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Genotypic Diversity and Drug Susceptibility Patterns among M. tuberculosis
Complex Isolates from South-Western Ghana
Article  in  PLoS ONE · July 2011
DOI: 10.1371/journal.pone.0021906 · Source: PubMed
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Genotypic Diversity and Drug Susceptibility Patterns
among M. tuberculosis Complex Isolates from South-
Western Ghana
Dorothy Yeboah-Manu1*, Adwoa Asante-Poku1, Thomas Bodmer2, David Stucki4,5, Kwadwo Koram1,
Frank Bonsu3, Gerd Pluschke5,6, Sebastien Gagneux4,5,6
1 Noguchi Memorial Institute for Medical Research, University of Ghana, Legon, Ghana, 2 University of Bern, Institute for Infectious Diseases, Bern, Switzerland, 3 Ghana
Health Services, Accra, Ghana, 4 MRC, National Institute for Medical Research, London, United Kingdom, 5 Swiss Tropical and Public Health Institute, Basel, Switzerland,
6 University of Basel, Basel, Switzerland
Abstract
Objective: The aim of this study was to use spoligotyping and large sequence polymorphism (LSP) to study the population
structure of M. tuberculosis complex (MTBC) isolates.
Methods: MTBC isolates were identified using standard biochemical procedures, IS6110 PCR, and large sequence
polymorphisms. Isolates were further typed using spoligotyping, and the phenotypic drug susceptibility patterns were
determined by the proportion method.
Result: One hundred and sixty-two isolates were characterised by LSP typing. Of these, 130 (80.25%) were identified as
Mycobacterium tuberculosis sensu stricto (MTBss), with the Cameroon sub-lineage being dominant (N = 59/130, 45.38%).
Thirty-two (19.75%) isolates were classified as Mycobacterium africanum type 1, and of these 26 (81.25%) were identified as
West-Africa I, and 6 (18.75%) as West-Africa II. Spoligotyping sub-lineages identified among the MTBss included Haarlem
(N = 15, 11.53%), Ghana (N = 22, 16.92%), Beijing (4, 3.08%), EAI (4, 3.08%), Uganda I (4, 3.08%), LAM (2, 1.54%), X (N = 1,
0.77%) and S (2, 1.54%). Nine isolates had SIT numbers with no identified sub-lineages while 17 had no SIT numbers. MTBss
isolates were more likely to be resistant to streptomycin (p,0.008) and to any drug resistance (p,0.03) when compared to
M. africanum.
Conclusion: This study demonstrated that overall 36.4% of TB in South-Western Ghana is caused by the Cameroon sub-
lineage of MTBC and 20% by M. africanum type 1, including both the West-Africa 1 and West-Africa 2 lineages. The diversity
of MTBC in Ghana should be considered when evaluating new TB vaccines.
Citation: Yeboah-Manu D, Asante-Poku A, Bodmer T, Stucki D, Koram K, et al. (2011) Genotypic Diversity and Drug Susceptibility Patterns among M. tuberculosis
Complex Isolates from South-Western Ghana. PLoS ONE 6(7): e21906. doi:10.1371/journal.pone.0021906
Editor: Philip Supply, Institut Pasteur de Lille, France
Received November 17, 2010; Accepted June 14, 2011; Published July 11, 2011
Copyright:  2011 Yeboah-Manu et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This investigation received financial support from the UNICEF/UNDP/World Bank/WHO special program for research and training in Tropical Diseases
for DY-M and the National Tuberculosis Program, Ghana. We also acknowledge the Leverhulme-Royal Society Africa Award for financial support. The funders had
no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: dyeboah-manu@noguchi.mimcom.org
Introduction which are resistant to first-line drugs especially isoniazid (INH) and
rifampicin (RIF). Such cases may either not be cured by the
Despite the World Health Organisation declaring tuberculosis current first-line treatment regimen or have a more expensive and
(TB) a global emergency in 1993, TB remains a major global long treatment course [5]. The tendency to acquire drug resistance
health problem. About 9 million new TB cases and 2 million may be influenced by the genetic and background of the strain
deaths occur each year. TB is the leading cause of adult mortality [6–8]. TB is caused mainly by a group of genetically closely related
caused by a single infectious agent worldwide [1–3]. Similar to species and sub-species together referred to as M. tuberculosis
other countries in sub-Saharan Africa, TB is a major public health complex (MTBC); however human TB is caused mainly by M.
problem in Ghana. In 2004, it was estimated that the prevalence tuberculosis sensu stricto (MTBss) and M. africanum. Based on
of all forms of TB was 376/100,000, with an annual incidence of biochemical analysis, M. africanum used to be subdivided into two
206 cases per 100, 000 populations. The annual risk of infection separate groups. However, genetic analyses have now indicated
with TB was estimated to be between 1–2%; deaths due to TB that M. africanum II, predominant in East-Africa is actually a
averaged 50/100,000 annually [1]. variant of M. tuberculosis. In this manuscript, M. africanum is defined
The backbone of TB control is case detection by smear as the one originally termed M. africanum 1 based on biochemical
microscopy and treatment of identified cases by the DOTS analysis, which is genetically sub- divided into West-African
strategy [4]. A threat to this strategy is the emergence of strains genotype 1 and II. While M. tuberculosis is globally distributed, M.
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Genetic Diversity in TB Isolates from Ghana
africanum is important cause of human TB in West-Africa. M Mycobacterial Isolation
africanum is responsible for up to 40% of pulmonary TB patients in All specimens after decontamination were cultured on two
some West-African countries [9–11]. DNA-DNA hybridisation Lowenstein-Jensen slopes; one with supplemented with 0.4%
and multi-locus sequencing analysis indicates that the members of sodium pyruvate to enhance the isolation of M. africanum and M.
MTBC share high genomic similarities [12]. In spite of this, bovis. The cultures were incubated at 37uC and were read weekly
various genetic methods have been developed for strain typing for growth for a maximal duration of 16 weeks.
which have been helpful for answering various epidemiological Preliminary identification of suspected mycobacterial isolates
questions and shed new light on the biology of the pathogen. was done by AFB staining and biochemical methods such as
Short- and long-term epidemiologic questions such as describing susceptibility to p-nitro benzoic acid (PNB) and to thiophene
transmission dynamics, identifying groups most at risk and risk carboxylic acid hydrazide (TCH), pyrazinamidase activity (PZA),
factors for transmission, estimating recent-versus-reactive disease nitrate reduction, niacin production [20].
and the extent of exogenous re-infection have been addressed
using these methods [13,14]. Genotyping of MTBC has also Drug Susceptibility Testing
helped in tracking the transmission links between individuals, and
The susceptibility pattern of all identified mycobacterial isolates
demonstrated instances in which epidemiologically linked people
to isoniazid (0.2 mg/ml), rifampicin (40 mg/ml), streptomycin
were in fact infected with unrelated strains [13,15,16]. Molecular
(4 mg/ml), and ethambutol (2 mg/ml) for all M. tuberculosis complex
methods that have been employed for strain differentiation among
primary isolates was determined phenotypically by the indirect
MTBC include Restriction Fragment Length Polymorphism
proportion method on L–J slants, as described previously [21].
(RFLP) analysis [9], spoligotyping [17] which detects variability
Drug resistance was expressed as the proportion of colonies that
within the direct repeat locus, Variable Number Tandem Repeats
grow on drug containing medium to drug-free medium and the
(VNTR) [18] and large sequence polymorphism (LSP) analysis
critical proportion for resistance was 1% for all drugs.
[11]. While VNTR and spoligotyping is usually used for
transmission and phylogenetic studies, LSP analysis is used for
species- and sub-species differentiation of MTBC and for DNA Extraction
phylogenetic analyses [9,13]. DNA extraction was done according to previously outlined
To date, only one study has used spoligoytping to study the protocol [22]. About two- 5 ml loop full of bacteria were heat killed
population structure of MTBC causing human TB in Ghana [19]. in 500 ml of an extraction mixture (50 mM Tris–HCl, 25 mM
This study reports the use of molecular methods to analyse a set of EDTA, and 5% monosodium glutamate). After cooling, 100 ml of
isolates cultured from sputum samples obtained from pulmonary TB a 50 mg/ml lysozyme solution was added and incubated with
patients attending various health facilities in two regions of Ghana. shaking for 2 h at 37uC. Sixty micro litres of 20 mg/ml proteinase
K solution in a 106 buffer [100 mM Tris–HCl, 50 mM EDTA,
5% sodium dodecyl sulphate (pH 7.8)] were then added and
Methods
incubated at 45uC overnight. The bacterial cell wall was fully
Specimen Collection and Patients’ Characteristics disrupted by adding 200 ml of 0.1 mm-diameter zirconia beads
Specimens included in this study were collected over a period of (BioSpec Products) to each sample and vortexing at full speed for
17 months (from October 2007 to March 2009) from sputum 4 min. Beads and undigested tissue fragments were removed by
AFB-positive pulmonary TB cases attending four main health centrifugation at 14,000 rpm for 3 minutes, and the supernatants
facilities; Agona Swedru Government Hospital, Winneba Gov- were transferred to fresh tubes for phenol-chloroform (Fluka)
ernment hospital, and St Gregory Catholic Clinic at Budumbura extraction. The DNA contained in the upper phase was
refugee camp,) covering three different districts in the Central precipitated with ethanol and re-suspended in 100 ml of water.
region and Effia-Nkwanta regional hospital in the western region
of Ghana before they were put on anti-TB drug. After informed Genotyping
consent was obtained, two sputum specimens were collected from Genotyping of MTBC isolates was done in a stepwise mode
each individual, and a structured questionnaire was used to obtain (table 1). All isolates included in the study were first identified as
standard demographic and epidemiologic data on patients. The belonging to MTBC by PCR targeting the insertion sequence IS6110
sputum specimens were either mixed with 1% cetylpyridinium as previously described [23]. Species were defined by analysing for
chloride and transported within seven days of collection to the large sequence polymorphisms (LSP) at the regions of difference (RD)
laboratory at the NMIMR or stored in a fridge and transported 9, 12 and 4 using published flanking primers [9,10]. Isolates that were
within 72 hours of collection on ice for Petroff decontamination identified as M. africanum were further typed for RD702 and RD711;
before cultivation [20]. Ethical clearance for the study was and the Cameroon lineage, which we assumed to be the most
approved by the institutional review board of the Noguchi dominant among the MTBss was defined by a deletion in RD726 also
Memorial Institute for Medical Research (Federalwide Assurance using flanking primers [10,11]. All the isolates that we confirmed as
number FWA00001824). The procedure for sampling in this study M. tuberculosis complex were further typed by spoligotyping [17].
was mainly the same as those used in routine management of TB Briefly the direct repeat region of each genome was amplified using
in Ghana. However, informed consent (written in the case of primers DRa (59-CCG AGA GGG GAC GGA AAC-39) and
literate participants and oral for those who cannot read) was biotinylated Drb (59-GGT TTT GGG TCT GAC GAC-39). The
sought from all participants before their inclusion in the study. In amplified DNA was tested for the presence of specific spacers by
the case of children below sixteen years, informed consent was hybridization with a set of 43 oligonucleotides derived from the
sought from their parents or guardians. The objectives and spacer sequences of M. tuberculosis H37Rv and M. bovis BCG P3 (the
benefits of the study were explained to them all. They were assured GenBank accession no. for the sequence of M. tuberculosis H37Rv is
of the confidentiality of all information collected from them. Z48304, and that for M. bovis BCG P3 is X57835). Bound fragments
Inconveniences of participation were explained to the participants were revealed by chemiluminescence after incubation with horse-
and they are free to join the study or exit at any time which will radish peroxidase-labeled streptavidin (Boehringer Mannheim). In
not in any way affect their treatment. order to prevent cross contamination, PCR amplifications and pre-
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Genetic Diversity in TB Isolates from Ghana
PCR procedures were conducted in physically separated rooms. the production of the specific 550 bp amplicon corresponding to a
Negative water controls were PCR amplified and included on each portion of the IS6110 DNA sequence (Figure 1a). The presence of
blot to identify any possible amplicon contamination. In addition, the main lineages within MTBC were analysed by large sequence
Positive controls (H37Rv and M. bovis BCG DNA) was amplified and polymorphism analysis at various regions of difference (RD). RD9
included on each blot. analysis by PCR identified 130/162 (80%) of the isolates as MTBss
defined by the detection of an intact PCR product (Figure 1b), and
Data Analysis among this group, RD726 PCR (Figure 1c) defined 59/130 (45%)
Spoligotypes were analysed as character types. The obtained as belonging to the Cameroon sub-lineage. 32/162 (20%) were
spoligotyping patterns were compared with those available in the classified as M. africanum type I by analysis of the RD9 region
international spoligotype database (SpolDB4) [24] containing 35,925 (Figure 1b); the majority of them 26 (81%) were identified by
spoligotypes comprising 39,295 isolates from 122 countries. A shared RD711 PCR (Figure 1d) as West-African I, and 6 (19%) as West-
type was defined as a spoligotyping pattern common to at least two African II by RD702 PCR (Figure 1e). Based on RD12 and RD4
isolates, and clades were assigned according to signatures described in analyses, no M. bovis was detected.
the database. Phylogenetic relationships among the isolates were
inferred from Spoligotyping using the MIRU-VNTR plus software. Spoligotyping Patterns
In addition we compared the diversity within the main lineages that is One hundred and sixty-one isolates comprising the 31 M.
MTBss and M. africanum I as well as between the main sublineages M. africanum and 130 MTBss isolates were spoligotyped, and the
africnaum West-African type I (WafrI) and the Cameroon family different lineages and corresponding spoligotype patterns are
(Euro-American). This was done by comparing both the number of indicated in Table 3. Even though we acknowledge limitation in
isolates and the number of different spoligotype patterns between the discriminatory ability of spoligotyping, we defined a cluster as
these groups. The significance difference among different categories spoligotypes that contained two or more isolates with identical
of specific demographic character as well as drug resistance and spoligotyping pattern in our analyses. Based on this definition,
isolate lineage were analysed by the chi squared test and Fisher’s clusters of between 2 and 41 isolates were observed in this study. In
exact test as appropriate using STATA, and the medians of the ages all, 56 distinct spoligotyping patterns were obtained; 39 and 16
of the various groups were analysed by Mann-Whitney U test.
different patterns were obtained from the MTBss and M. africanum
lineages, respectively. 27 different clusters involving 131 out of 161
Results (81.4%) of the isolates were observed, MTBss were more likely to
Study Population and Bacterial Samples be in spoligotyping clusters, with 111/130 (85.4%) isolates
clustered within 20 different spoligotypes, compared to 21/
One hundred and sixty-two isolates representing 70% of isolates
31(67.7%) of the M. africanum isolates grouped in 7 spoligotyping
(162/232) obtained from sputum samples consecutively collected from
patients suffering from pulmonary TB attending four main health clusters (OR: 2.78, 95%CI = 1.0004–7.35, p = 0.02). A large
facilities in the Central and Western regions of Ghana were analysed. cluster consisting of forty-one isolates (25.3%) shared a spoligo-
Age of patients enrolled ranged from 2 to 90 years, with a median age pattern defined in the latest spoligotype database (SpolDB4) with
of 38.5 years. Out of the 162 cases, the nationality of 160 was indicated. SIT number 61; these strains were identified by LSP involving the
12 were Liberians, two Togolese and 1 each of, Nigerians and Ivories, deletion of RD726 as belonging to the Cameroon sub-lineage.
respectively living in the Bujumbura refugee camp. The remaining 144 Comparing our isolate patterns with the SpolD4 database, 130/
patients were Ghanaians. Of the 161 TB cases who indicated their sex, 161 (80.7%) isolates had previously defined shared spoligotype
109 (67.7%) were male while 52 (32.3%) were female. numbers; while the remaining 31 isolates had unidentified
patterns. 14 of the isolates which gave newly identified
Prevalence of the different sub-species and lineages spoligotypes clustered into six groups of between 2 and 3 isolates.
The remaining 17 isolates gave unique patterns.
within the M. tuberculosis complex by LSP analysis
In addition to the Cameroon family, 8 additional spoligotyping
A total of 162 isolates were confirmed as belonging to MTBC
families were identified among the MTBss isolates that we tested.
(Table 2). All isolates had the insertion sequence IS6110 evident by
These are 15 isolates (11.53%) belonging to the Haarlem family,
22 isolates of the Ghana family (16.92%), 4 isolates (3.08%) each of
Table 1. PCR Procedures used for species and lineage ‘‘Beijing’’, Uganda I and EAI, respectively, LAM (2:1.54%), S
identification of M. tuberculosis complex isolates obtained in (2:1.54%) and X (1:0.77%). 9 isolates had SIT numbers with no
this study. identified sublineages while 16 had no SIT numbers.
Prevalence of drug resistance among the main lineages
Locus
Analyzed and sublineages
The drug susceptibility patterns of 92 of the 130 MTBss isolates
M. tuberculosis IS6110 RD4 RD9 RD12 RD702 RD711 RD726 and all the M. africanum isolates were analyzed by the proportion
complex
method. Table 2 specifies the level of resistance that was obtained
M. tuberculosis OTCF + + + + nd nd + among the main lineages and sublineages analysed in the study.
M. tuberculosis CF + + + + nd nd 2 While we did not find any difference in resistance to INH, RIF
M. africanum WAFri I + + 2 + + 2 nd and EMB, we found that MTBss (OR = 4.30, CI95% 1.33–18.10,
M. africanum WAFri II + + 2 + 2 + nd p,0.008) and the Cameroon sub-lineage (OR = 5.20, CI95%
1.27–30.22p,0.015) were more likely to be STR resistant when
PCR polymerase chain reaction; RD = regions of difference; compared to all M. africanum and the West-African I sublineage
+= locus intact; 2= locus deleted
respectively. Overall, the proportion of MTBss isolates resistant to
OTCF = Other than Cameroon family; CF = Cameroon family
WAfri = West-African type. nd = not determined. any of the tested drugs was higher when compared to all M.
doi:10.1371/journal.pone.0021906.t001 africanum (OR = 2.74, CI95% 1.01–8.24, P,0.03).
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Genetic Diversity in TB Isolates from Ghana
Table 2. The level of resistance obtained from the main lineages and sub-lineages that were tested in the study.
Tested Drug M. tuberculosis (n = 92) N (%) M. africanum (32) N (%) P value*
STR 35 (38%) 4 (12.5.1%) 0.008
INH 14 (15.2%) 2 (6.25%) 0.237
RIF 7 (7.6%) 1 (3.1%) 0.679
EMB 4 (4.3%) 1 (3.1%) 1.000
MDR 4 (4.3%) 0 (0%) 0.572
ANY RESISTANCE 40 (43.5%) 7 (21.9%) 0.030
West-African I (n = 26)
Tested Drug Cameroon Sub-lineage (n = 47) N (%) N (%) P value
STR 19 (40.4%) 3 (11.5%) 0.015
INH 9 (19.1%) 1 (3.8%) 0.086
RIF 5 (10.6%) 1 (3.8%) 0.412
EMB 4 (8.5%) 1(3.8%) 0.649
MDR 4 (8.5%) 0 (0%) 0.290
ANY RESISTANCE 22 (46.8%) 6(23.1%) 0.046
The resistance was measured by the proportion method.
*Where cells had values 5 or less the P value was computed using the Fisher’s exact test.
doi:10.1371/journal.pone.0021906.t002
Epidemiological Associations participants from whom M. africanum was isolated (median = 42,
Table 4 shows some demographic parameters we analysed. The range = 16–68) compared to that of MTBsss (median = 38.5,
median age of 48 female participants who indicated their age range = 2–90). Female and male TB patients were equally likely to
(29.8, range = 2–90)) was lower but not statistically significantly carry MTBss as opposed to M. africanum. 11 out of the 16
different from that of male participants (median = 41, range = 18– foreigners (68.8%) were male and only five were females, while
73). There was no significant difference in median age of 67.4% of the Ghanaians were males.
Figure 1. Polymerase chain reaction procedures used for the differentiation of the MTBC Amplicons obtained after various PCR
analysis performed in the study. A) IS6110; B–E = Large sequence polymorphism analysis of different regions of difference (RD) RD9 (b), 726(c),
711 (d) and 702 (e) showing deleted and intact genomic regions at the respective locus.
doi:10.1371/journal.pone.0021906.g001
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Genetic Diversity in TB Isolates from Ghana
Table 3. Spoligotyping profile for M. tuberculosis complex isolates from Ghana as defined by RDs.
aRD9 RD711 RD702 RD726 Spoligoprofileb Sub lineage No of isolates Spoldb4c
Undeld dele 1111111111111111111111000111111100001111111 Cameroon 41 61
Undel del 1111111111111101111111000111111100001111111 Cameroon 2 115
Undel del 1111111111111111111111000111111100001110111 Cameroon 1 403
Undel del 1111111111111111111111000111001100001111111 Cameroon 6 772
Undel del 1111111111111111111111001111111100001111111 Cameroon 1 1580
Undel del 1111111111110111111111000111111100001111111 2
Undel del 1111111111111111111111000000111100001111111 3
Undel del 1111111111111111111110000111111100001111111 1
Undel del 1111111111110101111111000111111100001111111 1
Undel del 1111111111111101111111000111111100001101101 1
Undel Undel 0000000000000000000000000000000000111111111 Beijing 4 1
Undel Undel 1001111000111111111111111111000010111111111 EAI 4 340
Undel Undel 1111111111111111111111011111111100001111111 Ghana 1 44
Undel Undel 1111111111111111111111111111111100001111111 Ghana 13 53
Undel Undel 1111111111111111111111111111111100001001111 Ghana 1 65
Undel Undel 1111111111111111111110111111111100001111111 Ghana 2 86
Undel Undel 1111111111111101111111111111111100001111111 Ghana 1 118
Undel Undel 1111111111111111111111101111111100001111111 Ghana 1 373
Undel Undel 1111111111111111111111111111011100001111111 Ghana 1 462
Undel Undel 1111111111110111111110111111111100001111111 Ghana 2 504
Undel Undel 1111111111111111111111101000000100001111111 Haarlem 2 45
Undel Undel 1111111111111111111111111111110100001111111 Haarlem 6 50
Undel Undel 1111111111111111111111111000000100001110111 Haarlem 1 62
Undel Undel 1001111111111111111111111111110100001111111 Haarlem 1 655
Undel Undel 1111111111111111111111111100000000000111111 Haarlem 3 1498
Undel Undel 1001111111111111111111111000000100001111111 Haarlem 2 1652
Undel Undel 1111111111111111111100001111111100001111111 LAM 2 42
Undel Undel 1111111100001111111111111111111100001111111 S 2 1223
Undel Undel 1110111111111111111111111111111100001110111 Uganda 1 4 848
Undel Undel 1110000000001111101111111111111100001101111 X3 3 70
Undel Undel 1110000000001111101111111111111100001110000 X3 6 200
Undel Undel 1111111111111111101111111111111100001111111 X 1 119
Undel Undel 1101111000000011111100001111111100001111111 2
Undel Undel 1101111111111111111111111111000010101111111 1
Undel Undel 1001111111101000011111111111110100001111111 1
Undel 1111111000011111111111111111000010111111011 1
Undel 1000000000001111111111000000000000111111111 1
Undel 0000000000000000000000001111111111110000111 1
Undel 1001111000111011111111111111000000111111111 1
Del Del Undel 1011111000001111111100001111111111110001111 West African I 7 319
Del Del Undel 1111111000001111111100001111111111110001111 West African I 4 331
Del Del Undel 1111111000001111111111111111111111110001111 West African I 2 428
Del Del Undel 1111111000001111111100001111111101110001111 West African I 1
Del Del Undel 1111111000001101111000001111111111110001111 West African I 1
Del Del Undel 1111111000000111111100001111111111100001111 West African I 1
Del Del Undel 1111111000001111111100001111111111100001111 West African I 2
Del Del Undel 1101111000000111111100001111111111110001111 West African I 1
Del Del Undel 1111111000001111111100000100000000000000000 West African I 3
Del Del Undel 1111110000001111111100000000000000000001111 West African I 1
Del Del Undel 1011111000001001111100001111111111110001111 West African I 2
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Genetic Diversity in TB Isolates from Ghana
Table 3. Cont.
aRD9 RD711 RD702 RD726 Spoligoprofileb Sub lineage No of isolates Spoldb4c
Del Del Undel 1111111000000111111100001111111111110001111 West African I 1
Del Undel del 1011110001111111111111111111111111111101111 West African II 2 318
Del Undel del 1111110001111111111111111111111111111101111 West African II 1 181
Del Undel del 1111110001111111111111111111111100000001111 West African II 1
Del Undel Del 1111110001111111111110001111111111111100111 West African II 1
aRD: Regions of difference.
b1, presence of the spacer; 0, absence of the spacer.
cSpoldb4 are the coded patterns in the international spoligotype database.
dUndel: Undeleted, eDel: Deleted.
doi:10.1371/journal.pone.0021906.t004
Discussion from TB patients attending various health facilities. Three main
methods which were used in this study namely, IS6110 PCR, RD-
This study sought to use various molecular methods in an PCR analysis and spoligotyping this also makes our study the first
African setting for the characterisation of MTBC isolates obtained to be conducted in which the same sets of isolates from Ghana are
analysed by RD-PCR and spoligotyping. This will provide the
basis for the design and implementation of in-depth molecular
Table 4. Demographics and main lineages of M. tuberculosis epidemiological studies in the country in future.
complex isolated from participants from whom sputum MTBC lineages that affect humans have been subdivided into
samples were analysed. six geographically linked phylogenetic lineages defined by both
SNPs and LSP analysis [11,12]. When Gagneux et al analysed a
collection of 875 MTBC isolates from patients originating from 80
Parameter Frequency n (%) countries using LSP analysis, one of the major observations was
Sex that two of the six lineages are dominantly found in West-Africa;
West-Africa I and West-Africa II. West-Africa I is predominantly
Males 109 (67.7%)
found around the Gulf of Guinea and West-Africa II is prevalent
Females 52 (32.3%)
in western West-Africa [25].
Nationality Our LSP analysis of 162 MTBC isolates from Ghana revealed
Ghanaian 144 (90.0%) that 20% belonged to M. africanum. Eighty-one percent of M.
Liberian 12 (7.5%) africanum isolates belonged to West-Africa I and 19% to West-
Africa II. M. africanum was first identified in 1968 in Senegal and
Other West-African Nationals 4 (2.5%)
was described biochemically as having characteristics between M.
Nationality and Sex
tuberculosis and M. bovis [26]. M africanum has been found in some
Ghana studies to cause up to 40% of human TB in West-Africa [25]. The
Females 47 (32.6%) observed percentage in the current study is higher than in a
Males 97 (67.4%) previous study, which found M. africanm type I to be up to 13%
Foreigners [27]. However, in that earlier study, mycobacterial characteriza-
tion was based solely on biochemical methods. In our analysis we
Females 5 (31.3%)
found isolates with discordant results between the biochemical
Males 11 (68.8%) analysis and the molecular identification we established (data not
M. africanum reported here). For example some of the isolates that tested
Males 20 (64.5%) positive for pyrazinamadase and negative for niacin accumulation
Females 11 (34.5%) were found to be M. tuberculosis rather than M. bovis. These
discordant findings were clarified by the RD-PCR analysis. This
M. tuberculosis
shows that reliance on biochemical methods for species differen-
Males 89 (68.5%) tiation is not only cumbersome but can also lead to mis-
Females 41 (31.5%) classification [28]. We therefore suggest that reference laboratories
M. africanum in endemic countries should establish genetic identification systems
Mean age 39.8615.3 to confirm results of biochemical differentiation methods or
abandon biochemical differentiation altogether. Also in Senegal it
Range 16–68
has been observed, that the proportion of M. africanum causing TB
Median 42
varies by region [29]. The same may be true for Ghana, as the
M. tuberculosis current study was conducted in the Central region of Ghana, while
Mean Age 39.7615.7 in the previous study isolates from the Greater-Accra region were
Range 2–90 analysed [27]. The proportion of M. africanum West-African I
lineage (.80%) of the total M. africanum isolates found in this study
Median 38.5
is high compared to the study reported by Goyal et al [19]. in
doi:10.1371/journal.pone.0021906.t003 which out of the 75 isolates whose pattern was indicated, 26%
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Genetic Diversity in TB Isolates from Ghana
were M. africanum and of this only 52% belonged to the M. Among the 161 isolates that we analysed by spoligotyping, 56
africanum West-African I lineage. The previous study collected distinct spoligotypes were identified, indicating a wide diversity
samples from the Ashanti region which is in the north central part among isolates obtained from a small region in Ghana.
of the country whilst the current study was conducted in the south- We found that MTBss isolates were more likely than the M.
western part of Ghana. This disparity could also confirm that even africanum isolates to be part of a spoligotyping cluster. This
within M. africanum endemic countries; there are regional observation could indicate an overall higher genetic diversity
variations in distribution. However, this need to be evaluated among M. africanum compared to MTBss in Ghana, similar to what
further in a population-based study as the sample sizes in both has been found in earlier publications from West Africa [9,31].
studies is small. The reason why M. africanum is common among This supports the hypothesis that M. africanum established itself in
MTBC isolates in humans in West-Africa but essentially absent in West Africa before the Euro-American M. tuberculosis lineage was
the rest of the world needs to be investigated further [25]. introduced during European exploration and colonization [34].
The outcomes of TB infections in humans are extremely Alternatively, MTBss might be more transmissible than M.
variable, ranging from lifelong latent infection to active disease africanum in Ghana. However, whether these spoligotyping clusters
with variable degrees of extra-pulmonary involvement. In addition represent linked transmission events will need to be confirmed by
to host and other environmental factors, this variability could be genotyping methods such as MIRU-VNTR which exhibit a higher
the result of genetic variation in infecting strains. There is discriminatory power. MIRU-VNTR typing as well as single
increasing evidence from experimental studies that points the nucleotide polymorphism analyses are currently being established
MTBss lineages differ in virulence and immunogenicity [30]. It in our laboratory in Ghana.
has been suggested that M. africanum is less virulent than MTBss,
We conclude that molecular methods are more robust and
since a study in The Gambia demonstrated that although MTBss
specific than the classical biochemical test for MTBC species
and M. africanum infected cases were equally able to transmit
determination and that such techniques can and should be
infections to household contacts, more contacts infected with
established more widely in countries of sub-Saharan Africa. Ghana
MTBss progressed to active disease [31]. In this work we evaluated
is one of the few countries which harbour both lineages of M.
the effect of strain genetic background and the occurrence of drug
africanum (i.e. West-Africa I and West-Africa II). Given the current
resistance by comparing the proportion of phenotypic drug
efforts in TB vaccine development, strain diversity should be
resistance between the different MTBC lineages. We found that
considered when evaluating new vaccine candidates in areas
MTBss was more likely to be resistant to any of the tested drugs
where M. africanum is prevalent.
when compared to M. africanum, this association was primarily
driven by resistance to STR. Drug resistance has been often
associated with the Beijing lineage for reasons that remain unclear Acknowledgments
[32]. Our finding that M. africanum was less likely to be resistant to We thank Ms Emelia Danso, Head and staff of the Bacteriology
STR suggests putative interaction between drug resistant and Department, and Mr David Mensah of Epidemiology department of
strain genetic background. There is mounting evidence that NMIMR for their contributions to the study. We also acknowledge Dr
different lineages of MTBC can be associated with different drug- Bouke de Jong for various discussions before setting spoligotyping in our
resistance conferring mutations [7,32], perhaps indicating an laboratory; and the numerous laboratory staff of Ghana Health service in
interaction between the strain genetic background and particular the Central and Western regions in patients’ recruitment.
drug resistance mutations [33]. A study conducted in Ghana using
DNA sequencing detected significant variations in the proportion Author Contributions
of INH resistance-conferring mutations in different MTBC Conceived and designed the experiments: DYM FB KK GP SG.
lineages. While there was a significantly higher proportion of katG Performed the experiments: DYM AAP TB DS. Analyzed the data:
315 mutations in MTBss, M. africanum West-African I strains were DYM AAP SG KK. Contributed reagents/materials/analysis tools: FB.
more likely to harbour a mutation in the promotor region of inhA Wrote the paper: DYM GP SG.
[6]. Future work in our laboratory will try to confirm these results.
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