynh Mai Tuberculosis Laboratory, National Institute of Hygiene and Epidemiology No 1 Yersin Street, Hai Ba Trung District Hanoi, 10000 Vietnam Tel: +84 983110183 Email: [email protected]
Word count: 2679 words (Tables 4, Figure 1)
Highlights
TB/HIV co-infection in Vietnam was associated with high rates of TB drug resistance and likely community transmission of drug resistant strains.
The vast majority of isoniazid resistant strains in Vietnam have high-level isoniazid resistance in HIV (+) patients.
Beijing is the predominant lineage among TB/HIV co-infected patients; but EAI-5 is specifically overrepresented compared to HIV (-) patients.
The variety of drug resistance profiles and phylogenetic diversity of strains do not suggest high rates of nosocomial transmission.
ABSTRACT Background: Mycobacterium tuberculosis strain diversity and drug resistance among people living with human immunodeficiency virus (HIV) in Vietnam have not been described previously. Methods: We examined M. tuberculosis isolates from TB/HIV co-infected patients in Ho Chi Minh City, Vietnam. Drug susceptibility testing (DST), spoligotyping and 24-locus Mycobacterial Interspersed Repetitive Unit (MIRU-24 typing) were performed, and the rpoB, katG, inhA and inhA promoter, rpsL, rrs and embB genes were sequenced in all drug resistant isolates identified. Results: In total, 84/200 (42.0%) strains demonstrated “any drug resistance”; 17 (8.5%) were multi-drug resistant (MDR). Streptomycin resistance was present in 80 (40.0%) isolates; 95.2% (80/84) with “any drug resistance” and 100% with MDR. No rifampicin monoresistance was detected. Of the rifampicin resistant strains 16/18 (88.9%) had mutations in the 81-bp Rifampicin Resistance Defining Region (RRDR)
of the rpoB gene. Isoniazid resistance was mostly associated with Ser315Thr mutations in the katG gene (15/17; 88.2%). Beijing (49.0%) and East African Indian (EAI) lineage strains (35.0%; 56/70 EAI-5) were most common. Conclusion: TB/HIV co-infection in Vietnam was associated with high rates of TB drug resistance, although we were unable to differentiate new from retreatment cases.
Keywords: Mycobacterium tuberculosis; drug resistance, tuberculosis; epidemiology; TB/HIV co-infection; Vietnam
Word
count:
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BACKGROUND Tuberculosis (TB) and human immunodeficiency virus (HIV) infection have mutually detrimental effects that complicate patient management and global TB control efforts (1). Of the estimated 10.4 million new TB cases identified worldwide in 2015, 1.2 million (11%) were co-infected with HIV, with 0.4 million TB-related deaths occurring in people living with HIV (2). The Asia Pacific region, which contributes more than 60% of all TB cases worldwide, reports relatively low TB/HIV co-infection rates compared to sub-Saharan Africa (2). Although HIV has not had a major detrimental impact on TB control in the Asia-Pacific region, its future burden remains uncertain given poor disease visibility and unreliable data; less than two thirds of TB patients are routinely tested for HIV (3). In Vietnam, the health outcomes of TB/HIV co-infected patients have improved in recent years, with concerted bi-directional screening and better access to anti-retroviral therapy (ART) (4). However, rising rates of drug resistant TB have become a major concern (4).
In 2015 Vietnam ranked 15th among high-burden TB countries and 16th among highburden multi-drug resistant (MDR)-TB countries in the world, based on the absolute number of incident cases (2). MDR-TB was reported in 4% of new and 25% of retreatment TB cases (2). MDR-TB can be acquired as a result of inadequate treatment of patients initially infected with a fully or partially drug susceptible strain (secondary or acquired drug resistance) or can occur following direct transmission of an MDR strain (primary or transmitted drug resistance) (5). Nosocomial transmission in health care facilities, is a particular concern among HIV-infected patients with immunodeficiency (6). When interpreting data reported to the World Health Organization (WHO) a common assumption has been that resistance rates among new
TB cases provide an accurate indication of primary (transmitted) drug resistance. However, recent modeling and strain typing data suggest that in the absence of routine drug susceptibility testing (DST) the majority of MDR-TB cases diagnosed at retreatment represent primary (transmitted) drug resistance, especially in the AsiaPacific region (7).
Despite the rapid expansion MDR-TB services and national efforts to identify and treat MDR-TB cases in Vietnam, case numbers continue to rise (8). MDR-TB/HIV co-infection is a particular concern given the poor treatment outcomes achieved and the risk of MDR-TB transmission within health care settings (9). While the roll-out of the Xpert MTB RIF® test in Vietnam has improved screening for MDR-TB (10), concerns about potential false negatives in strains with mutations outside the 81-bp Rifampicin Resistance Defining Region (RRDR) persist (11). In addition, our understanding of M. tuberculosis strain diversity, transmission dynamics and drug resistance profiles in TB/HIV co-infected patients in Vietnam remains limited. This study examined the M. tuberculosis strain diversity and described the frequency and molecular mechanisms of resistance against first-line TB drugs in TB/HIV co-infected patients in Ho Chi Minh City, Vietnam.
METHODS We conducted a retrospective laboratory-based analysis of 200 M. tuberculosis isolates from TB/HIV co-infected patients sent to the Pham Ngoc Thach Hospital mycobacterial reference laboratory in Ho Chi Minh city, Vietnam. We included consecutive culture positive isolates routinely collected from 2009-2014, during
periods that no other studies were performed. Demographic data, including age, gender and residential province were collected from the laboratory database.
Study setting Ho Chi Minh City is the most populous metropolis in Vietnam, with an estimated population of 8.2 million (12). Pham Ngoc Thach Hospital is the largest (750-bed) lung disease hospital in Ho Chi Minh City and treated over 7,600 TB patients in 2016. The hospital serves as a tertiary referral center for TB patients and people living with HIV throughout southern Vietnam (12). The hospital has a WHO accredited regional mycobacterial reference laboratory (13). Pham Ngoc Thach Hospital was one of the first centers to provide ART and is the largest center providing treatment to HIVinfected and TB/HIV co-infected patients in Vietnam.
Drug susceptibility testing Primary mycobacterial cultures were grown on solid Lowenstein–Jensen (LJ) media (bioMérieux SA, Marcy-l’Étoile, France), or liquid culture (BACTECTM or MGITTM 960; Becton Dickinson & Co., Franklin Lakes, NJ). All isolates identified as M. tuberculosis underwent phenotypic DST against isoniazid, rifampicin, streptomycin and ethambutol by the standard 1% proportion method (14). Cultures were grown on solid LJ media with the following critical drug concentrations: isoniazid (0.2 μg/mL), rifampin (40 μg/mL), streptomycin (4 μg/mL), and ethambutol (2 μg/mL).
Spoligotyping, MIRU-24 typing and gene sequencing Spoligotyping was performed according to standard protocol (15) and phylogenetic lineages
were
assigned
using
international
SIVITWEB databases
(http://www.pasteur-guadeloupe.fr:8081/SITVIT_ONLINE/) (http://cgi2.cs.rpi.edu/~bennek/SPOTCLUST.html).
and
24-locus
SPOTCLUST Mycobacterial
Interspersed Repetitive Unit (MIRU-24 typing) was performed based on Supply’s protocol (2006) and strain lineage using MIRU-24 typing was assigned using the miru-vntrplus.org online database. DNA extraction was performed from M. tuberculosis colonies suspended in 200 μL of TE buffer 1X (1mM EDTA, 10mM Tris HCl). Extracted DNA was stored in cryotubes at -200C for polymerase chain reaction (PCR) analysis. PCR was performed as previously described (15) with M. tuberculosis H37Rv as a positive control. The rpoB, katG, inhA and inhA promoter, rpsL, rrs and embB genes were amplified and analysed by fragment analysis (First BASE Laboratories Sdn Bhd, Malaysia). Nine fragments of 6 genes known to be involved in M. tuberculosis drug resistance were studied: embB gene (1 fragment), rpsL (1 fragment), rrs (2 fragments), rpoB (1 fragment), katG (2 fragments), inhA gene and its promoter. Table S1 (on-line supplement) specifies the target regions sequenced and the primers used. All drug resistant and ten randomly selected drug susceptible isolates were sequenced. A consensus sequence for each fragment was created using the Bioedit Sequence Alignment Editor and Sequence Scanner software. Fragment sequences were compared with H37Rv (GenBank NC.000962.3; http://www.ncbi.nlm.nih.gov/gene) to assess the presence of mutations.
Statistical analysis and ethics approval We performed descriptive data analysis using STATA version 13 (StataCorp, College Station, TX, USA). Continuous variables were expressed as the median or mean ± standard deviation (SD) and categorical variables as the number and percentage. The study was approved by the Pham Ngoc Thach Hospital Ethics Review Committee
(approved on 9 Oct 2014) and the Human Research Ethics Committee at the University of Sydney (Project No 2015/522).
RESULTS The majority (63.8%) of TB/HIV co-infected cases were young adults (25-34 years of age); predominantly males (86.5%) (Table 1). Sputum smear microscopy results were recorded in only 46.5% of the cases; 38.7% being sputum smear-positive. In total, 84 (42.0%) isolates had in vitro resistance to at least one anti-TB drug; 17 (8.5%) were MDR and 10 (5.0%) demonstrated pan-resistance to all first-line drugs tested (Figure 1). Resistance to streptomycin was most common, being present in 40.0% of all isolates tested; in 95.2% (80/84) of isolates with “any drug resistance” and in all MDR isolates. No rifampicin monoresistance was detected. Ethambutol resistance was only observed among pan-resistant isolates.
Table 2 presents all mutations observed in phenotypically drug resistant M. tuberculosis isolates (including instances where the same mutation was not associated with phenotyic resistance in our study). Mutations in the katG and inhA (including inhA promoter) genes were found in 100% and 20% of the 49 INH resistant isolates, respectively. Interestingly, the most common katG mutation (463 G→T; Arg463Leu) was observed in isoniazid resistant and susceptible isolates and is known to be phylogenetically informative (16). Only one mutation was found in the open reading frame of the inhA gene (Ser94Ala), whereas the promoter inhA region had mutations at positions -15 (14%) and -191 (5%).
There were 17 MDR, 1 rifampicin and streptomycin poly-resistant and 0 rifampicin mono-resistant isolates. All 18 isolates that were phenotypically resistant to rifampicin had mutations in the rpoB gene; 2 isolates had more than one amino acid substitution (Ser531Leu and Thr481Ala; Ser531Leu and Arg529Gln). The most frequent mutation (531 C→T; Ser531Leu) was present in 72% of all rifampicin resistant isolates. Among isolates with phenotypic rifampicin resistance (16/18; 89%) had mutations in the RRDR (codon 507-533). In total 3 rifampicin resistant isolates had mutations outside the RRDR, two at codon 572 and one at codon 481; one had an additional mutation inside the RRDR. No rpoB mutations were detected in the 10 susceptible isolates tested.
Multiple polymorphisms were found in the embB gene; occurring both in phenotypically susceptible and resistant isolates (Table 2). Mutations at codon 378 (A→C, Glu-Ala) were common, however, it has been recognised as a phylogentically informative mutation (16) and only 2/10 (20.0%) strains with this mutation had phenotypic resistance to ethambutol (Table 2). Both rpsL and rrs genes were sequenced to explore mutations associated with streptomycin resistance. Mutations in the rpsL gene were present in 48/80 (60%) streptomycin resistant isolates; mostly at codons 43 (44%) and 88 (13%). One strain had mutations in rpsL (Lys43Arg) and at codon 1401 of the rrs gene, which is associated with resistance to second-line injectables. Mutations in the in rrs gene was less common, being present in 12/80 (15%) streptomycin resistant isolates. No rpsL or rrs gene mutations were found in strains without phenotypic streptomycin resistance.
Table 3 shows the M. tuberculosis strain diversity and strain-associated phenotypic drug resistance observed. Beijing lineage strains were most prevalent (49%) followed by East African-Indian (EAI) lineage strains (35%). Minority strains included Haarlem, LAM, MANU, T and U. The majority (60%) of strains with “any drug resistance”, and 71% of MDR strains, belonged to the Beijing lineage. Among EIA lineage strains, sub-lineage EAI-5 accounted for 56/70 (80%) of the isolates, including all MDR strains. Neither spoligotyping nor MIRU-24 was able to adequately differentiate Beijing lineage strains, but the variable drug resistance profiles observed did not suggest a large transmission cluster.
Table 4 reflects drug resistance mutations observed in patients with MDR-TB, focusing on Beijing and EAI lineage strains. Table S2 includes all lineages and mutations detected in drug resistance genes. Two mutations, rpoB Ser531Leu and katG Ser315Thr were found in the majority of MDR isolates, irrespective of strain background. No strain-specific mutation patterns could be identified. Of 17 MDR isolates, 15 (88%) had a Ser315Thr substitution in the katG gene and 2 (12%) had a 15 (C-T) substitution in the inhA gene promotor. In the rpoB gene, 12 (71%) MDR isolates had a Ser531Leu and 3 (18%) a His526Tyr substitution. One MDR isolate had a single mutation outside the RRDR region (at codon 572) and would have been missed by the Xpert MTB RIF® test.
DISCUSSION We found streptomycin resistance to be common in TB/HIV co-infected patients in Ho Chi Minh city and nearly universally present in drug resistant cases. This observation is similar to previous findings in Vietnam (17, 18) and recent
observations in Mongolia, where MDR-TB transmission is driven by Beijing lineage strains with pan-resistance to all first-line drugs (19). In Vietnam, injectable drugs are generally preferred and streptomycin has long been favorite drug that is easily available across the counter. Within the TB control program streptomycin was only recently (in 2016) replaced by ethambutol as the fourth drug given during the intensive phase of standard first-line treatment. Mutations in codons 43 and 88 of the rpsL gene accounted for 60% of streptomycin resistant isolates. Mutations in the rrs gene were mostly at codons 514 and 516, with only one mutation in region 1400 (1401) which has been associated with in vitro resistance to all second-line injectable drugs (20).
In contrast to reports that rifampicin mono-resistance is common among TB/HIV coinfected patients (21), we detected no rifampicin mono-resistance. Intermittent TB treatment has never been used in TB/HIV co-infected patients in Vietnam (22), neither has rifabutin been used as prophylaxis against Mycobacterium aviumintracellulare (MAC) in severely immune compromised HIV patients. Two rifampicin resistant isolates, one MDR, would have been missed by the GeneXpert MTB/RIF® assay. The Ile572Phe mutation is well characterized (23), while the mutation at codon 481 has been reported once before (24). Demonstration that an Ile491Phe mutation underwent rapid clonal expansion in Swaziland (11) illustrates the potential for diagnostic selection and epidemic spread of strains with mutations that fall outside the RRDR region detected by the GeneXpert MTB/RIF® assay. Ser531Leu is generally the most common substitution associated with rifampicin resistance (25), also among non-HIV infected patients in Vietnam (13). Although mutations in codon 516 are not always associated with phenotypic rifampicin
resistance (26), we did not detect any mutations in the 10 drug susceptible isolates tested.
Among isoniazid resistant isolates mutations at codon 463 were most common, but this was also found in all 10 drug susceptible isolates and has been associated with retained wild-type katG activity (26, 27). Many isoniazid resistant isolates in our study had a Ser315Thr substitution that has been consistently associated with highlevel INH resistance (28). Very few mutations were found in the inhA gene or its promotor region, supporting observations that the vast majority of isoniazid resistant strains in Vietnam have mutations in the katG gene and are associated with high-level isoniazid resistance (13). Resistance to ethambutol was uncommon and never observed in isolation. Mutations at codon 378 (A→C, Glu-Ala) were common, however, it has been recognised as a phylogentically informative mutation (16) and only 2/10 (20.0%) strains with this mutation had phenotypic resistance to ethambutol. The role of mutations in codon 306 is uncertain, since it is commonly detected in ethambutol resistant and susceptible isolates (29-31). Mutations in embB codons 306, 406 and 497 have all been associated with low level ethambutol resistance (32). This may have been missed by the single 2 μg/mL breakpoint used in our study. It has also been demonstrated that M. tuberculosis might develop a pre-resistant state characterized by ethambutol MICs below the critical threshold, with such preresistant strains acquiring additional resistance in a stepwise manner (33).
Beijing lineage strains accounted for nearly half of all isolates and the majority of MDR strains. This is consistent with previous findings from non-HIV infected patients in Vietnam (34, 35). Beijing strains are highly prevalent in most Asian
countries and also predominate among TB/HIV patients in parts of Africa (36). Among EAI lineage strains sub-lineage 5 was over represented, while sub-lineage EAI4_VNM has traditionally been most common among non-HIV-infected TB patients in Vietnam (34, 37). It is interesting to consider if this difference might be explained by HIV co-infection, or indicates possible transmission among people living with HIV in Ho Chi Minh City. Mutations at codon 531 in rpoB gene and codon 315 in katG gene showed a strong correlation with Beijing genotype and with multidrug resistance (38).
Our study was limited by the fact that it only included a limited number of TB/HIV co-infected patients diagnosed in Ho Chi Minh city, which may not be representative of all TB/HIV co-infected patients in Vietnam (39). Although a limited number of consecutive isolates were evaluated, we believe it should provide a fairly representative overview of drug resistance mutations and M. tuberculosis strain diversity among TB/HIV co-infected patients in Ho Chi Minh city. It would have been of interest to assess M. tuberculosis isolates from new and retreatment TB cases, but this information was not available in the laboratory database. In addition, the added value of this distinction has been challenged (7). The predominance of young males in our study reflects the fact the HIV epidemic in Vietnam primarily affects injecting drug users and men who have sex with men (4) which limit its generalizability to settings like sub-Saharan Africa where HIV transmission is mostly heterosexual and affects equal numbers of women (40).
In conclusion, M. tuberculosis isolates from TB/HIV co-infected patients in Ho Chi Ming City showed high rates of drug resistance with a predominance of Beijing
lineage strains. More detailed genomic analysis is needed to explore transmission patterns and mutations that may affect resistance to second-line anti-TB drugs.
DECLARATIONS Funding : Funding from the NHMRC Centre for Research Excellence in Tuberculosis and the Australian Award Scholarship for TQM are also gratefully acknowledged. Competing Interests: No conflict of interest Ethical Approval: Approved by the Ethics Review Committee at the PNT Hospital, Vietnam (approved on 9 Oct 2014), and the Human Research Ethics Committee at the University of Sydney, Australia (Project No 2015/522)
Conflict of interests: None
ACKNOWLEDGEMENTS The authors thank doctors and nurses from the Pham Ngoc Thach TB and Lung Disease Hospital for their help with data collection; Dr. Thai Thanh Truc from the University of Medicine and Pharmacy at Ho Chi Minh city for assistance in data analysis.
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Figure 1: Resistance against first-line TB drugs among TB/HIV co-infected patients in Ho Chi Minh City, Vietnam (2009-2014)
TB – tuberculosis; HIV – human immunodeficiency virus; MDR – multidrug resistant (resistant to at least isoniazid and rifampicin). *Pan-resistant: resistant to all 4 first line drugs tested including isoniazid, rifampicin, ethambutol and streptomycin; pyrazinamide resistance was not determined
Table 1: Demographic characteristics and bacteriological profiles of TB/HIV co-infected patients in Ho Chi Minh City, Vietnam Characteristic Gender (N=200) Male Age distribution (N=174) Median Age and Range (years) Age category 17-24 25-34 35-44 >45
n (%) 173 (86.5) 31 (17-57) 13 (7.5) 111 (63.8) 38 (21.8) 12 (6.9)
Patient location (N=200) Ho Chi Minh urban
181 (90.5)
Sputum smear microscopy (N=93)* Positive
36 (38.7)
Drug susceptibility testing (N=200) Any drug resistance Resistance to a single drug Mono H Mono R Mono S Resistance to multiple classes of drugs, but not MDR HS RS Multidrug resistant (MDR) HR HRS HRSE
84 (42.0) 4 (2.0) 0 34 (17.0) 28 (14.0) 1 (0.5) 0 7 (3.5) 10 (5.0)
*Sputum smear microscopy results were recorded in only 93 patients TB – tuberculosis; HIV – human immunodeficiency virus; MDR – multidrug resistant (resistant to at least isoniazid and rifampicin); H – isoniazid; R – rifampicin; E – ethambutol; S - streptomycin
Table 2: Mutations in phenotypically drug resistant M. tuberculosis isolated from TB/HIV co-infected patients in Ho Chi Minh City, Vietnam Codon with mutation
Nucleotide substitution
Isoniazid resistant (N=49) katG gene 131 C→A 190 G→A 315 G→C 315 AGC-ACA 463 G→T 569 C→T 614 G→A inhA gene 94 T→G inhA promotor region 15 C→T 191 C→T Rifampicin resistant (N=18) rpoB gene 481## A→G 516 A→T 526 C→T 526 A→G 526 CAC-CTA 529 G→A 531 C→T 572## A→T
Amino acid alteration
Any DR n (%) N=84**
Mono-DR n (%) H S N=4 N=34
Poly-DR n (%)
MDR n (%)
Susceptible* n (%)
HS N=28
RS N=1
HRS N=7
HRSE N=10
N=10#
Pro-Gln Glu Ser-Thr Ser-Thr Arg- Leu Pro-Ser Ala-Thr
1 (1.2) 1 (1.2) 36 (42.9) 2 (2.4) 74 (88.0) 1 (1.2) 1 (1.2)
0 0 2 (50.0) 0 3 (75.0) 0 0
1 (2.9) 1 (2.9) 0 0 29 (85.3) 0 0
0 0 19 (67.9) 2 (7.1) 25 (89.3) 1 (3.6) 0
0 0 0 0 1 (100) 0 0
0 0 6 (85.7) 0 7 (100) 0 1 (14.3)
0 0 9 (90.0) 0 9 (90.0) 0 0
0 0 0 0 10 (100) 0 0
Ser-Ala
1 (1.2)
0
0
0
0
0
1 (10.0)
0
NA NA
12 (14.3) 4 (4.8)
2 (50.0) 1 (75.0)
3 (8.8) 2 (5.9)
4 (14.3) 1 (3.6)
1 (100) 0
1 (14.3) 0
1 (10.0) 0
0 0
Thr-Ala Asp-Val His-Tyr His-Arg His-Leu Arg-Gln Ser-Leu Ile-Phe
1 (1.2) 1 (1.2) 1 (1.2) 1 (1.2) 1 (1.2) 1 (1.2) 13 (15.5) 2 (2.4)
0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 1 (3.6)
0 0 0 0 0 0 1 (100) 0
0 0 0 0 0 0 7 (100) 0
1 (10.0) 1 (10.0) 1 (10.0) 1 (10.0) 1 (10.0) 1 (10.0) 5 (50.0) 1 (10.0)
0 0 0 0 0 0 0 0
Ethambutol resistant (N=10) embB gene 306 G→A 306 A→G 330 T→C 354 A→C 355 G→A 378 A→C 405 G→T 406 G→A 439 G→A 454 G→A 497 A→G 534 C→T 571 G→A Streptomycin resistant (N=80) rpsL gene 43 A→G 88 A→G 88 A→C rrs gene 514 A→C 516 C→T 1401 A→G
Met-Ile Met-Val Phe-Ser Asp-Ala Leu Glu-Ala Glu-Asp Gly-Ser Ala-Thr Ala-Thr Gln-Arg Asp Ala-Thr
5 (6.0) 5 (6.0) 1 (1.2) 1 (1.2) 1 (1.2) 17 (20.2) 1 (1.2) 1 (1.2) 2 (2.4) 2 (2.4) 3 (3.6) 9 (10.8) 1 (1.2)
0 0 0 0 0 3 (75.0) 0 0 0 0 0 0 0
0 0 0 1 (2.9) 1 (2.9) 4 (11.8) 0 0 0 0 0 2 (5.9) 1 (2.9)
1 (3.6) 1 (3.6) 0 0 0 8 (25.0) 0 0 2 (7.1) 0 0 3 (10.7) 0
0 0 0 0 0 0 0 0 0 0 0 0 0
2 (28.6) 1 (14.3) 0 0 0 0 0 0 0 2 (28.6) 1 (14.3) 3 (42.9) 0
2 (20.0) 3 (30.0) 1 (10.0) 0 0 2 (20.0) 1 (10.0) 1 (10.0) 0 0 2 (20.0) 1 (10.0) 0
0 0 0 0 1 (100) 7 (70.0) 0 0 0 0 0 0 0
Lys-Arg Lys-Arg Lys- Thr
37 (44.0) 8 (9.5) 3 (3.6)
0 0 0
11 (32.4) 1 (2.9) 0
12 (42.9) 5 (17.9) 3 (10.7)
1 (100.0) 0 0
6 (85.7) 0 0
7 (70.0) 2 (20.0) 0
0 0 0
NA NA NA
6 (7.1) 4 (4.8) 1 (1.2)
0 0 0
5 (14.7) 4 (11.8) 0
0 0 0
0 0 0
1 (14.3) 0 1 (14.3)
0 0 0
0 0 0
NA – not applicable; DR – drug resistant; MDR – multidrug resistant; *Some mutations were detected in drug susceptible “control isolates” as well; ** From 200 tested; #10 randomly selected fully drug susceptible strains; ##Not contained in the 81-bp Rifampicin Resistance Defining Region (RRDR) of the rpoB gene
Table 3: M. tuberculosis strain diversity and strain-associated phenotypic drug resistance in TB/HIV co-infected patients in Ho Chi Minh City, Vietnam
MIRU-24 strain lineage
MIRU-24 sublineage
Beijing
Bejing
EAI
EAI1, EAI1_SOM EAI2_Manila EAI4_VNM EAI5
Haarlem
H3
LAM
LAM9
MANU
MANU_Ancestor, MANU2
T
T1, T2, T3:T2
U
U
Total (N)
Spoligotype
All
Any DR
n (%)
n (%)
H
S
HS
RS
HRS
98 (49.0)
50 (59.8)
0
19(55.9)
18 (64.3)
1 (100)
5 (71.4)
48, Orphan*
3 (1.5)
0
0
0
0
0
0
0
19
1 (0.5)
1 (1.1)
0
1 (2.9)
0
0
0
0
139, 1901, 564, Orphan
10 (5.0)
2 (2.2)
0
0
2 (7.1)
0
0
0
126, 139, 152, 234, 236, 413, 517, 564, 618, 894, 1372, 1609, Orphan*
56 (28.0)
20 (24.0)
2 (50.0)
7 (20.7)
6 (21.5)
0
2 (28.6)
3 (30.0)
50, 335, 742
5 (2.5)
2 (2.2)
0
0
2 (7.1)
0
0
0
42
1 (0.5)
0
0
0
0
0
0
0
5 (2.5)
1 (1.1)
0
1 (2.9)
0
0
0
0
53, 73, 393, Orphan*
9 (4.5)
4 (4.8)
1 (25.0)
3 (8.8)
0
0
0
0
619, Orphan*
12 (6.0)
4 (4.8)
1 (25.0)
3 (8.8)
0
0
0
0
200
84
4
34
28
1
7
10
1
523, 1634, 1690
Mono-DR
Poly-DR
MDR HRSE 7 (70.0)
MIRU-24 - mycobacterial interspersed repetitive units 24 loci; TB – tuberculosis; HIV – human immunodeficiency virus; Mono-DR – Mono-drug resistant (resistant to a single first line drug); Poly-DR – Poly-drug resistant (resistant to two or more first-line drugs but not to both isoniazid and rifampicin); MDR – multidrug resistant (resistant to at least isoniazid and rifampicin); H – isoniazid; R – rifampicin; E – ethambutol; S – streptomycin; Phenotypic resistance parameters used: isoniazid (0.2 μg/ml), rifampin (40 μg/ml), streptomycin (4 μg/ml), and ethambutol (2 μg/ml); *Orphan = unique spoligotype
Table 4: Association between dominant M. tuberculosis strain lineages and drug resistance mutations in TB/HIV co-infected patients with MDR-TB
Gene
katG
inhA rpoB
embB
rpsL rrs
Mutated codon Beijing (N=12) 1 (8.3) 1 (8.3) 9 (75.0) 1 (8.3) 0 1 (8.3) 0 11 (91.7) 0 2 (16.7) 8 (66.7) 1 (8.3) 0 1 (8.3) 0 4 (33.3) 1 (8.3) 1 (8.3) 0 2 (16.7) 1 (8.3) 0 0 1 (8.3) 1 (8.3) 1 (8.3) 9 (75.0) 2 (16.7) 1 (8.3) 1 (8.3) 0 11 (91.7)
315 463* 315 & 463* 315, 463*, 613 & 614 No mutation -15** -15** & 94 No mutation 516 526 531 572 481 & 531 526 & 529 No mutation 306 330 497 534+ 306 & 454*** 306 & 534 378* & 405 378* & 406 497 & 534+ 497 & 558 No mutation 43 88 No mutation 514 1401* No mutation
Strain lineage n (%) EAI5 (N=5) 0 1 (20.0) 4 (80.0) 0 0 0 1 (20.0) 4 (80.0) 1 (20.0) 1 (20.0) 2 (40.0) 0 1 (20.0) 0 0 1 (20.0) 0 0 2 (40.0) 0 0 1 (20.0) 1 (20.0) 0 0 0 4 (80.0) 0 1 (20.0) 0 (8.7) 1 (20.0) 4 (80.0)
Total (N=17) 1 (5.9) 2 (11.8) 13 (76.5) 1 (5.9) 0 2 (11.8) 0 15 (88.2) 1 (5.9) 3 (17.6) 10 (58.8) 1 (5.9) 1 (5.9) 1 (5.9) 0 5 (29.4) 1 (5.9) 1 (5.9) 2 (11.8) 2 (11.8) 1 (5.9) 1 (5.9) 1 (5.9) 1 (5.9) 1 (5.9) 1 (5.9) 13(76.5) 2 (11.8) 2 (11.8) 1 (5.9) 1 (5.9) 15 (88.2)
TB – tuberculosis; HIV – human immunodeficiency virus; MDR – multidrug resistant (resistant to at least isoniazid and rifampicin) +synonymous mutation; *Mutation not associated with resistance to the relevant drug; ** Mutation in the promoter region; *** New allele substitution
26
S2213-7165(17)30126-1 http://dx.doi.org/doi:10.1016/j.jgar.2017.07.003 JGAR 450
To appear in: Received date: Revised date: Accepted date:
8-3-2017 22-6-2017 12-7-2017
Please cite this article as: Trinh Quynh Mai, Nguyen Thi Van Anh, Nguyen Tran Hien, Nguyen Huu Lan, Do Chau Giang, Pham Thi Thu Hang, Nguyen Thi Ngoc Lan, Ben J.Marais, Vitali Sintchenko, Drug resistance and Mycobacterium tuberculosis strain diversity in TB/HIV co-infected patients in Ho Chi Minh city, Vietnam (2010), http://dx.doi.org/10.1016/j.jgar.2017.07.003 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Drug resistance and Mycobacterium tuberculosis strain diversity in TB/HIV coinfected patients in Ho Chi Minh city, Vietnam Trinh Quynh Mai1,2,3, Nguyen Thi Van Anh1, Nguyen Tran Hien1, Nguyen Huu Lan4, Do Chau Giang4, Pham Thi Thu Hang4, Nguyen Thi Ngoc Lan4, Ben J Marais2, Vitali Sintchenko2,3
1
National Institute of Hygiene and Epidemiology, Hanoi, Vietnam; 2Sydney Medical
School and Marie Bashir Institute for Infectious Diseases and Biosecurity, The University of Sydney, Australia; 3Centre for Infectious Disease and Microbiology – Public Health, ICPMR, Westmead Hospital, Sydney, Australia; 4Pham Ngoc Thach TB and Lung Disease Hospital, Ho Chi Minh City, Vietnam.
Corresponding author Trinh Quynh Mai Tuberculosis Laboratory, National Institute of Hygiene and Epidemiology No 1 Yersin Street, Hai Ba Trung District Hanoi, 10000 Vietnam Tel: +84 983110183 Email: [email protected]
Word count: 2679 words (Tables 4, Figure 1)
Highlights
TB/HIV co-infection in Vietnam was associated with high rates of TB drug resistance and likely community transmission of drug resistant strains.
The vast majority of isoniazid resistant strains in Vietnam have high-level isoniazid resistance in HIV (+) patients.
Beijing is the predominant lineage among TB/HIV co-infected patients; but EAI-5 is specifically overrepresented compared to HIV (-) patients.
The variety of drug resistance profiles and phylogenetic diversity of strains do not suggest high rates of nosocomial transmission.
ABSTRACT Background: Mycobacterium tuberculosis strain diversity and drug resistance among people living with human immunodeficiency virus (HIV) in Vietnam have not been described previously. Methods: We examined M. tuberculosis isolates from TB/HIV co-infected patients in Ho Chi Minh City, Vietnam. Drug susceptibility testing (DST), spoligotyping and 24-locus Mycobacterial Interspersed Repetitive Unit (MIRU-24 typing) were performed, and the rpoB, katG, inhA and inhA promoter, rpsL, rrs and embB genes were sequenced in all drug resistant isolates identified. Results: In total, 84/200 (42.0%) strains demonstrated “any drug resistance”; 17 (8.5%) were multi-drug resistant (MDR). Streptomycin resistance was present in 80 (40.0%) isolates; 95.2% (80/84) with “any drug resistance” and 100% with MDR. No rifampicin monoresistance was detected. Of the rifampicin resistant strains 16/18 (88.9%) had mutations in the 81-bp Rifampicin Resistance Defining Region (RRDR)
of the rpoB gene. Isoniazid resistance was mostly associated with Ser315Thr mutations in the katG gene (15/17; 88.2%). Beijing (49.0%) and East African Indian (EAI) lineage strains (35.0%; 56/70 EAI-5) were most common. Conclusion: TB/HIV co-infection in Vietnam was associated with high rates of TB drug resistance, although we were unable to differentiate new from retreatment cases.
Keywords: Mycobacterium tuberculosis; drug resistance, tuberculosis; epidemiology; TB/HIV co-infection; Vietnam
Word
count:
188
BACKGROUND Tuberculosis (TB) and human immunodeficiency virus (HIV) infection have mutually detrimental effects that complicate patient management and global TB control efforts (1). Of the estimated 10.4 million new TB cases identified worldwide in 2015, 1.2 million (11%) were co-infected with HIV, with 0.4 million TB-related deaths occurring in people living with HIV (2). The Asia Pacific region, which contributes more than 60% of all TB cases worldwide, reports relatively low TB/HIV co-infection rates compared to sub-Saharan Africa (2). Although HIV has not had a major detrimental impact on TB control in the Asia-Pacific region, its future burden remains uncertain given poor disease visibility and unreliable data; less than two thirds of TB patients are routinely tested for HIV (3). In Vietnam, the health outcomes of TB/HIV co-infected patients have improved in recent years, with concerted bi-directional screening and better access to anti-retroviral therapy (ART) (4). However, rising rates of drug resistant TB have become a major concern (4).
In 2015 Vietnam ranked 15th among high-burden TB countries and 16th among highburden multi-drug resistant (MDR)-TB countries in the world, based on the absolute number of incident cases (2). MDR-TB was reported in 4% of new and 25% of retreatment TB cases (2). MDR-TB can be acquired as a result of inadequate treatment of patients initially infected with a fully or partially drug susceptible strain (secondary or acquired drug resistance) or can occur following direct transmission of an MDR strain (primary or transmitted drug resistance) (5). Nosocomial transmission in health care facilities, is a particular concern among HIV-infected patients with immunodeficiency (6). When interpreting data reported to the World Health Organization (WHO) a common assumption has been that resistance rates among new
TB cases provide an accurate indication of primary (transmitted) drug resistance. However, recent modeling and strain typing data suggest that in the absence of routine drug susceptibility testing (DST) the majority of MDR-TB cases diagnosed at retreatment represent primary (transmitted) drug resistance, especially in the AsiaPacific region (7).
Despite the rapid expansion MDR-TB services and national efforts to identify and treat MDR-TB cases in Vietnam, case numbers continue to rise (8). MDR-TB/HIV co-infection is a particular concern given the poor treatment outcomes achieved and the risk of MDR-TB transmission within health care settings (9). While the roll-out of the Xpert MTB RIF® test in Vietnam has improved screening for MDR-TB (10), concerns about potential false negatives in strains with mutations outside the 81-bp Rifampicin Resistance Defining Region (RRDR) persist (11). In addition, our understanding of M. tuberculosis strain diversity, transmission dynamics and drug resistance profiles in TB/HIV co-infected patients in Vietnam remains limited. This study examined the M. tuberculosis strain diversity and described the frequency and molecular mechanisms of resistance against first-line TB drugs in TB/HIV co-infected patients in Ho Chi Minh City, Vietnam.
METHODS We conducted a retrospective laboratory-based analysis of 200 M. tuberculosis isolates from TB/HIV co-infected patients sent to the Pham Ngoc Thach Hospital mycobacterial reference laboratory in Ho Chi Minh city, Vietnam. We included consecutive culture positive isolates routinely collected from 2009-2014, during
periods that no other studies were performed. Demographic data, including age, gender and residential province were collected from the laboratory database.
Study setting Ho Chi Minh City is the most populous metropolis in Vietnam, with an estimated population of 8.2 million (12). Pham Ngoc Thach Hospital is the largest (750-bed) lung disease hospital in Ho Chi Minh City and treated over 7,600 TB patients in 2016. The hospital serves as a tertiary referral center for TB patients and people living with HIV throughout southern Vietnam (12). The hospital has a WHO accredited regional mycobacterial reference laboratory (13). Pham Ngoc Thach Hospital was one of the first centers to provide ART and is the largest center providing treatment to HIVinfected and TB/HIV co-infected patients in Vietnam.
Drug susceptibility testing Primary mycobacterial cultures were grown on solid Lowenstein–Jensen (LJ) media (bioMérieux SA, Marcy-l’Étoile, France), or liquid culture (BACTECTM or MGITTM 960; Becton Dickinson & Co., Franklin Lakes, NJ). All isolates identified as M. tuberculosis underwent phenotypic DST against isoniazid, rifampicin, streptomycin and ethambutol by the standard 1% proportion method (14). Cultures were grown on solid LJ media with the following critical drug concentrations: isoniazid (0.2 μg/mL), rifampin (40 μg/mL), streptomycin (4 μg/mL), and ethambutol (2 μg/mL).
Spoligotyping, MIRU-24 typing and gene sequencing Spoligotyping was performed according to standard protocol (15) and phylogenetic lineages
were
assigned
using
international
SIVITWEB databases
(http://www.pasteur-guadeloupe.fr:8081/SITVIT_ONLINE/) (http://cgi2.cs.rpi.edu/~bennek/SPOTCLUST.html).
and
24-locus
SPOTCLUST Mycobacterial
Interspersed Repetitive Unit (MIRU-24 typing) was performed based on Supply’s protocol (2006) and strain lineage using MIRU-24 typing was assigned using the miru-vntrplus.org online database. DNA extraction was performed from M. tuberculosis colonies suspended in 200 μL of TE buffer 1X (1mM EDTA, 10mM Tris HCl). Extracted DNA was stored in cryotubes at -200C for polymerase chain reaction (PCR) analysis. PCR was performed as previously described (15) with M. tuberculosis H37Rv as a positive control. The rpoB, katG, inhA and inhA promoter, rpsL, rrs and embB genes were amplified and analysed by fragment analysis (First BASE Laboratories Sdn Bhd, Malaysia). Nine fragments of 6 genes known to be involved in M. tuberculosis drug resistance were studied: embB gene (1 fragment), rpsL (1 fragment), rrs (2 fragments), rpoB (1 fragment), katG (2 fragments), inhA gene and its promoter. Table S1 (on-line supplement) specifies the target regions sequenced and the primers used. All drug resistant and ten randomly selected drug susceptible isolates were sequenced. A consensus sequence for each fragment was created using the Bioedit Sequence Alignment Editor and Sequence Scanner software. Fragment sequences were compared with H37Rv (GenBank NC.000962.3; http://www.ncbi.nlm.nih.gov/gene) to assess the presence of mutations.
Statistical analysis and ethics approval We performed descriptive data analysis using STATA version 13 (StataCorp, College Station, TX, USA). Continuous variables were expressed as the median or mean ± standard deviation (SD) and categorical variables as the number and percentage. The study was approved by the Pham Ngoc Thach Hospital Ethics Review Committee
(approved on 9 Oct 2014) and the Human Research Ethics Committee at the University of Sydney (Project No 2015/522).
RESULTS The majority (63.8%) of TB/HIV co-infected cases were young adults (25-34 years of age); predominantly males (86.5%) (Table 1). Sputum smear microscopy results were recorded in only 46.5% of the cases; 38.7% being sputum smear-positive. In total, 84 (42.0%) isolates had in vitro resistance to at least one anti-TB drug; 17 (8.5%) were MDR and 10 (5.0%) demonstrated pan-resistance to all first-line drugs tested (Figure 1). Resistance to streptomycin was most common, being present in 40.0% of all isolates tested; in 95.2% (80/84) of isolates with “any drug resistance” and in all MDR isolates. No rifampicin monoresistance was detected. Ethambutol resistance was only observed among pan-resistant isolates.
Table 2 presents all mutations observed in phenotypically drug resistant M. tuberculosis isolates (including instances where the same mutation was not associated with phenotyic resistance in our study). Mutations in the katG and inhA (including inhA promoter) genes were found in 100% and 20% of the 49 INH resistant isolates, respectively. Interestingly, the most common katG mutation (463 G→T; Arg463Leu) was observed in isoniazid resistant and susceptible isolates and is known to be phylogenetically informative (16). Only one mutation was found in the open reading frame of the inhA gene (Ser94Ala), whereas the promoter inhA region had mutations at positions -15 (14%) and -191 (5%).
There were 17 MDR, 1 rifampicin and streptomycin poly-resistant and 0 rifampicin mono-resistant isolates. All 18 isolates that were phenotypically resistant to rifampicin had mutations in the rpoB gene; 2 isolates had more than one amino acid substitution (Ser531Leu and Thr481Ala; Ser531Leu and Arg529Gln). The most frequent mutation (531 C→T; Ser531Leu) was present in 72% of all rifampicin resistant isolates. Among isolates with phenotypic rifampicin resistance (16/18; 89%) had mutations in the RRDR (codon 507-533). In total 3 rifampicin resistant isolates had mutations outside the RRDR, two at codon 572 and one at codon 481; one had an additional mutation inside the RRDR. No rpoB mutations were detected in the 10 susceptible isolates tested.
Multiple polymorphisms were found in the embB gene; occurring both in phenotypically susceptible and resistant isolates (Table 2). Mutations at codon 378 (A→C, Glu-Ala) were common, however, it has been recognised as a phylogentically informative mutation (16) and only 2/10 (20.0%) strains with this mutation had phenotypic resistance to ethambutol (Table 2). Both rpsL and rrs genes were sequenced to explore mutations associated with streptomycin resistance. Mutations in the rpsL gene were present in 48/80 (60%) streptomycin resistant isolates; mostly at codons 43 (44%) and 88 (13%). One strain had mutations in rpsL (Lys43Arg) and at codon 1401 of the rrs gene, which is associated with resistance to second-line injectables. Mutations in the in rrs gene was less common, being present in 12/80 (15%) streptomycin resistant isolates. No rpsL or rrs gene mutations were found in strains without phenotypic streptomycin resistance.
Table 3 shows the M. tuberculosis strain diversity and strain-associated phenotypic drug resistance observed. Beijing lineage strains were most prevalent (49%) followed by East African-Indian (EAI) lineage strains (35%). Minority strains included Haarlem, LAM, MANU, T and U. The majority (60%) of strains with “any drug resistance”, and 71% of MDR strains, belonged to the Beijing lineage. Among EIA lineage strains, sub-lineage EAI-5 accounted for 56/70 (80%) of the isolates, including all MDR strains. Neither spoligotyping nor MIRU-24 was able to adequately differentiate Beijing lineage strains, but the variable drug resistance profiles observed did not suggest a large transmission cluster.
Table 4 reflects drug resistance mutations observed in patients with MDR-TB, focusing on Beijing and EAI lineage strains. Table S2 includes all lineages and mutations detected in drug resistance genes. Two mutations, rpoB Ser531Leu and katG Ser315Thr were found in the majority of MDR isolates, irrespective of strain background. No strain-specific mutation patterns could be identified. Of 17 MDR isolates, 15 (88%) had a Ser315Thr substitution in the katG gene and 2 (12%) had a 15 (C-T) substitution in the inhA gene promotor. In the rpoB gene, 12 (71%) MDR isolates had a Ser531Leu and 3 (18%) a His526Tyr substitution. One MDR isolate had a single mutation outside the RRDR region (at codon 572) and would have been missed by the Xpert MTB RIF® test.
DISCUSSION We found streptomycin resistance to be common in TB/HIV co-infected patients in Ho Chi Minh city and nearly universally present in drug resistant cases. This observation is similar to previous findings in Vietnam (17, 18) and recent
observations in Mongolia, where MDR-TB transmission is driven by Beijing lineage strains with pan-resistance to all first-line drugs (19). In Vietnam, injectable drugs are generally preferred and streptomycin has long been favorite drug that is easily available across the counter. Within the TB control program streptomycin was only recently (in 2016) replaced by ethambutol as the fourth drug given during the intensive phase of standard first-line treatment. Mutations in codons 43 and 88 of the rpsL gene accounted for 60% of streptomycin resistant isolates. Mutations in the rrs gene were mostly at codons 514 and 516, with only one mutation in region 1400 (1401) which has been associated with in vitro resistance to all second-line injectable drugs (20).
In contrast to reports that rifampicin mono-resistance is common among TB/HIV coinfected patients (21), we detected no rifampicin mono-resistance. Intermittent TB treatment has never been used in TB/HIV co-infected patients in Vietnam (22), neither has rifabutin been used as prophylaxis against Mycobacterium aviumintracellulare (MAC) in severely immune compromised HIV patients. Two rifampicin resistant isolates, one MDR, would have been missed by the GeneXpert MTB/RIF® assay. The Ile572Phe mutation is well characterized (23), while the mutation at codon 481 has been reported once before (24). Demonstration that an Ile491Phe mutation underwent rapid clonal expansion in Swaziland (11) illustrates the potential for diagnostic selection and epidemic spread of strains with mutations that fall outside the RRDR region detected by the GeneXpert MTB/RIF® assay. Ser531Leu is generally the most common substitution associated with rifampicin resistance (25), also among non-HIV infected patients in Vietnam (13). Although mutations in codon 516 are not always associated with phenotypic rifampicin
resistance (26), we did not detect any mutations in the 10 drug susceptible isolates tested.
Among isoniazid resistant isolates mutations at codon 463 were most common, but this was also found in all 10 drug susceptible isolates and has been associated with retained wild-type katG activity (26, 27). Many isoniazid resistant isolates in our study had a Ser315Thr substitution that has been consistently associated with highlevel INH resistance (28). Very few mutations were found in the inhA gene or its promotor region, supporting observations that the vast majority of isoniazid resistant strains in Vietnam have mutations in the katG gene and are associated with high-level isoniazid resistance (13). Resistance to ethambutol was uncommon and never observed in isolation. Mutations at codon 378 (A→C, Glu-Ala) were common, however, it has been recognised as a phylogentically informative mutation (16) and only 2/10 (20.0%) strains with this mutation had phenotypic resistance to ethambutol. The role of mutations in codon 306 is uncertain, since it is commonly detected in ethambutol resistant and susceptible isolates (29-31). Mutations in embB codons 306, 406 and 497 have all been associated with low level ethambutol resistance (32). This may have been missed by the single 2 μg/mL breakpoint used in our study. It has also been demonstrated that M. tuberculosis might develop a pre-resistant state characterized by ethambutol MICs below the critical threshold, with such preresistant strains acquiring additional resistance in a stepwise manner (33).
Beijing lineage strains accounted for nearly half of all isolates and the majority of MDR strains. This is consistent with previous findings from non-HIV infected patients in Vietnam (34, 35). Beijing strains are highly prevalent in most Asian
countries and also predominate among TB/HIV patients in parts of Africa (36). Among EAI lineage strains sub-lineage 5 was over represented, while sub-lineage EAI4_VNM has traditionally been most common among non-HIV-infected TB patients in Vietnam (34, 37). It is interesting to consider if this difference might be explained by HIV co-infection, or indicates possible transmission among people living with HIV in Ho Chi Minh City. Mutations at codon 531 in rpoB gene and codon 315 in katG gene showed a strong correlation with Beijing genotype and with multidrug resistance (38).
Our study was limited by the fact that it only included a limited number of TB/HIV co-infected patients diagnosed in Ho Chi Minh city, which may not be representative of all TB/HIV co-infected patients in Vietnam (39). Although a limited number of consecutive isolates were evaluated, we believe it should provide a fairly representative overview of drug resistance mutations and M. tuberculosis strain diversity among TB/HIV co-infected patients in Ho Chi Minh city. It would have been of interest to assess M. tuberculosis isolates from new and retreatment TB cases, but this information was not available in the laboratory database. In addition, the added value of this distinction has been challenged (7). The predominance of young males in our study reflects the fact the HIV epidemic in Vietnam primarily affects injecting drug users and men who have sex with men (4) which limit its generalizability to settings like sub-Saharan Africa where HIV transmission is mostly heterosexual and affects equal numbers of women (40).
In conclusion, M. tuberculosis isolates from TB/HIV co-infected patients in Ho Chi Ming City showed high rates of drug resistance with a predominance of Beijing
lineage strains. More detailed genomic analysis is needed to explore transmission patterns and mutations that may affect resistance to second-line anti-TB drugs.
DECLARATIONS Funding : Funding from the NHMRC Centre for Research Excellence in Tuberculosis and the Australian Award Scholarship for TQM are also gratefully acknowledged. Competing Interests: No conflict of interest Ethical Approval: Approved by the Ethics Review Committee at the PNT Hospital, Vietnam (approved on 9 Oct 2014), and the Human Research Ethics Committee at the University of Sydney, Australia (Project No 2015/522)
Conflict of interests: None
ACKNOWLEDGEMENTS The authors thank doctors and nurses from the Pham Ngoc Thach TB and Lung Disease Hospital for their help with data collection; Dr. Thai Thanh Truc from the University of Medicine and Pharmacy at Ho Chi Minh city for assistance in data analysis.
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Figure 1: Resistance against first-line TB drugs among TB/HIV co-infected patients in Ho Chi Minh City, Vietnam (2009-2014)
TB – tuberculosis; HIV – human immunodeficiency virus; MDR – multidrug resistant (resistant to at least isoniazid and rifampicin). *Pan-resistant: resistant to all 4 first line drugs tested including isoniazid, rifampicin, ethambutol and streptomycin; pyrazinamide resistance was not determined
Table 1: Demographic characteristics and bacteriological profiles of TB/HIV co-infected patients in Ho Chi Minh City, Vietnam Characteristic Gender (N=200) Male Age distribution (N=174) Median Age and Range (years) Age category 17-24 25-34 35-44 >45
n (%) 173 (86.5) 31 (17-57) 13 (7.5) 111 (63.8) 38 (21.8) 12 (6.9)
Patient location (N=200) Ho Chi Minh urban
181 (90.5)
Sputum smear microscopy (N=93)* Positive
36 (38.7)
Drug susceptibility testing (N=200) Any drug resistance Resistance to a single drug Mono H Mono R Mono S Resistance to multiple classes of drugs, but not MDR HS RS Multidrug resistant (MDR) HR HRS HRSE
84 (42.0) 4 (2.0) 0 34 (17.0) 28 (14.0) 1 (0.5) 0 7 (3.5) 10 (5.0)
*Sputum smear microscopy results were recorded in only 93 patients TB – tuberculosis; HIV – human immunodeficiency virus; MDR – multidrug resistant (resistant to at least isoniazid and rifampicin); H – isoniazid; R – rifampicin; E – ethambutol; S - streptomycin
Table 2: Mutations in phenotypically drug resistant M. tuberculosis isolated from TB/HIV co-infected patients in Ho Chi Minh City, Vietnam Codon with mutation
Nucleotide substitution
Isoniazid resistant (N=49) katG gene 131 C→A 190 G→A 315 G→C 315 AGC-ACA 463 G→T 569 C→T 614 G→A inhA gene 94 T→G inhA promotor region 15 C→T 191 C→T Rifampicin resistant (N=18) rpoB gene 481## A→G 516 A→T 526 C→T 526 A→G 526 CAC-CTA 529 G→A 531 C→T 572## A→T
Amino acid alteration
Any DR n (%) N=84**
Mono-DR n (%) H S N=4 N=34
Poly-DR n (%)
MDR n (%)
Susceptible* n (%)
HS N=28
RS N=1
HRS N=7
HRSE N=10
N=10#
Pro-Gln Glu Ser-Thr Ser-Thr Arg- Leu Pro-Ser Ala-Thr
1 (1.2) 1 (1.2) 36 (42.9) 2 (2.4) 74 (88.0) 1 (1.2) 1 (1.2)
0 0 2 (50.0) 0 3 (75.0) 0 0
1 (2.9) 1 (2.9) 0 0 29 (85.3) 0 0
0 0 19 (67.9) 2 (7.1) 25 (89.3) 1 (3.6) 0
0 0 0 0 1 (100) 0 0
0 0 6 (85.7) 0 7 (100) 0 1 (14.3)
0 0 9 (90.0) 0 9 (90.0) 0 0
0 0 0 0 10 (100) 0 0
Ser-Ala
1 (1.2)
0
0
0
0
0
1 (10.0)
0
NA NA
12 (14.3) 4 (4.8)
2 (50.0) 1 (75.0)
3 (8.8) 2 (5.9)
4 (14.3) 1 (3.6)
1 (100) 0
1 (14.3) 0
1 (10.0) 0
0 0
Thr-Ala Asp-Val His-Tyr His-Arg His-Leu Arg-Gln Ser-Leu Ile-Phe
1 (1.2) 1 (1.2) 1 (1.2) 1 (1.2) 1 (1.2) 1 (1.2) 13 (15.5) 2 (2.4)
0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 1 (3.6)
0 0 0 0 0 0 1 (100) 0
0 0 0 0 0 0 7 (100) 0
1 (10.0) 1 (10.0) 1 (10.0) 1 (10.0) 1 (10.0) 1 (10.0) 5 (50.0) 1 (10.0)
0 0 0 0 0 0 0 0
Ethambutol resistant (N=10) embB gene 306 G→A 306 A→G 330 T→C 354 A→C 355 G→A 378 A→C 405 G→T 406 G→A 439 G→A 454 G→A 497 A→G 534 C→T 571 G→A Streptomycin resistant (N=80) rpsL gene 43 A→G 88 A→G 88 A→C rrs gene 514 A→C 516 C→T 1401 A→G
Met-Ile Met-Val Phe-Ser Asp-Ala Leu Glu-Ala Glu-Asp Gly-Ser Ala-Thr Ala-Thr Gln-Arg Asp Ala-Thr
5 (6.0) 5 (6.0) 1 (1.2) 1 (1.2) 1 (1.2) 17 (20.2) 1 (1.2) 1 (1.2) 2 (2.4) 2 (2.4) 3 (3.6) 9 (10.8) 1 (1.2)
0 0 0 0 0 3 (75.0) 0 0 0 0 0 0 0
0 0 0 1 (2.9) 1 (2.9) 4 (11.8) 0 0 0 0 0 2 (5.9) 1 (2.9)
1 (3.6) 1 (3.6) 0 0 0 8 (25.0) 0 0 2 (7.1) 0 0 3 (10.7) 0
0 0 0 0 0 0 0 0 0 0 0 0 0
2 (28.6) 1 (14.3) 0 0 0 0 0 0 0 2 (28.6) 1 (14.3) 3 (42.9) 0
2 (20.0) 3 (30.0) 1 (10.0) 0 0 2 (20.0) 1 (10.0) 1 (10.0) 0 0 2 (20.0) 1 (10.0) 0
0 0 0 0 1 (100) 7 (70.0) 0 0 0 0 0 0 0
Lys-Arg Lys-Arg Lys- Thr
37 (44.0) 8 (9.5) 3 (3.6)
0 0 0
11 (32.4) 1 (2.9) 0
12 (42.9) 5 (17.9) 3 (10.7)
1 (100.0) 0 0
6 (85.7) 0 0
7 (70.0) 2 (20.0) 0
0 0 0
NA NA NA
6 (7.1) 4 (4.8) 1 (1.2)
0 0 0
5 (14.7) 4 (11.8) 0
0 0 0
0 0 0
1 (14.3) 0 1 (14.3)
0 0 0
0 0 0
NA – not applicable; DR – drug resistant; MDR – multidrug resistant; *Some mutations were detected in drug susceptible “control isolates” as well; ** From 200 tested; #10 randomly selected fully drug susceptible strains; ##Not contained in the 81-bp Rifampicin Resistance Defining Region (RRDR) of the rpoB gene
Table 3: M. tuberculosis strain diversity and strain-associated phenotypic drug resistance in TB/HIV co-infected patients in Ho Chi Minh City, Vietnam
MIRU-24 strain lineage
MIRU-24 sublineage
Beijing
Bejing
EAI
EAI1, EAI1_SOM EAI2_Manila EAI4_VNM EAI5
Haarlem
H3
LAM
LAM9
MANU
MANU_Ancestor, MANU2
T
T1, T2, T3:T2
U
U
Total (N)
Spoligotype
All
Any DR
n (%)
n (%)
H
S
HS
RS
HRS
98 (49.0)
50 (59.8)
0
19(55.9)
18 (64.3)
1 (100)
5 (71.4)
48, Orphan*
3 (1.5)
0
0
0
0
0
0
0
19
1 (0.5)
1 (1.1)
0
1 (2.9)
0
0
0
0
139, 1901, 564, Orphan
10 (5.0)
2 (2.2)
0
0
2 (7.1)
0
0
0
126, 139, 152, 234, 236, 413, 517, 564, 618, 894, 1372, 1609, Orphan*
56 (28.0)
20 (24.0)
2 (50.0)
7 (20.7)
6 (21.5)
0
2 (28.6)
3 (30.0)
50, 335, 742
5 (2.5)
2 (2.2)
0
0
2 (7.1)
0
0
0
42
1 (0.5)
0
0
0
0
0
0
0
5 (2.5)
1 (1.1)
0
1 (2.9)
0
0
0
0
53, 73, 393, Orphan*
9 (4.5)
4 (4.8)
1 (25.0)
3 (8.8)
0
0
0
0
619, Orphan*
12 (6.0)
4 (4.8)
1 (25.0)
3 (8.8)
0
0
0
0
200
84
4
34
28
1
7
10
1
523, 1634, 1690
Mono-DR
Poly-DR
MDR HRSE 7 (70.0)
MIRU-24 - mycobacterial interspersed repetitive units 24 loci; TB – tuberculosis; HIV – human immunodeficiency virus; Mono-DR – Mono-drug resistant (resistant to a single first line drug); Poly-DR – Poly-drug resistant (resistant to two or more first-line drugs but not to both isoniazid and rifampicin); MDR – multidrug resistant (resistant to at least isoniazid and rifampicin); H – isoniazid; R – rifampicin; E – ethambutol; S – streptomycin; Phenotypic resistance parameters used: isoniazid (0.2 μg/ml), rifampin (40 μg/ml), streptomycin (4 μg/ml), and ethambutol (2 μg/ml); *Orphan = unique spoligotype
Table 4: Association between dominant M. tuberculosis strain lineages and drug resistance mutations in TB/HIV co-infected patients with MDR-TB
Gene
katG
inhA rpoB
embB
rpsL rrs
Mutated codon Beijing (N=12) 1 (8.3) 1 (8.3) 9 (75.0) 1 (8.3) 0 1 (8.3) 0 11 (91.7) 0 2 (16.7) 8 (66.7) 1 (8.3) 0 1 (8.3) 0 4 (33.3) 1 (8.3) 1 (8.3) 0 2 (16.7) 1 (8.3) 0 0 1 (8.3) 1 (8.3) 1 (8.3) 9 (75.0) 2 (16.7) 1 (8.3) 1 (8.3) 0 11 (91.7)
315 463* 315 & 463* 315, 463*, 613 & 614 No mutation -15** -15** & 94 No mutation 516 526 531 572 481 & 531 526 & 529 No mutation 306 330 497 534+ 306 & 454*** 306 & 534 378* & 405 378* & 406 497 & 534+ 497 & 558 No mutation 43 88 No mutation 514 1401* No mutation
Strain lineage n (%) EAI5 (N=5) 0 1 (20.0) 4 (80.0) 0 0 0 1 (20.0) 4 (80.0) 1 (20.0) 1 (20.0) 2 (40.0) 0 1 (20.0) 0 0 1 (20.0) 0 0 2 (40.0) 0 0 1 (20.0) 1 (20.0) 0 0 0 4 (80.0) 0 1 (20.0) 0 (8.7) 1 (20.0) 4 (80.0)
Total (N=17) 1 (5.9) 2 (11.8) 13 (76.5) 1 (5.9) 0 2 (11.8) 0 15 (88.2) 1 (5.9) 3 (17.6) 10 (58.8) 1 (5.9) 1 (5.9) 1 (5.9) 0 5 (29.4) 1 (5.9) 1 (5.9) 2 (11.8) 2 (11.8) 1 (5.9) 1 (5.9) 1 (5.9) 1 (5.9) 1 (5.9) 1 (5.9) 13(76.5) 2 (11.8) 2 (11.8) 1 (5.9) 1 (5.9) 15 (88.2)
TB – tuberculosis; HIV – human immunodeficiency virus; MDR – multidrug resistant (resistant to at least isoniazid and rifampicin) +synonymous mutation; *Mutation not associated with resistance to the relevant drug; ** Mutation in the promoter region; *** New allele substitution
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