Background
Despite the existence of anti-tuberculosis drugs for the last 60 years, tuberculosis (TB) continues to be a major threat worldwide. In 2009, WHO estimated the global incidence of TB with 9.4 million cases. Most of the estimated number of TB cases occurred in Asia (55%) and Africa (30%). The 22 high burden tuberculosis countries account for 81% of all estimated cases worldwide [
1]. Ethiopia ranks seventh among the world’s 22 high-burden tuberculosis countries. The country had 314,267 TB cases in 2007, with an estimated incidence rate of 378 cases per 100,000 population [
2]. According to the Ministry of Health hospital statistics data, tuberculosis is one of the leading causes of morbidity, the fourth most common cause of hospital admission, and the second most common cause of hospital death in Ethiopia [
3]. Additionally, the countrywide anti-TB drug resistance survey conducted in 2005 showed that the prevalence of multidrug resistant TB (MDR, resistance to at least isoniazid [INH] and rifampicin [RMP]) was 1.6% and 11.8% among new cases and previously treated TB cases, respectively [
4]. These data show that the TB epidemic is a significant public health threat in Ethiopia.
Molecular strain typing (genotyping) has contributed significantly to the understanding of TB epidemiology and has helped to improve TB control by providing information on transmission dynamics [
5], determining the importance of reactivation versus exogenous reinfection [
6], investigating/confirming outbreaks [
7], confirmation of laboratory cross contamination [
8] and to identify the clonal spread of successful clones, including multi-drug-resistant ones [
9]. Furthermore, molecular typing has revealed that the MTBC has a diverse population structure with manifold lineages that show large differences in their geographical occurrence and, also, in their pathobiological properties e.g. development and spread of drug resistance [
10].
In Ethiopia, few molecular epidemiological studies have been done so far only in the capital city, Addis Ababa [
11‐
13]. Recent data are only available from an MDR strain targeted study from year 2006. However, the strains were investigated by spoligotyping only, allowing neither for high resolution phylogenetic strain classification nor for analysis of transmission dynamics [
11].
In this study, we used a combination of mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) typing and spoligotyping methods to investigate a large collection of MTBC strains isolated from patients living in Amhara region, Northwest Ethiopia. In contrast to classical molecular typing methods such as IS
6110 DNA fingerprint and spoligotyping, 24-loci MIRU-VNTR genotyping allows for a high-resolution discrimination of isolates for epidemiological studies and a valid phylogenetic strain classification [
14]. The data obtained allow for new insights into population structure and transmission dynamics, thus also revealing urgently needed data to improve TB control in Ethiopia.
Methods
Study design, area and study period
A total of 260 smear positive pulmonary tuberculosis patients diagnosed at Gondar Hospital, Gondar Health Center, Metemma Hospital, Bahir Dar Hospital and Debre Markos Hospital between March 2009 and July 2009 were included in this study. For all study subjects, information on the socio-demographic data, history of previous tuberculosis treatment, HIV status and the drug susceptibility patterns of the
M.
tuberculosis isolates was available. The single morning sputum sample and 5 ml venous blood sample were collected prior to commencing TB treatment. A structured questionnaire was used to classify patients into new and previously treated tuberculosis cases and to collect socio-demographic data of the study subjects. Specimens were stored and transported to the Institute of Medical Microbiology and Epidemiology of Infectious Diseases, University Hospital of Leipzig, Germany as described previously [
15] for culture and drug susceptibility testing. The study was approved by the research and publication committee of University of Gondar, Ethiopia. Written informed consent was obtained from all study subjects.
Culture and drug susceptibility testing (DST)
Isolation, identification and DST were performed as described previously [
16]. Briefly, specimens were processed and cultured according to the Deutsches Institut für Normung (DIN) recommendations [
17] using Lowenstein Jensen (L-J) media, Gottsacker media and the BacT/ALERT 3D system. Isolates were identified by DNA hybridization technology (GenoType® MTBC; Hain Lifescience, Nehren, Germany) following the manufacturer’s instructions. DST for first line drugs including isoniazid, rifampicin, streptomycin, ethambutol and pyrazinamide was performed by BacT/ALERT 3D system (BioMerieux, S.A, France) according to the methods developed for MB/BacT system [
18,
19]. DST for second line drugs including fluoroquinolones (FLQ) (ofloxacin & moxifloxacin) and aminoglycocides (AM)/cyclic peptides (CM) (capreomycin, viomicin/kanamycin and amikacin) was performed using DNA hybridization technology on nitrocellulose strips (GenoType® MTBDRsl; Hain Lifescience, Nehren, Germany) following the manufacturer’s instructions. Patients’ serum samples were screened for HIV-1 and HIV-2 using Vironostika HIV Uni-Form II Ag/Ab enzyme-linked immunosorbent assay (ELISA) kit (Bio-Merieux, Boxtel, The Netherlands) following the manufacturer’s instructions.
DNA was extracted from all isolates by heating mycobacterial pellets obtained from liquid culture, suspended in 200 μL 10 mM Tris–HCl, 1 mM EDTA (pH 7.0) buffer at 95°C for 20 minutes followed by 15 minutes sonication in a sonicating water bath. The suspension was centrifuged at 15,000 rpm for 1 minute, and the supernatant was stored at -20°C until used.
Genotyping
All isolates were analyzed by spoligotyping technique as described previously [
20] and by 24 - loci MIRU-VNTR genotyping technique as described previously [
14]. Briefly, for MIRU-VNTR genotyping, 24 loci were amplified by using the MIRU-VNTR typing kit (Genoscreen, Lille, France). Analyses of the PCR products were performed by using the Rox-labeled MapMarker 1,500 size standard (BioVentures, Inc., Murfreesboro, VT) for mix 5 and 1000 size standard (BioVentures, Inc., Murfreesboro, VT) for other mixes (mix 1–4, and mix 6–8), and using the ABI 3130 XL sequencer with 16 capillaries (Applied Biosystems, Foster City, CA). Sizing of the PCR fragments and assignment of the various VNTR alleles were done by using the GeneMapper software version 4.0 (Applied Biosystems, Foster City, CA).
The MIRU-VNTR 24-loci profiles and spoligotyping patterns were used to classify the strains into main phylogenetic lineages by using the reference strain collection and identification tools available online at
http://www.miru-vntrplus.org[
21]. Briefly, a stepwise identification procedure was carried out as follows. The strains were first classified by the simple match approach that is based on the best match with strains of the reference database. The cut of distance for lineage assignment was set to 0.17. In a second step, phylogenetic tree identification was carried out. Additionally, for each MIRU-VNTR 24-loci pattern a unique MLVA 15–9 code was assigned by using the MIRU-VNTRplus nomenclature.
Cluster analyses of molecular typing data were performed with the Bionumerics software (version 6.6; Applied Maths, Sint-Martens-Latem, Belgium) according to the manufacturers’ instructions. Similarities of genotyping patterns among strains were calculated by using the categorical coefficient. A dendrogram was generated by using the unweighted pair group method with arithmetic averages (UPGMA). Minimum spanning tree analysis was done based on MIRU-VNTR typing data by using the categorical coefficient. For the cluster analysis, a cluster was defined as a minimum of two strains harbouring identical DNA genotyping patterns (using composite data, MIRU-VNTR 24-loci and spoligotyping) from different patients belonging to the study subjects. The recent transmission index (RTI) was calculated as (number of clustered patients - number of clusters)/total number of patients. Determination of the discriminatory power of the genotyping methods (MIRU-VNTR 24-loci typing and Spoligotyping) was calculated using the Hunter-Gaston Discriminatory Index (HGDI) as previously described [
22].
Statistical analysis
All laboratory data were entered, cleared and analyzed using SPSS version 13 statistical package software (SPSS Inc., Chicago, IL). Categorical data were compared by the chi-square test or the fisher exact test, when expected cell sizes (n) were smaller than 5. Two models were constructed in a logistic regression analysis using clusters and anti-TB drug resistance as the respective outcome variables. In order to determine independent risk factors, odds ratios (OR) and 95% confidence intervals (CI) were calculated by using logistic regression analysis for demographic (gender, age, address and religion), epidemiologic (previous treatment and HIV status), and microbiological variables (drug resistance, and infection by M. tuberculosis lineages). P-values less than 0.05 were considered statistically significant.
Discussion
Recent advances in molecular strain typing such as the development of 24-loci MIRU-VNTR typing provide a powerful tool to analyze MTBC population structure and transmission dynamics locally and on the global level, which provides valuable information for the development of effective tuberculosis control policy. In this study, we present the first in-depth analysis of the population structure of M. tuberculosis strains in Northwest Ethiopia based on high-resolution MIRU-VNTR 24-loci typing and spoligotyping. Our data confirm a highly diverse population structure that comprises, thirteen phylogenetic lineages, four of which were not described before. Furthermore, our data indicate a high rate of recent transmission, of which the spread of resistant and MDR strains is of special importance.
While homoplasy is a true phenomenon within the evolution of TB, spoligotyping has been shown to provide invalid phylogenetic classifications by suggesting homoplasy too often [
25]. In the contrary, the MIRU-VNTR 24-loci typing method applied in our study has the advantage to allow for high-resolution genotyping needed for molecular epidemiological studies and, simultaneously, for valid phylogenetic strain classification enabling screening for new phylogenetic lineages/clonal complexes [
14].
Using this method, 90.6% of the strains investigated were classified into various
M.
tuberculosis complex lineages; of which, 58.9% were described before and 31.6% were newly described in this study. We documented that
M.
tuberculosis Dehli/CAS is the predominant phylogenetic lineage in Ethiopia, accounting for 39% of investigated strains. Similarly, a previously published study from the capital city of Ethiopia showed that 43.5% of the strains were of the CAS lineage [
11], and a study from Sudan [
26] also showed that
M.
tuberculosis Dehli/CAS is the predominant lineage (49%) of investigated strains. The Dehli/CAS lineage is essentially localized in the Central Asia and Middle-East, more specifically in India [
27]. Two hypotheses could explain the presence of high Dehli/CAS lineage in Ethiopia: (i) the large Indian and Chinese communities in Ethiopia due to the growing economic partnerships between Ethiopia and the two Asian countries, India and China may have contributed in the introduction of this lineage; or (ii) this lineage could have emerged from Ethiopia and migrated through Asia, this hypothesis is in agreement with the suggestion that East Africa is the origin of
M.
tuberculosis complex species [
28].
Additionally, we confirmed the presence of previously undefined phylogenetic lineages named as Ethiopia_3, Ethiopia_1, Ethiopia_H37RV-like and Ethiopia_2 that were clearly defined by tree based, as well as by minimum spanning tree-based analysis. However, comparison with other studies is hampered by the fact that they are mainly based on IS6110 DNA fingerprint and/or spoligotyping analysis hindering a valid analysis of the population structure and standardized comparisons based on MIRU-VNTR nomenclature. Thus, the actual picture of M. tuberculosis population diversity in African, high-incidence settings is largely incomplete and needs a systematic investigation with phylogenetic useful genotyping methods.
This study also showed a significant association between infection with strains of the Haarlem lineage and multi-drug resistance, resistance to all first line anti-TB drugs and resistance to each first line anti-TB drugs including INH, RMP, STM, EMB and PZA. Similarly, a previous study from Tunisia showed that the Haarlem family genotype has a similar relationship with drug resistance and rapid clonal expansion [
29]. From TB-control point of view, it is relevant to understand whether specific genotype families are overrepresented among drug-resistant cases and, in particular, if these resistant strains are successfully transmitted within the community. In this study, HIV infection was not significantly associated with resistance to anti-TB drugs. The high HIV prevalence in the study subjects did not appear to be a significant risk factor selectively driving drug resistance development and transmission. This might be due to the fact that HIV infection increases the susceptibility of the population for both drug susceptible and drug resistant
M.
tuberculosis strains.
Clustering is a marker for recent transmission [
30‐
32]. By using degree of recent TB transmission in a study population, one can estimate the efficacy of the TB control program [
30]. Both high TB incidence and the current drug-resistance rates in Ethiopia are indicative of defects of the TB control program [
2,
4,
16]. Supporting this suggestion, we found a high rate of clustering, 45.1% of the total strains investigated. This is in agreement with the previous reports from the capital city of Ethiopia that showed clustering rate of 41.2% [
13] and 48.1% [
12].
Even more important, we confirm an elevated clustering rate in drug resistant strains in general as well as for MDR strains. Similarly, there was a significant association between recent transmission and patients with the history of previous TB treatment, infection with INH resistant strains, STM resistant strains, EMB resistant strains, strains resistant to one or more first line anti-TB drugs and patients with strains resistant to all first line anti-TB drugs. This might be due to the fact that, in Ethiopia there is no culture and drug susceptibility testing facility for routine diagnosis of drug resistance, thus, drug resistant-TB is only diagnosed after prolonged treatment with first-line anti-TB drugs and clinical recognition that treatment has failed. Treatment of drug-resistant TB with standard first line drugs, instead of a regimen designed according to the resistance pattern has several potential adverse consequences: patients remain on inadequate treatment longer, increasing the risk of treatment failure or death; selection of drug resistant strains and patients remain infectious, promoting transmission to close contacts [
33]. These data indicate a successful transmission of drug resistant and MDR strains in the community, a situation that needs to be carefully monitored in the future to determine extensive transmission of resistant strains early enough to avoid more significant problems for TB control as already eminent in several parts of Eastern Europe or South Africa [
34,
35].
Interestingly, we present evidence of significant association between recent transmission and the Dehli/CAS, Ethiopia_3, TUR and Ethiopia_H37Rv like strain infections. Similarly, Gagneux et al. have recently proposed that the major
M.
tuberculosis lineages have evolved so as to become adapted to specific host genetic backgrounds and are much more likely to transmit and cause disease among patients of the same ethnicity [
36].
Acknowledgements
We acknowledge Elisabeth Krawczyk for her kind assistance during culture and drug susceptibility testing, Tanja Ubben for her assistance for genotyping by MIRU-VNTR typing and spoligotyping, Julia Zallet for her assistance during spoligotyping. We also give our appreciation to all data collectors and study participants from all study areas in Northwest Ethiopia. The work presented in this paper was made possible by funding from the German Federal Ministry of Education and Research (BMBF, PtJ-Bio, 0315883). This study was also supported by Institute of Medical Microbiology and Epidemiology of Infectious Diseases, University Hospital of Leipzig, Germany; German Academic Exchange Service (DAAD); Molecular Mycobacteriology, Research Centre Borstel, Borstel, Germany; and University of Gondar, Ethiopia.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
BT was the primary researcher, conceived the study, designed, participated in sample collection, performed laboratory experiments, conducted data analysis, strain classification, cluster analysis and drafted the manuscript for publication. JB participated in the interpretation of the results and reviewed the initial and final manuscript. MM participated in performing cluster analysis and reviewed the initial and final manuscript. FE, US and AR reviewed the initial and final manuscript. SN Participated in strain classification, cluster analysis, interpretation of the results and reviewed the initial and final manuscript. All authors read and approved the final manuscript.