Genotypic Diversity and Drug Susceptibility Patterns
among M. tuberculosis Complex Isolates from SouthWestern 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 sublineage 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: ß 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
which are resistant to first-line drugs especially isoniazid (INH) and
rifampicin (RIF). Such cases may either not be cured by the
current first-line treatment regimen or have a more expensive and
long treatment course [5]. The tendency to acquire drug resistance
may be influenced by the genetic and background of the strain
[6–8]. TB is caused mainly by a group of genetically closely related
species and sub-species together referred to as M. tuberculosis
complex (MTBC); however human TB is caused mainly by M.
tuberculosis sensu stricto (MTBss) and M. africanum. Based on
biochemical analysis, M. africanum used to be subdivided into two
separate groups. However, genetic analyses have now indicated
that M. africanum II, predominant in East-Africa is actually a
variant of M. tuberculosis. In this manuscript, M. africanum is defined
as the one originally termed M. africanum 1 based on biochemical
analysis, which is genetically sub- divided into West-African
genotype 1 and II. While M. tuberculosis is globally distributed, M.
Introduction
Despite the World Health Organisation declaring tuberculosis
(TB) a global emergency in 1993, TB remains a major global
health problem. About 9 million new TB cases and 2 million
deaths occur each year. TB is the leading cause of adult mortality
caused by a single infectious agent worldwide [1–3]. Similar to
other countries in sub-Saharan Africa, TB is a major public health
problem in Ghana. In 2004, it was estimated that the prevalence
of all forms of TB was 376/100,000, with an annual incidence of
206 cases per 100, 000 populations. The annual risk of infection
with TB was estimated to be between 1–2%; deaths due to TB
averaged 50/100,000 annually [1].
The backbone of TB control is case detection by smear
microscopy and treatment of identified cases by the DOTS
strategy [4]. A threat to this strategy is the emergence of strains
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Genetic Diversity in TB Isolates from Ghana
africanum is important cause of human TB in West-Africa. M
africanum is responsible for up to 40% of pulmonary TB patients in
some West-African countries [9–11]. DNA-DNA hybridisation
and multi-locus sequencing analysis indicates that the members of
MTBC share high genomic similarities [12]. In spite of this,
various genetic methods have been developed for strain typing
which have been helpful for answering various epidemiological
questions and shed new light on the biology of the pathogen.
Short- and long-term epidemiologic questions such as describing
transmission dynamics, identifying groups most at risk and risk
factors for transmission, estimating recent-versus-reactive disease
and the extent of exogenous re-infection have been addressed
using these methods [13,14]. Genotyping of MTBC has also
helped in tracking the transmission links between individuals, and
demonstrated instances in which epidemiologically linked people
were in fact infected with unrelated strains [13,15,16]. Molecular
methods that have been employed for strain differentiation among
MTBC include Restriction Fragment Length Polymorphism
(RFLP) analysis [9], spoligotyping [17] which detects variability
within the direct repeat locus, Variable Number Tandem Repeats
(VNTR) [18] and large sequence polymorphism (LSP) analysis
[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
phylogenetic analyses [9,13].
To date, only one study has used spoligoytping to study the
population structure of MTBC causing human TB in Ghana [19].
This study reports the use of molecular methods to analyse a set of
isolates cultured from sputum samples obtained from pulmonary TB
patients attending various health facilities in two regions of Ghana.
Mycobacterial Isolation
All specimens after decontamination were cultured on two
Lowenstein-Jensen slopes; one with supplemented with 0.4%
sodium pyruvate to enhance the isolation of M. africanum and M.
bovis. The cultures were incubated at 37uC and were read weekly
for growth for a maximal duration of 16 weeks.
Preliminary identification of suspected mycobacterial isolates
was done by AFB staining and biochemical methods such as
susceptibility to p-nitro benzoic acid (PNB) and to thiophene
carboxylic acid hydrazide (TCH), pyrazinamidase activity (PZA),
nitrate reduction, niacin production [20].
Drug Susceptibility Testing
The susceptibility pattern of all identified mycobacterial isolates
to isoniazid (0.2 mg/ml), rifampicin (40 mg/ml), streptomycin
(4 mg/ml), and ethambutol (2 mg/ml) for all M. tuberculosis complex
primary isolates was determined phenotypically by the indirect
proportion method on L–J slants, as described previously [21].
Drug resistance was expressed as the proportion of colonies that
grow on drug containing medium to drug-free medium and the
critical proportion for resistance was 1% for all drugs.
DNA Extraction
DNA extraction was done according to previously outlined
protocol [22]. About two- 5 ml loop full of bacteria were heat killed
in 500 ml of an extraction mixture (50 mM Tris–HCl, 25 mM
EDTA, and 5% monosodium glutamate). After cooling, 100 ml of
a 50 mg/ml lysozyme solution was added and incubated with
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
incubated at 45uC overnight. The bacterial cell wall was fully
disrupted by adding 200 ml of 0.1 mm-diameter zirconia beads
(BioSpec Products) to each sample and vortexing at full speed for
4 min. Beads and undigested tissue fragments were removed by
centrifugation at 14,000 rpm for 3 minutes, and the supernatants
were transferred to fresh tubes for phenol-chloroform (Fluka)
extraction. The DNA contained in the upper phase was
precipitated with ethanol and re-suspended in 100 ml of water.
Methods
Specimen Collection and Patients’ Characteristics
Specimens included in this study were collected over a period of
17 months (from October 2007 to March 2009) from sputum
AFB-positive pulmonary TB cases attending four main health
facilities; Agona Swedru Government Hospital, Winneba Government hospital, and St Gregory Catholic Clinic at Budumbura
refugee camp,) covering three different districts in the Central
region and Effia-Nkwanta regional hospital in the western region
of Ghana before they were put on anti-TB drug. After informed
consent was obtained, two sputum specimens were collected from
each individual, and a structured questionnaire was used to obtain
standard demographic and epidemiologic data on patients. The
sputum specimens were either mixed with 1% cetylpyridinium
chloride and transported within seven days of collection to the
laboratory at the NMIMR or stored in a fridge and transported
within 72 hours of collection on ice for Petroff decontamination
before cultivation [20]. Ethical clearance for the study was
approved by the institutional review board of the Noguchi
Memorial Institute for Medical Research (Federalwide Assurance
number FWA00001824). The procedure for sampling in this study
was mainly the same as those used in routine management of TB
in Ghana. However, informed consent (written in the case of
literate participants and oral for those who cannot read) was
sought from all participants before their inclusion in the study. In
the case of children below sixteen years, informed consent was
sought from their parents or guardians. The objectives and
benefits of the study were explained to them all. They were assured
of the confidentiality of all information collected from them.
Inconveniences of participation were explained to the participants
and they are free to join the study or exit at any time which will
not in any way affect their treatment.
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Genotyping
Genotyping of MTBC isolates was done in a stepwise mode
(table 1). All isolates included in the study were first identified as
belonging to MTBC by PCR targeting the insertion sequence IS6110
as previously described [23]. Species were defined by analysing for
large sequence polymorphisms (LSP) at the regions of difference (RD)
9, 12 and 4 using published flanking primers [9,10]. Isolates that were
identified as M. africanum were further typed for RD702 and RD711;
and the Cameroon lineage, which we assumed to be the most
dominant among the MTBss was defined by a deletion in RD726 also
using flanking primers [10,11]. All the isolates that we confirmed as
M. tuberculosis complex were further typed by spoligotyping [17].
Briefly the direct repeat region of each genome was amplified using
primers DRa (59-CCG AGA GGG GAC GGA AAC-39) and
biotinylated Drb (59-GGT TTT GGG TCT GAC GAC-39). The
amplified DNA was tested for the presence of specific spacers by
hybridization with a set of 43 oligonucleotides derived from the
spacer sequences of M. tuberculosis H37Rv and M. bovis BCG P3 (the
GenBank accession no. for the sequence of M. tuberculosis H37Rv is
Z48304, and that for M. bovis BCG P3 is X57835). Bound fragments
were revealed by chemiluminescence after incubation with horseradish peroxidase-labeled streptavidin (Boehringer Mannheim). In
order to prevent cross contamination, PCR amplifications and pre2
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Genetic Diversity in TB Isolates from Ghana
PCR procedures were conducted in physically separated rooms.
Negative water controls were PCR amplified and included on each
blot to identify any possible amplicon contamination. In addition,
Positive controls (H37Rv and M. bovis BCG DNA) was amplified and
included on each blot.
the production of the specific 550 bp amplicon corresponding to a
portion of the IS6110 DNA sequence (Figure 1a). The presence of
the main lineages within MTBC were analysed by large sequence
polymorphism analysis at various regions of difference (RD). RD9
analysis by PCR identified 130/162 (80%) of the isolates as MTBss
defined by the detection of an intact PCR product (Figure 1b), and
among this group, RD726 PCR (Figure 1c) defined 59/130 (45%)
as belonging to the Cameroon sub-lineage. 32/162 (20%) were
classified as M. africanum type I by analysis of the RD9 region
(Figure 1b); the majority of them 26 (81%) were identified by
RD711 PCR (Figure 1d) as West-African I, and 6 (19%) as WestAfrican II by RD702 PCR (Figure 1e). Based on RD12 and RD4
analyses, no M. bovis was detected.
Data Analysis
Spoligotypes were analysed as character types. The obtained
spoligotyping patterns were compared with those available in the
international spoligotype database (SpolDB4) [24] containing 35,925
spoligotypes comprising 39,295 isolates from 122 countries. A shared
type was defined as a spoligotyping pattern common to at least two
isolates, and clades were assigned according to signatures described in
the database. Phylogenetic relationships among the isolates were
inferred from Spoligotyping using the MIRU-VNTR plus software.
In addition we compared the diversity within the main lineages that is
MTBss and M. africanum I as well as between the main sublineages M.
africnaum West-African type I (WafrI) and the Cameroon family
(Euro-American). This was done by comparing both the number of
isolates and the number of different spoligotype patterns between
these groups. The significance difference among different categories
of specific demographic character as well as drug resistance and
isolate lineage were analysed by the chi squared test and Fisher’s
exact test as appropriate using STATA, and the medians of the ages
of the various groups were analysed by Mann-Whitney U test.
Spoligotyping Patterns
One hundred and sixty-one isolates comprising the 31 M.
africanum and 130 MTBss isolates were spoligotyped, and the
different lineages and corresponding spoligotype patterns are
indicated in Table 3. Even though we acknowledge limitation in
the discriminatory ability of spoligotyping, we defined a cluster as
spoligotypes that contained two or more isolates with identical
spoligotyping pattern in our analyses. Based on this definition,
clusters of between 2 and 41 isolates were observed in this study. In
all, 56 distinct spoligotyping patterns were obtained; 39 and 16
different patterns were obtained from the MTBss and M. africanum
lineages, respectively. 27 different clusters involving 131 out of 161
(81.4%) of the isolates were observed, MTBss were more likely to
be in spoligotyping clusters, with 111/130 (85.4%) isolates
clustered within 20 different spoligotypes, compared to 21/
31(67.7%) of the M. africanum isolates grouped in 7 spoligotyping
clusters (OR: 2.78, 95%CI = 1.0004–7.35, p = 0.02). A large
cluster consisting of forty-one isolates (25.3%) shared a spoligopattern defined in the latest spoligotype database (SpolDB4) with
SIT number 61; these strains were identified by LSP involving the
deletion of RD726 as belonging to the Cameroon sub-lineage.
Comparing our isolate patterns with the SpolD4 database, 130/
161 (80.7%) isolates had previously defined shared spoligotype
numbers; while the remaining 31 isolates had unidentified
patterns. 14 of the isolates which gave newly identified
spoligotypes clustered into six groups of between 2 and 3 isolates.
The remaining 17 isolates gave unique patterns.
In addition to the Cameroon family, 8 additional spoligotyping
families were identified among the MTBss isolates that we tested.
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
‘‘Beijing’’, Uganda I and EAI, respectively, LAM (2:1.54%), S
(2:1.54%) and X (1:0.77%). 9 isolates had SIT numbers with no
identified sublineages while 16 had no SIT numbers.
Results
Study Population and Bacterial Samples
One hundred and sixty-two isolates representing 70% of isolates
(162/232) obtained from sputum samples consecutively collected from
patients suffering from pulmonary TB attending four main health
facilities in the Central and Western regions of Ghana were analysed.
Age of patients enrolled ranged from 2 to 90 years, with a median age
of 38.5 years. Out of the 162 cases, the nationality of 160 was indicated.
12 were Liberians, two Togolese and 1 each of, Nigerians and Ivories,
respectively living in the Bujumbura refugee camp. The remaining 144
patients were Ghanaians. Of the 161 TB cases who indicated their sex,
109 (67.7%) were male while 52 (32.3%) were female.
Prevalence of the different sub-species and lineages
within the M. tuberculosis complex by LSP analysis
A total of 162 isolates were confirmed as belonging to MTBC
(Table 2). All isolates had the insertion sequence IS6110 evident by
Table 1. PCR Procedures used for species and lineage
identification of M. tuberculosis complex isolates obtained in
this study.
Prevalence of drug resistance among the main lineages
and sublineages
Locus
Analyzed
RD4 RD9
RD12 RD702 RD711 RD726
M. tuberculosis OTCF +
+
+
+
nd
nd
+
M. tuberculosis CF
+
+
+
+
nd
nd
2
M. africanum WAFri I +
+
2
+
+
2
nd
M. africanum WAFri II +
+
2
+
2
+
nd
M. tuberculosis
complex
IS6110
The drug susceptibility patterns of 92 of the 130 MTBss isolates
and all the M. africanum isolates were analyzed by the proportion
method. Table 2 specifies the level of resistance that was obtained
among the main lineages and sublineages analysed in the study.
While we did not find any difference in resistance to INH, RIF
and EMB, we found that MTBss (OR = 4.30, CI95% 1.33–18.10,
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
compared to all M. africanum and the West-African I sublineage
respectively. Overall, the proportion of MTBss isolates resistant to
any of the tested drugs was higher when compared to all M.
africanum (OR = 2.74, CI95% 1.01–8.24, P,0.03).
PCR polymerase chain reaction; RD = regions of difference;
+ = locus intact; 2 = locus deleted
OTCF = Other than Cameroon family; CF = Cameroon family
WAfri = West-African type. nd = not determined.
doi:10.1371/journal.pone.0021906.t001
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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
Tested Drug
Cameroon Sub-lineage (n = 47) N (%)
West-African I (n = 26)
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
participants from whom M. africanum was isolated (median = 42,
range = 16–68) compared to that of MTBsss (median = 38.5,
range = 2–90). Female and male TB patients were equally likely to
carry MTBss as opposed to M. africanum. 11 out of the 16
foreigners (68.8%) were male and only five were females, while
67.4% of the Ghanaians were males.
Epidemiological Associations
Table 4 shows some demographic parameters we analysed. The
median age of 48 female participants who indicated their age
(29.8, range = 2–90)) was lower but not statistically significantly
different from that of male participants (median = 41, range = 18–
73). There was no significant difference in median age of
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.
a
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
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
RD9
RD711
RD702
1
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.
a
Spoligoprofileb
Sub lineage
No of isolates
Undel
1111111000000111111100001111111111110001111
West African I
1
del
1011110001111111111111111111111111111101111
West African II
2
318
Undel
del
1111110001111111111111111111111111111101111
West African II
1
181
Del
Undel
del
1111110001111111111111111111111100000001111
West African II
1
Del
Undel
Del
1111110001111111111110001111111111111100111
West African II
1
RD9
RD711
RD702
Del
Del
Del
Undel
Del
RD726
Spoldb4c
a
RD: Regions of difference.
1, presence of the spacer; 0, absence of the spacer.
Spoldb4 are the coded patterns in the international spoligotype database.
d
Undel: Undeleted, eDel: Deleted.
doi:10.1371/journal.pone.0021906.t004
b
c
from TB patients attending various health facilities. Three main
methods which were used in this study namely, IS6110 PCR, RDPCR analysis and spoligotyping this also makes our study the first
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
epidemiological studies in the country in future.
MTBC lineages that affect humans have been subdivided into
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
countries using LSP analysis, one of the major observations was
that two of the six lineages are dominantly found in West-Africa;
West-Africa I and West-Africa II. West-Africa I is predominantly
found around the Gulf of Guinea and West-Africa II is prevalent
in western West-Africa [25].
Our LSP analysis of 162 MTBC isolates from Ghana revealed
that 20% belonged to M. africanum. Eighty-one percent of M.
africanum isolates belonged to West-Africa I and 19% to WestAfrica II. M. africanum was first identified in 1968 in Senegal and
was described biochemically as having characteristics between M.
tuberculosis and M. bovis [26]. M africanum has been found in some
studies to cause up to 40% of human TB in West-Africa [25]. The
observed percentage in the current study is higher than in a
previous study, which found M. africanm type I to be up to 13%
[27]. However, in that earlier study, mycobacterial characterization was based solely on biochemical methods. In our analysis we
found isolates with discordant results between the biochemical
analysis and the molecular identification we established (data not
reported here). For example some of the isolates that tested
positive for pyrazinamadase and negative for niacin accumulation
were found to be M. tuberculosis rather than M. bovis. These
discordant findings were clarified by the RD-PCR analysis. This
shows that reliance on biochemical methods for species differentiation is not only cumbersome but can also lead to misclassification [28]. We therefore suggest that reference laboratories
in endemic countries should establish genetic identification systems
to confirm results of biochemical differentiation methods or
abandon biochemical differentiation altogether. Also in Senegal it
has been observed, that the proportion of M. africanum causing TB
varies by region [29]. The same may be true for Ghana, as the
current study was conducted in the Central region of Ghana, while
in the previous study isolates from the Greater-Accra region were
analysed [27]. The proportion of M. africanum West-African I
lineage (.80%) of the total M. africanum isolates found in this study
is high compared to the study reported by Goyal et al [19]. in
which out of the 75 isolates whose pattern was indicated, 26%
Discussion
This study sought to use various molecular methods in an
African setting for the characterisation of MTBC isolates obtained
Table 4. Demographics and main lineages of M. tuberculosis
complex isolated from participants from whom sputum
samples were analysed.
Parameter
Frequency n (%)
Sex
Males
109 (67.7%)
Females
52 (32.3%)
Nationality
Ghanaian
144 (90.0%)
Liberian
12 (7.5%)
Other West-African Nationals
4 (2.5%)
Nationality and Sex
Ghana
Females
47 (32.6%)
Males
97 (67.4%)
Foreigners
Females
5 (31.3%)
Males
11 (68.8%)
M. africanum
Males
20 (64.5%)
Females
11 (34.5%)
M. tuberculosis
Males
89 (68.5%)
Females
41 (31.5%)
M. africanum
Mean age
39.8615.3
Range
16–68
Median
42
M. tuberculosis
Mean Age
39.7615.7
Range
2–90
Median
38.5
doi:10.1371/journal.pone.0021906.t003
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Genetic Diversity in TB Isolates from Ghana
were M. africanum and of this only 52% belonged to the M.
africanum West-African I lineage. The previous study collected
samples from the Ashanti region which is in the north central part
of the country whilst the current study was conducted in the southwestern part of Ghana. This disparity could also confirm that even
within M. africanum endemic countries; there are regional
variations in distribution. However, this need to be evaluated
further in a population-based study as the sample sizes in both
studies is small. The reason why M. africanum is common among
MTBC isolates in humans in West-Africa but essentially absent in
the rest of the world needs to be investigated further [25].
The outcomes of TB infections in humans are extremely
variable, ranging from lifelong latent infection to active disease
with variable degrees of extra-pulmonary involvement. In addition
to host and other environmental factors, this variability could be
the result of genetic variation in infecting strains. There is
increasing evidence from experimental studies that points the
MTBss lineages differ in virulence and immunogenicity [30]. It
has been suggested that M. africanum is less virulent than MTBss,
since a study in The Gambia demonstrated that although MTBss
and M. africanum infected cases were equally able to transmit
infections to household contacts, more contacts infected with
MTBss progressed to active disease [31]. In this work we evaluated
the effect of strain genetic background and the occurrence of drug
resistance by comparing the proportion of phenotypic drug
resistance between the different MTBC lineages. We found that
MTBss was more likely to be resistant to any of the tested drugs
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
[32]. Our finding that M. africanum was less likely to be resistant to
STR suggests putative interaction between drug resistant and
strain genetic background. There is mounting evidence that
different lineages of MTBC can be associated with different drugresistance conferring mutations [7,32], perhaps indicating an
interaction between the strain genetic background and particular
drug resistance mutations [33]. A study conducted in Ghana using
DNA sequencing detected significant variations in the proportion
of INH resistance-conferring mutations in different MTBC
lineages. While there was a significantly higher proportion of katG
315 mutations in MTBss, M. africanum West-African I strains were
more likely to harbour a mutation in the promotor region of inhA
[6]. Future work in our laboratory will try to confirm these results.
Among the 161 isolates that we analysed by spoligotyping, 56
distinct spoligotypes were identified, indicating a wide diversity
among isolates obtained from a small region in Ghana.
We found that MTBss isolates were more likely than the M.
africanum isolates to be part of a spoligotyping cluster. This
observation could indicate an overall higher genetic diversity
among M. africanum compared to MTBss in Ghana, similar to what
has been found in earlier publications from West Africa [9,31].
This supports the hypothesis that M. africanum established itself in
West Africa before the Euro-American M. tuberculosis lineage was
introduced during European exploration and colonization [34].
Alternatively, MTBss might be more transmissible than M.
africanum in Ghana. However, whether these spoligotyping clusters
represent linked transmission events will need to be confirmed by
genotyping methods such as MIRU-VNTR which exhibit a higher
discriminatory power. MIRU-VNTR typing as well as single
nucleotide polymorphism analyses are currently being established
in our laboratory in Ghana.
We conclude that molecular methods are more robust and
specific than the classical biochemical test for MTBC species
determination and that such techniques can and should be
established more widely in countries of sub-Saharan Africa. Ghana
is one of the few countries which harbour both lineages of M.
africanum (i.e. West-Africa I and West-Africa II). Given the current
efforts in TB vaccine development, strain diversity should be
considered when evaluating new vaccine candidates in areas
where M. africanum is prevalent.
Acknowledgments
We thank Ms Emelia Danso, Head and staff of the Bacteriology
Department, and Mr David Mensah of Epidemiology department of
NMIMR for their contributions to the study. We also acknowledge Dr
Bouke de Jong for various discussions before setting spoligotyping in our
laboratory; and the numerous laboratory staff of Ghana Health service in
the Central and Western regions in patients’ recruitment.
Author Contributions
Conceived and designed the experiments: DYM FB KK GP SG.
Performed the experiments: DYM AAP TB DS. Analyzed the data:
DYM AAP SG KK. Contributed reagents/materials/analysis tools: FB.
Wrote the paper: DYM GP SG.
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