Highlights
- •Spoligotyping and the LSP analyses showed the lineage 4, including the LAM family, as the major M. tuberculosis genotype (78.8%) in Lusaka, Zambia.
- •The LAM11_ZWE (SIT 59) subfamily was the most prevalent genotype and did not show predilection for multidrug resistance (MDR).
- •The CAS1-Kili (SIT 21) and LAM1 (SIT 20) subfamilies significantly associated with MDR-TB (p = 0.0001 and p = 0.001, respectively).
- •Three isolates were identified as M. bovis, indicating human infection with this zoonotic species.
- •The first TB case infected with the Beijing genotype was identified in Zambia.
Abstract
Objectives
The burden of multidrug-resistant tuberculosis (MDR-TB) has been reported to be increasing in Zambia. The reasons for the increase are still unclear. This study determined the diversity of Mycobacterium tuberculosis genotypes among isolates in Lusaka, the capital city, and investigated their association with MDR-TB.
Methods
Spoligotyping, large sequence polymorphism (LSP) analysis, and sequencing of MDR associated genes were performed on a total of 274 M. tuberculosis clinical isolates stored at the University Teaching Hospital from 2013 to 2017. Of these, 134 were MDR-TB while 126 were pan-susceptible.
Results
Spoligotyping showed the LAM family as the most predominant genotype (149/274, 54.4%) followed by the CAS family (44/274, 16.1%), T family (39/274, 14.2%), and minor proportions of X, S, Harleem, EAI and Beijing spoligofamilies were identified. Three M. bovis isolates were also observed. Among those, CAS1-Kili (SIT 21) and LAM1 (SIT 20) subfamilies showed a propensity for MDR-TB with p = 0.0001 and p = 0.001, respectively.
Conclusions
This phenomenon might explain the future increase in the MDR-TB burden caused by specific lineages in Zambia. Therefore, it is recommended that the National TB control program in the country complements conventional control strategies with molecular analysis for monitoring and surveillance of MDR-TB epidemiology.
Keywords
Introduction
Worldwide, an estimated 10 million people fell ill with tuberculosis (TB) in 2018. Of this figure, most TB cases occurred in the WHO regions of South-East Asia (44%), Africa (24%), and the Western Pacific (18%) (
World Health Organization, 2019
). An estimated 1.2 million deaths occurred due to TB among HIV-negative people and 251 000 deaths among HIV-positive people (World Health Organization, 2019
). Globally, TB is one of the top ten causes of death and the leading cause from a single infectious agent above HIV/AIDS (World Health Organization, 2019
). With early case detection and adequate health care, TB is curable and preventable. The emergence of multidrug-resistant (MDR) Mycobacterium tuberculosis strains, simultaneously resistant to rifampicin (RIF) and isoniazid (INH), is among complex factors impeding the control of TB. Globally, 3.4% of new TB cases and 18% of previously treated cases had MDR-TB or RIF-resistant TB (MDR/RR-TB) (World Health Organization, 2019
).In Zambia, a high TB burden nation, the proportions of MDR-TB have risen from 0.3% among new cases and 8.1% among previously treated cases in 2014 to 2.8% in new cases and 18% in previously treated cases in 2018 (
World Health Organization, 2019
). Local studies have also reported an emerging threat of MDR-TB in the country. For instance, a survey in Zambian prisons reported the MDR-TB rate was 9.5% (Habeenzu et al., 2007
). Another study that reviewed TB national data observed an increasing trend (four-fold between 2000 and 2011) of MDR-TB cases being detected by the national TB program (NTP) (Kapata et al., 2013
). Presently, the factors associated with this increase are not clear.Although evidence on the association between MDR-TB and HIV infection is scanty, suggestions linking HIV infection with primary MDR-TB has been made (
Suchindran et al., 2009
). Researchers in sub-Sahara Africa have speculated that in populations where HIV infection is coupled with socioeconomic challenges, poor treatment adherence and lack of access to proper treatment may contribute to the development of acquired drug-resistant TB (Sanchez-Padilla et al., 2012
). Secondly, people living with HIV/AIDS are likely to be exposed to MDR-TB patients due to increased hospitalizations in settings with inadequate infection control standards resulting in nosocomial MDR-TB infections (Mesfin et al., 2014
). Furthermore, biological studies have demonstrated poor absorption of RIF and INH in patients with HIV/AIDS (- Mesfin Y.M.
- Hailemariam D.
- Biadglign S.
- Kibret K.T.
Association between HIV/AIDS and multi-drug resistance tuberculosis: a systematic review and meta-analysis.
PLoS One. 2014; 9 (e82235)https://doi.org/10.1371/journal.pone.0082235
Patel et al., 1995
), leading to drug resistance due to sub-therapeutic drug concentrations. Zambia is one of the HIV-endemic countries, and that might have an association with the incremental increase of MDR-TB. However, to identify the associated factors of the increase of MDR-TB, we should first know the genotypes of the prevalent M. tuberculosis strains and their population structures in the area.The establishment of molecular tools has improved the understanding of the circulating strains of M. tuberculosis in various geographical locations. Spoligotyping (spacer oligonucleotide typing) is a widely employed molecular method for studying M. tuberculosis genotypic structures. It is a PCR based method and employs a reverse hybridization rationale by determining the presence or absence of the 43 specific DNA spacer sequencers in the direct repeat (DR) region of M. tuberculosis (
Kamerbeek et al., 1997
). Spoligotype data can be encoded in a numerical format and interpreted using an international database, SITVITWEB (Demay et al., 2012
).Using spoligotyping, the global mapping of major genotype families of M. tuberculosis, such as Beijing, Haarlem, T, X, S, East African-Indian (EAI), Latin American-Mediterranean (LAM), and Central Asian (CAS) families has been achieved (
Costa et al., 2013
). Some of these genotypes have been linked to drug resistance, hypervirulence, and increased transmissibility (Glynn et al., 2002
). For instance, a subfamily of the LAM genotype (LAM4) was reported as the leading cause of extensively drug-resistant (XDR) TB in KwaZulu Natal, South Africa (Pillay and Sturm, 2007
).In line with the national TB guidelines, the mainstay for routine examination of presumptive TB patients in Zambia is Xpert MTB/RIF and smear microscopy. TB culture and drug susceptibility testing (DST) is recommended for patients with poor treatment outcomes such as treatment failure and relapse cases (
Ministry of Health, 2017
). In Zambia, TB culture laboratories do not routinely genotype recovered M. tuberculosis isolates. To our knowledge, two local studies have described M. tuberculosis genotypes in two districts of Zambia, namely, Ndola (Mulenga et al., 2010b
) and Namwala (Malama et al., 2014
). Genotype profiles of M. tuberculosis strains circulating in other parts of the country are unknown, and the possible correlation of these genotypes with drug resistance is still un-investigated. In this study, genotypic structures of M. tuberculosis strains in Lusaka, the capital city of Zambia, and their association with anti-TB drugs were investigated to design effective MDR-TB control strategies.Materials and methods
Study settings and design
This was a collaborative study between the University Teaching Hospital (UTH) in Lusaka, Zambia, and Hokkaido University in Japan. The study utilized M. tuberculosis isolates routinely collected over a period of five years (2013–2017) at the UTH in Lusaka. The isolates were previously recovered from different pulmonary TB patients, mainly living in Lusaka city, followed by phenotypic DST at the TB culture laboratory in the UTH. The TB culture laboratory participates in a TB DST proficiency testing scheme offered by the Uganda Supranational TB reference laboratory.
All the available clinical M. tuberculosis isolates with determined DST profiles in the laboratory information system were revived from −80 °C storage freezers at the UTH in Lusaka, followed by DNA extraction. Extracted DNA samples were then transported to Hokkaido University in Japan for molecular examination. TB genotypes were established using spoligotyping and large sequence polymorphism (LSP), and sequencing of drug-resistant associated genes was conducted to validate phenotypic resistance to RIF and INH and determine associated mutations.
Drug susceptibility test
Phenotypic DST to the first-line anti-TB drugs was previously performed using BACTEC™ 960 MGIT™ (Mycobacteria Growth Indicator Tube) system (Becton, Dickinson and Company, Franklin Lakes, NJ, USA) in a Biosafety Level 3 (BSL3) laboratory. The critical drug concentrations of 1.0, 0.1, 1.0, and 5.0 μg/mL were employed for RIF, INH, streptomycin (STR), and ethambutol (EMB), respectively, as recommended by the kit manufacturer (Becton, Dickinson, and Company). M. tuberculosis H37Rv strain was routinely utilized as a control strain.
DNA extraction
DNA was prepared for PCR by transferring 1 mL culture broth from MGIT tube into cryovials, followed by heating the aliquots at 90 °C for ten minutes in a dry heating block.
Spoligotyping
Spoligotyping was performed on all isolates, as described by
Kamerbeek et al., 1997
. Briefly, a PCR-based reverse hybridization method was used in which the direct repeat (DR) region of M. tuberculosis was amplified using a pair of primers. The PCR products were hybridized to a set of 43 oligonucleotide probes corresponding to each spacer on the DR region and covalently bound to the membrane. Obtained spoligo-patterns were then compared to the SITVIT-Web database (http://www.pasteur-guadeloupe.fr:8081/SITVIT_ONLINE, http://www.pasteur-guadeloupe.fr:8081/SITVIT2/files/SITVIT-KBBN_report_310313.xls) for identification of spoligo families and spoligotype international types (SIT) (Demay et al., 2012
).Large Sequence Polymorphism (LSP)
Isolates that could not be classified by spoligotyping were further analyzed with a PCR-based method using specific primers for the expected region of difference (RD) for each lineage, as reported by
Gagneux et al., 2006
.Sequencing of rpoB, katG genes, and inhA regulatory region
PCR was performed with 20 μL of a mixture comprising 25 mM deoxy-ribonucleotide triphosphate (dNTP), 5 M betaine, 10 μM of each primer described by
Poudel et al., 2012
, 1 U of GoTaq DNA polymerase (Promega Corp, Madison, WI, USA), GoTaq buffer (Promega Corp) and 1 μL of DNA template. The reaction was carried out in a thermal cycler (Bio Rad Laboratories, Hercules, CA, USA) under the following conditions; pre-heating at 96 °C for one minute, 35 cycles for denaturation at 96 °C for ten seconds, attachment at 55 °C for ten seconds, and elongation at 72 °C for 30 s and a final extension at 72 °C for five minutes. Electrophoresis was then conducted using 2% agarose gel to separate the PCR products. DNA fragments were recovered from the gel and applied for sequencing according to the manufacturer’s instructions with the BigDye Terminator v3.1 cycle sequencing kit (Life Technologies Corp, Carlsbad, CA, USA) on an ABI 3500 Genetic Analyzer (Life Technologies Corp). The obtained sequences were compared to wild type sequences of M. tuberculosis H37RV using BioEdit software version 7.0.9 (Hall, 1999
).Ethical clearance
The ethical approval to conduct this study was granted by The University of Zambia Biomedical Research Ethics Committee, while The Zambia National Health Research Ethics Committee approved the transfer of mycobacterial DNA to Hokkaido University (Japan) for molecular analysis.
Data analysis
Using VassarStats (http://vassarstats.net/) (
Lowry, 1998
), an online tool, Fisher’s exact test and a chi-square test were computed to determine significance for observed differences. A p-value equal to or less than 0.05 was considered significant.Lowry R. VassarStats. Vassar College, NY USA; 1998–2020; http://vassarstats.net/: (Accessed 19 June 2020).
Results
Phenotypic drug susceptibility profile
This study analyzed a total of 274 clinical M. tuberculosis isolates, of which 134 were MDR while six and three were INH and RIF mono-resistant isolates, respectively (Table 1). Among the MDR-TB isolates, 31 were resistant to INH and RIF only, while 37 were resistant to all the four first-line drugs. One hundred and twenty-six isolates were pan susceptible (Table 1).
Table 1Phenotypic drug susceptibility profile for M. tuberculosis isolates.
| Characteristics | Resistance Patterns | No. (%) |
|---|---|---|
| MDR | INH + RIF | 31 (11.3) |
| INH + RIF + EMB | 24 (8.7) | |
| INH + RIF + STR | 42 (15.3) | |
| INH + RIF + EMB + STR | 37 (13.5) | |
| non-MDR | INH | 4 (1.5) |
| INH + STR | 2 (0.7) | |
| RIF | 1 (0.4) | |
| RIF + STR | 2 (0.7) | |
| STR | 4 (1.5) | |
| EMB | 1 (0.4) | |
| Pan-susceptible | None | 126 (46.0) |
| Total | 274 (100) |
a Susceptible for the four 1st-line anti-TB drugs; INH, RIF, EMB, and STR.
Genetic diversity of M. tuberculosis isolates
From the 274 isolates, 64 spoligotype patterns were observed and classified into major M. tuberculosis lineages; lineages 1, 2, 3, and 4 (
Gagneux et al., 2006
), as shown in Table 2. Adding to that, three M. bovis (1.1%, 3/143) isolates were also identified among the clinical samples. Unclassified spoligotype patterns by the SITVIT WEB database were identified by the recognition rule proposed by Filliol et al., 2002
or by LSP analysis. The observed frequencies of major spoligofamilies were the LAM family 54.4% (149/274), the CAS family 16.1% (44/274), and the T family 14.2% (39/274). In minor proportions, X, EAI, Harleem, S, and Beijing spoligofamilies were identified. Among the LAM family, 24 out of 29 spoligotypes, including orphans, showed a specific pattern of LAM11_ZWE (spacer deletion: 21–24, 27–30, and 33–36) and made up a majority (77.2%, 115/149) of the LAM family isolates (Table 2).Table 2Diversity of M. tuberculosis subfamilies in Lusaka, Zambia.
   |
SIT: Spoligo International Type by SITVIT WEB and/or SITVIT-KBBN_report_310313, LSP: large sequence polymorphism.
*Percentages of each spoligotype were shown when it is larger than 1.0 % (number of isolates > 2). **Used genotyping method other than spoligotyping.
Distribution of M. tuberculosis SITs among MDR-TB and non-MDR isolates
The majority of CAS1-Kili (SIT 21) isolates (33/40, 82.5%) were MDR and more predominant among total MDR-TB isolates (33/134, 24.6%) than non-MDR isolates (7/140, 5.0%) (p = 0.0001). Similarly, most of LAM1 (SIT 20) were MDR (16/18, 88.9%) and was more predominant among MDR-TB isolates compared to non-MDR isolates (11.9% vs. 1.4%, p = 0.001). Other families, including LAM11-ZWE (SIT 815), LAM11-ZWE (SIT 59), and T1 (SIT 53), did not show statistically significant differences in distribution between MDR-TB and non-MDR isolates (Table 3).
Table 3Correlation between observed M. tuberculosis SITs and MDR-TB.
| Spoligotype | MDR-TB isolates N = 134 | Non-MDR isolates N = 140 | Significance | ||
|---|---|---|---|---|---|
| SIT | CLADE | Frequency no. (%) | Frequency no. (%) | Odds ratio (95% CI) | p-value |
| 59 | LAM11-ZWE | 29 (21.6) | 19 (13.6) | 1.8 (0.4–3.3) | 0.1 |
| 815 | LAM11-ZWE | 14(10.4) | 15 (10.7) | 0.9 (0.4−2.1) | 0.9 |
| 20 | LAM1 | 16 (11.9) | 2 (1.4) | 9.4 (2.1–41.5) | 0.001a |
| 42 | LAM9 | 4 (3.0) | 8 (5.7) | 0.5 (0.1−1.7) | 0.4 |
| 21 | CAS1-Kili | 33 (24.6) | 7 (5.0) | 6.2 (2.6−14.6) | 0.0001a |
| 3284 | CAS1-Kili | 1 (0.8) | 0 | – | – |
| 2277 | CAS | 1 (0.8) | 0 | – | – |
| 25 | CAS1-Delh | 0 | 1 (0.7) | – | – |
| 137 | X2 | 7 (5.2) | 7 (5.0) | – | – |
| 476 | X2 | 0 | 2 (1.4) | – | – |
| 119 | X1 | 0 | 2 (1.4) | – | – |
| 1080 | X1 | 0 | 1 (0.7) | – | – |
| 53 | T1 | 8 (6.0) | 9 (6.4) | – | – |
| 52 | T2 | 4 (3.0) | 0 | – | – |
| 51 | T1 | 0 | 2 (1.4) | – | – |
| 117 | T2 | 0 | 1 (0.7) | – | – |
| 154 | T1 | 0 | 1 (0.7) | – | – |
| 373 | T1 | 0 | 3 (2.1) | – | – |
| 102 | T1 | 0 | 2 (1.4) | – | – |
| 245 | T1 | 0 | 1 (0.7) | – | – |
| 317 | T2 | 2 (1.5) | 1 (0.7) | – | – |
| 2067 | T1 | 0 | 1 (0.7) | – | – |
| 73 | T | 1 (0.8) | 0 | – | – |
| 34 | S | 1 (0.8) | 3 (2.1) | – | – |
| 2173 | LAM11-ZWE | 0 | 6 (4.3) | – | – |
| 812 | LAM11-ZWE | 0 | 1 (0.7)) | – | – |
| 811 | LAM11-ZWE | 1 (0.8) | 2 (1.4) | – | – |
| 814 | LAM11-ZWE | 0 | 2 (1.4) | – | – |
| 184 | LAM11-ZWE | 0 | 4 (2.9) | – | – |
| 2488 | LAM11-ZWE | 0 | 2 (1.4) | – | – |
| 1607 | LAM11-ZWE | 0 | 1 (0.7) | – | – |
| 33 | LAM3 | 0 | 2 (1.4) | – | – |
| 1468 | LAM11-ZWE | 0 | 2 (1.4) | – | – |
| 1471 | LAM11-ZWE | 0 | 1 (0.7) | – | – |
| 2017 | LAM11-ZWE | 0 | 1 (0.7) | – | – |
| 2265 | LAM11-ZWE | 0 | 1 (0.7) | – | – |
| 719 | LAM3 | 0 | 1 (0.7) | – | – |
| 10 | EAI8-MDG | 1 (0.8) | 0 | – | – |
| 702 | EAI6-BGD1 | 0 | 3 (2.1) | – | – |
| 129 | EAI6-BGD1 | 0 | 2 (1.4) | – | – |
| 1 | Beijing | 0 | 1 (0.7) | – | – |
| 482 | BOV_1 | 0 | 1 (0.7) | – | – |
| 594 | BOV_1 | 0 | 2 (1.4) | – | – |
| – | Orphansb | 11 (8.2) | 17 (12.1) | – | – |
–: Not applicable; a positive correlation with MDR-TB, bOrphans by SITVIT WEB search.
Frequencies of drug resistance-conferring mutations among M. tuberculosis isolates
Of the 134 MDR-TB isolates and three mono RIF resistant isolates, Ser531Leu was the most frequent substitution across all the SITs. CAS1-Kili (SIT 21) exhibited a high diversity of mutations, especially at codon His 526. Four isolates from LAM11_ZWE (SIT 59) carried double mutations of Asp516Tyr and Leu511Arg (Table 4).
Table 4Frequency of observed RIF-resistance associated mutations in rpoB RRDR among the four main SITs.
| Amino acid change | LAM11-ZWE SIT 59 n = 29 No. (%) | LAM11-ZWE SIT 815 n = 14 No. (%) | LAM1 SIT 20 n = 16 No. (%) | CAS1-Kili SIT 21 n = 33 No. (%) |
|---|---|---|---|---|
| Ser531Leu | 15 (51.7) | 12 (85.7) | 15 (93.8) | 10 (30.3) |
| Ser531Phe | – | 1 (7.1) | – | – |
| Ser531Trp | 5 (17.2) | – | – | – |
| His526Tyr | – | 1 (7.1) | – | 8 (24.2) |
| His526Asp | – | – | – | 7 (21.2) |
| His526Leu | – | – | – | 1 (3.0) |
| His526Gln and Leu533Pro | – | – | – | 1 (3.0) |
| His526Arg and Ser509Ile | – | – | – | 1 (3.0) |
| Ser522Val | – | – | – | 1 (3.0) |
| Asn518 deletion | – | – | – | |
| Asp516Val | 2 (6.9) | – | – | – |
| Asp516Phe | 1 (3.5) | – | – | – |
| Asp516Tyr | – | – | 1 (6.2) | – |
| Asp516Tyr and Leu511Arg | 4 (13.8) | – | – | – |
| Asp516 deletion | – | – | – | 4 (12.1) |
| Leu511Arg | 1 (3.5) | – | – | – |
| Ser531Trp and Glu504Ala | 1 (3.5) | – | – | – |
Regarding INH resistance-conferring mutations, all isolates except one carried a mutation at codon Ser315 in katG, and the majority (136/139) was Ser315Thr. Two isolates had a mutation in the inhA promoter region (C-15 T) (
Solo et al., 2020
).Discussion
Diversity of M. tuberculosis genotypes
In line with what has been previously reported in Zambia, this study identified the LAM family (54.4%) as the most prevalent spoligofamily among the examined isolates from Lusaka (
Mulenga et al., 2010b
, Malama et al., 2014
). The CAS family was second at 16.1%, and T was third at 14.2% (Table 2). Ten isolates could not be identified by the SITVIT WEB database; however, upon the application of LSP and classification by the spoligotype patterns, they were grouped into lineages 4 and 1, as shown in Table 2. Both spoligotyping and LSP analysis confirmed the predominance of the lineage 4 strain.The LAM genotype is dominant among countries in the Southern region of Africa, including Zimbabwe reported at 47.2% (
Chihota et al., 2007
), Mozambique at 37% (Viegas et al., 2010
), Angola at 64.8% (Perdigão et al., 2017
), and Malawi at 44.0% (Mallard et al., 2010
). The SITs identified at high prevalence were LAM11_ZWE (SIT 59), LAM11_ZWE (SIT 815), and LAM1 (SIT 20) (Table 2). Apart from Zambia, LAM11-ZWE has been reported to be a dominant subfamily in Zimbabwe (Chihota et al., 2007
). The origin of this subfamily (LAM11_ZWE) has been speculated to be Portugal (Chihota et al., 2018
). This genotype may have entered Southern Africa either through Angola or Mozambique, as both countries were colonized by Portugal. Zambia shares a common border with both Angola and Mozambique, and there were long-standing civil wars in these two neighboring countries, which forced many immigrants to seek refuge in Zambia. Countries in this region are also linked by shared ethnicity and trade.Another genotype identified at a relatively high proportion in the current study is the CAS family (16.1%). This rate is similar to what was reported in the Namwala District of Zambia (15%) by
Malama et al., 2014
. Among the CAS family, CAS1-Kili (SIT 21) 40/44 was the main subfamily observed in the current study. CAS1-Kili (SIT 21) has been reported as the predominant subfamily in Tanzania (25.9%) and reported to have adapted to the local population in that country (Mbugi et al., 2016
). The population of this spoligofamily has been reported to be expanding in neighboring Malawi, increasing by 12.2% between 2006 and 2008 (Glynn et al., 2010
). On the other hand, Mulenga et al., 2010b
observed a very low prevalence of the CAS family (0.7%) in Zambia's Ndola district. While the distribution of M. tuberculosis genotypes described in Ndola by Mulenga et al., 2010a
, Mulenga et al., 2010b
and in Namwala by Malama et al., 2014
may be restricted to those locations, the observations in the current study might have a national representation because the general population travels from different parts of the country to Lusaka for trade, administrative activity and social visits.The cosmopolitan nature of Lusaka city may also explain the observation of a Beijing isolate in the current study (Table 2). To our knowledge, the current study is the first to report the Beijing genotype in Zambia. Although the patient was a Zambian, the strain had likely been introduced by Asians, given the recent rapid increase in immigration from East Asia, including China. The appearance of the Beijing genotype among the studied population is worrisome as this strain has been associated with hypervirulence, drug resistance, and evasion of the Bacillus Calmette - Guerin (BCG) vaccine in some settings, including South Africa (
Hanekom et al., 2011
). The Beijing isolates identified in the current study exhibited a mono-resistance to streptomycin.Another spoligofamily identified at a relatively high proportion in the current study was the T family (14.2%). This spoligofamily has been observed to be the second most prevalent family in Zambia and Zimbabwe (
Chihota et al., 2018
). Specifically, T1 (SIT 53) was the most prevalent subfamily (Table 2), and among the neighboring countries, this SIT has been reported at a relatively high frequency in Angola (13.6%) (Perdigão et al., 2017
).Furthermore, the present study, as well as data reported by
Malama et al., 2014
, identified M. bovis among isolates from human populations. Again, the heterogenic nature of Lusaka's population may account for the three M. bovis isolates observed in the current study as people living in rural parts of the country frequently visit the capital city for various activities. Although the frequency of cases observed in the current study is low (1.1%), Malama et al., 2014
identified two M. bovis isolates from 33 samples providing a 6% prevalence of M. bovis among TB patients in the Namwala district. This figure is close to the 9% prevalence of M. bovis reported in the city of Amsterdam (the Netherlands) before the era of pasteurization of milk products (Majoor et al., 2011
). Within Zambia, Pandey et al., 2013
reported an 18.7% prevalence of M. bovis in cow milk sampled from tuberculin positive cattle. As M. bovis is intrinsically resistant against one of the first-line anti-TB drugs, pyrazinamide, identifying this species is important to treat patients properly (Bwalya et al., 2018
). Further studies involving genotyping of M. tuberculosis strains are required in other parts of the country to determine the burden of bovine TB and understand its transmission dynamics.Correlation of SITs with MDR-TB
Besides the identification of M. tuberculosis genotypes, this study investigated an association between spoligotypes and MDR-TB. Interestingly, a positive correlation was observed between MDR-TB and two genotypes, namely CAS1-Kili (SIT 21) and LAM1 (SIT 20) (Table 3).
CAS1-Kili (SIT 21) was more prevalent among MDR-TB isolates than susceptible isolates (24.6% versus 5.0%, p = 0.0001). In fact, 33 of the 40 isolates of this subfamily identified in this study were MDR. Elsewhere, CAS1-Kili (SIT 21) was found to be associated with MDR-TB in Ethiopia (
Agonafir et al., 2010
). In India, all the five MDR-TB identified in the state of Madhya Pradesh exclusively belonged to the CAS family, and investigators attributed the burden of MDR-TB in that state to the CAS genotype (Gupta et al., 2019
). Similarly, we attribute the increasing burden of MDR-TB in Zambia to the growing population of M. tuberculosis families with the propensity to developing MDR. On the contrary, CAS1-Kili (SIT 21) did not demonstrate an association with drug resistance in Tanzania. (Kibiki et al., 2007
).LAM1 (SIT 20) was another subfamily that showed association with MDR-TB (p = 0.001) in the current study (Table 3). Among neighboring countries, this SIT is prevalent in Namibia (78.5%), and Angola (18.2%) (
Perdigão et al., 2017
), but drug susceptibility data for this SIT was scarce in these two neighboring countries. Elsewhere, the LAM1 (SIT 20) genotype has been associated with MDR-TB in Portugal (Perdigão et al., 2014
).Apart from the two SITs discussed above, other genotypes identified in this study, including the predominant strains LAM11-ZWE (SIT 59) and (SIT 815), did not demonstrate a correlation with MDR-TB (Table 3). These results suggest that a lack of predilection for acquiring MDR by the predominant strains might explain the current low MDR-TB rates in Zambia. Similar assertions were presented before in Uganda when the predominant strain in that country (T2 SIT135) showed a negative correlation with anti-TB drug resistance (
Lukoye et al., 2014
). On the other hand, an earlier study conducted in Zambia, which only utilized phenotypic data, attributed the low MDR-TB burden in the country to a successful DOTS (Directly Observed Therapy Short Course) program (Mulenga et al., 2010a
). There is a need for NTP in Zambia to combine conventional TB epidemiological monitoring with molecular analysis to fully understand MDR-TB dynamics in the country.Frequencies of rpoB drug resistance-conferring mutations among MDR-TB isolates
Ser531Leu was the most prevalent mutation found in rpoB among the MDR-TB isolates in this study (Table 4). These results are consistent with what has been observed by similar studies (
Hillemann et al., 2005
, Poudel et al., 2012
, Prim et al., 2015
, San et al., 2018
). On the contrary, Lipin et al., 2007
reported unusual findings of the predominance of the Asp516Val mutation (75%) among the LAM family in Russia. Notably, in the current study, four isolates belonging to the LAM11_ZWE (SIT 59) subfamily exhibited double mutations of Asp516Tyr and Leu511Arg in the rpoB gene. Compensatory mutations have been explained to alleviate bacteria's fitness cost (Comas et al., 2012
) and increase their transmissibility (de Vos et al., 2013
). Therefore, this finding might suggest a clonal expansion of a LAM11_ZWE (SIT 59) MDR clone in Lusaka. In the LAM1 (SIT 20) subfamily, which showed a significant association with MDR-TB, a high ratio of Ser531Leu, 15 out of 16 isolates (93.8%), was observed. This higher ratio than expected might suggest a clonal expansion of a specific MDR strain, possibly possessing some compensatory mechanisms (Comas et al., 2012
, de Vos et al., 2013
). Other clusters sharing the same rare mutations, five isolates with Ser531Trp in LAM11_ZWE (SIT 59) and four isolates with Asp516 deletion in CAS1_Kili (SIT 21) might also be suggesting primary MDR-TB cases (Table 4). An in-depth analysis of those strains seems to be needed to elucidate transmission dynamics.Apart from the Ser531Leu mutation, His526Tyr was prevalent among CAS1_Kili (SIT 21) isolates. The diversity of mutations among this SIT was relatively high compared to other SITs, especially at the position His 526. Two of them were found accompanying secondary mutations within RRDR (Table 4). This specific lineage might tend to have point mutations and thus acquire drug resistance like the Beijing lineage (
Glynn et al., 2002
, San et al., 2018
). Further analysis employing whole genome sequencing will help to characterize this lineage.This study has provided baseline information on the composition of different MTB genotypes and their association with MDR-TB in Lusaka. Certain strains tend to acquire drug resistance, and some of those were shown to have already spread and caused MDR-TB, primarily in the studied area. To control such dangerous strains, an effective monitoring system using a genotyping method is necessary. The current study's data has highlighted the specific need for policy formulation towards the attainment of universal TB drug susceptibility testing by the national TB control program and surveillance of TB genotypes circulating in the country.
Study limitations and future directions
A sampling of M. tuberculosis clinical isolates from a TB culture laboratory might have an inclination towards a particular category of patient. The current study only investigated clinical isolates whose availability was influenced by the national TB testing policy algorithm (MoH, 2017). Therefore, population-based studies are needed in Zambia to validate our findings, and multicenter studies are also needed to examine transmission links of MDR-TB in the country and the region at large. In the next step, authors plan to utilize more specific epidemiological methods such as Mycobacterial Interspersed Repetitive Units-Variable Number of Tandem Repeats (MIRU-VNTR) and whole-genome sequencing to explore epidemiologic transmission dynamics of specific genotypes which have shown an association with MDR-TB in the current study.
Conclusion
Similar to previous reports, this study identified the LAM family (54.4%) at a relatively high proportion among isolates from Lusaka. The predominant SITs were LAM11-ZWE (SIT 59) and (SIT 815), and they did not show correlation with MDR-TB, a characteristic which possibly explains the current low MDR-TB status in Zambia. On the other hand, CAS1-Kili (SIT 21) and LAM1 (SIT 20) showed a predilection for acquiring MDR. An increase in the population of these genotypes may be responsible for the future increase in the MDR-TB burden in the country. Therefore, to arrest the emerging MDR-TB burden in Zambia, the NTP needs to complement conventional control strategies with molecular analysis to allow population-based molecular epidemiological surveys in Zambia.
Conflict of interests
None of the authors declared competing interests.
Funding
This work was supported in part by a grant from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (MEXT)for the Joint Research Program of the Research Center for Zoonosis Control, Hokkaido University, and in part by the Japan Agency for Medical Research and Development (AMED) under Grant Number JP20jk0210005,JP20jm0110021, JP20wm0125008 to YS and partially by RONPAKU fellowship R11522 by the Japanese Society for the Promotion of Science (JSPS) to ESS.
Acknowledgments
This was a collaborative study between Hokkaido University (Japan) and the University Teaching Hospital in Lusaka, Zambia.
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Article info
Publication history
Published online: October 11, 2020
Accepted:
October 6,
2020
Received in revised form:
October 4,
2020
Received:
July 8,
2020
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