Evaluation of Customised Lineage-Specific Sets of MIRUVNTR Loci for Genotyping Mycobacterium tuberculosis
Complex Isolates in Ghana
Adwoa Asante-Poku1,2,3, Michael Selasi Nyaho1,4, Sonia Borrell2,3, Iñaki Comas5,6, Sebastien Gagneux2,3,
Dorothy Yeboah-Manu1*
1 Bacteriology Department, Noguchi Memorial institute For Medical Research, University of Ghana, Legon, Ghana, 2 Department of Medical Parasitology and Infection
Biology, Swiss Tropical and Public Health Institute, Basel, Switzerland, 3 University of Basel, Basel, Switzerland, 4 Biochemistry Department, University of Ghana, Legon,
Ghana, 5 Genomics and Health Unit, Centre for Public Health Research, Valencia, Spain, 6 CIBER (Centros de Investigación Biomédica en Red) in Epidemiology and Public
Health, Madrid, Spain
Abstract
Background: Different combinations of variable number of tandem repeat (VNTR) loci have been proposed for genotyping
Mycobacterium tuberculosis complex (MTBC). Existing VNTR schemes show different discriminatory capacity among the six
human MTBC lineages. Here, we evaluated the discriminatory power of a ‘‘customized MIRU12’’ loci format proposed
previously by Comas et al. based on the standard 24 loci defined by Supply et al. for VNTR-typing of MTBC in Ghana.
Method: One hundred and fifty-eight MTBC isolates classified into Lineage 4 and Lineage 5 were used to compare a
customized lineage-specific panel of 12 MIRU-VNTR loci (‘‘customized MIRU-120) to the standard MIRU-15 genotyping
scheme. The resolution power of each typing method was determined based on the Hunter-Gaston- Discriminatory Index
(HGDI). A minimal set of customized MIRU-VNTR loci for typing Lineages 4 (Euro-American) and 5 (M. africanum West African
1) strains from Ghana was defined based on the cumulative HGDI.
Results and Conclusion: Among the 106 Lineage 4 strains, the customized MIRU-12 identified a total of 104 distinct
genotypes consisting of 2 clusters of 2 isolates each (clustering rate 1.8%), and 102 unique strains while standard MIRU-15
yielded a total of 105 different genotypes, including 1 cluster of 2 isolates (clustering rate: 0.9%) and 104 singletons. Among,
52 Lineage 5 isolates, customized MIRU-12 genotyping defined 51 patterns with 1 cluster of 2 isolates (clustering rate: 0.9%)
and 50 unique strains whereas MIRU-15 classified all 52 strains as unique. Cumulative HGDI values for customized MIRU-12
for Lineages 4 and 5 were 0.98 respectively whilst that of standard MIRU-15 was 0.99. A union of loci from the customised
MIRU-12 and standard MIRU-15 revealed a set of customized eight highly discriminatory loci: 4052, 2163B, 40, 4165, 2165,
10,16 and 26 with a cumulative HGDI of 0.99 for genotyping Lineage 4 and 5 strains from Ghana.
Citation: Asante-Poku A, Nyaho MS, Borrell S, Comas I, Gagneux S, et al. (2014) Evaluation of Customised Lineage-Specific Sets of MIRU-VNTR Loci for Genotyping
Mycobacterium tuberculosis Complex Isolates in Ghana. PLoS ONE 9(3): e92675. doi:10.1371/journal.pone.0092675
Editor: Igor Mokrousov, St. Petersburg Pasteur Institute, Russian Federation
Received November 8, 2013; Accepted February 25, 2014; Published March 25, 2014
Copyright: ß 2014 Asante-Poku 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 study was supported financially by the UNICEF/UNDP/World Bank/WHO special program for research and training in Tropical Diseases for DY-M
and the Leverhulme-Royal Society Africa (DY-M and SG) and NTP Ghana. 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.ug.edu.gh
TB also depends on understanding the patterns and dynamics of
transmission which is useful for the implementation of public
health measures to reduce sources of infection [4,5].
A number of molecular markers are available for differentiating
members of the Mycobacterium tuberculosis complex (MTBC) for
conventional epidemiological investigations of TB outbreaks and
to assess risk factors associated with recent transmissions [6,7].
Mycobacterial interspersed repetitive unit-variable number of
tandem repeats (MIRU-VNTR) typing, have overcome most of
the shortcomings of IS6110 RFLP [8–10], and have now replaced
this older technique as the new gold standard for molecular
epidemiological investigation of TB. MIRU-VNTR typing which
uses genomic diversity at different VNTR loci can have a
Introduction
Tuberculosis (TB) is a major public health problem worldwide,
causing 8.8 million new cases and more than 1.4 million deaths
each year [1]. The main strategy for controlling TB, especially in
low resourced countries, is case detection and treatment using the
directly observed treatment short course (DOTS) strategy [2]. The
conventional indicators used for assessing TB control programmes
focuses on the proportion of patients with new sputum smear
positive pulmonary disease that are cured by the end of treatment
or whose sputum microscopy becomes negative after the first 2
months of treatment [3]. Such indicators ignore equally important
aspects of TB control such as the duration of infectivity, the
frequency of reactivation, and the risk of progression among the
infected contacts, or the risk of transmission. Thus the control of
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Evaluation of Customised MIRU-VNTR Loci in Ghana
Table 1. List of MIRU-VNTRs used for the assay.
LOCUS
ALIAS
15
L4
L5
424
Mtub04
X
X
X
577
ETRC
X
X
X
580
MIRU04
X
802
MIRU40
X
X
X
960
MIRU10
X
X
X
1644
MIRU16
X
1955
Mtub21
X
X
X
2163b
QUB11b
X
X
X
2165
ETRA
X
X
X
2401
Mtub30
X
X
2531
MIRU23
2996
MIRU26
3007
MIRU27
3192
MIRU31
X
3690
Mtub39
X
X
4052
QUB26
X
X
X
4156
QUB4156
X
X
X
X
X
X
X
X
doi:10.1371/journal.pone.0092675.t001
discrimination index within one lineage were not necessarily the
ones with the highest discriminatory power in other lineages.
Based on the allelic diversity of individual MIRU-VNTR locus,
Comas et al. [30] suggested different combinations of MIRUVNTR loci that offered high resolution for the different MTBC
lineages. These combinations offered two main advantages over
the existing one; it maximized allelic diversity for a given MTBC
lineage and allowed for cost effective analyses [30].
Here we evaluated this concept in the Ghanaian setting and
compared the standard MIRU-15 to two lineage-specific 12-loci
sets (here referred to as ‘‘customized MIRU-12’’), one for Lineage
4 and one for Lineage 5, which are the most frequent MTBC
lineages in Ghana [27–28,49].
cumulative resolution comparable to that of IS6110 RFLP analysis
depending on the combination of loci analysed [11–17].
Several combinations of MIRU- VNTR loci have been
published with initial methods relying on only a few loci, which
turned out to have low discriminatory power among MTBC
isolates [18,37–39]. Subsequently, a standard MIRU-12 loci set
with discriminatory power close to IS6110-RFLP was proposed for
molecular epidemiological studies in TB [19–21]. More recently,
this initial MIRU-12 set was replaced by the standard MIRU-15
set, and currently, standard MIRU- 24 loci set [34] has been
proposed for optimal discrimination of closely related strains. The
standard MIRU15 set which includes six of the previous MIRU12 with nine additional loci has been recommended as the
standard for routine molecular epidemiology of TB, including
outbreak investigations and population-based transmission studies.
MIRU-24 set comprises the same 15 loci plus an additional nine
provide additional information aimed at phylogenetic and
population genetic aspects of MTBC.
The usage of the standard MIRU-15 and MIRU-24 has helped
to gain insight into the transmission dynamics of MTBC.
However, the initial selection of these loci was to some extent
biased towards strains belonging to Lineage 4 (Euro-American
lineage) [34]. The inability of the proposed loci led to new
customized sets for Lineage 2 strains that include the clinically
relevant Beijing family of strains [18]. However, the humanassociated MTBC includes 6 additional lineages [22,23,45–47],
which show a strong phylogeographic structure [24–26]. As
observed for Lineage 2 strains, this might suggest that the usage of
high discriminatory MIRU-VNTR loci may be sub-optimal in
areas such as Ghana where about 20% of all TB cases are caused
by Lineages 5 and 6 of MTBC (also known as M. africanum West
Africa 1 and 2) [27–28].
Comas et al. [30] using 108 global MTBC strains [30] showed
that the majority of the loci included in standard MIRU-24 had a
variable discriminatory power across the different MTBC lineages.
Moreover, the MIRU-VNTR loci that exhibited the highest
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Materials and Methods
Ethics Statement
Ethical clearance for this study was obtained from the IRB of
the Noguchi Memorial Institute for Medical Research, which has
a Federal wide Assurance number FWA00001824. The procedure
for sampling in this study was basically the same as those outlined
by the National Tuberculosis Programme for the routine
management of TB in Ghana. Informed consent both written (in
the case of literate participants) and oral (for illiterates) was sought
from all participants before their inclusion in the study. Consent
was sought from their parents or guardians on behalf of children
below sixteen years. As per the guidelines of the institutional
review board of the Noguchi Memorial Institute for Medical
Research, the objectives and benefits of the study were explained
to all participants and they were assured of the confidentiality of all
information collected from during the study.
Isolate Selection and Lineage Classification
A total of 178 MTBC isolates consecutively selected from a pool
of retrospective samples were included in the study. Specimens
included in this study were collected consecutively over a period of
2
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Table 2. Cumulative HGI and clustering rate for Lineage 4 successive addition of individual MIRU-VNTR Loci.
3
Locus
VNTR
Loci
Alias
No. of Clusters
No. of clustered isolates
No. of isolates in individual cluster
Clustering Rate (%)
Individual HGI
Cumulative HGI
1
4052
QUB26
9
96
2–34
82.1
0.829
0.829
2
2163B
QUB11B
23
88
2–9
61.3
0.804
0.966
3
802
MIRU40
20
53
2–8
31.1
0.742
0.988
4
2165
ETRA
13
28
2–4
14.2
0.702
0.996
5
960
MIRU10
8
19
2–4
10.4
0.689
0.997
6
4156
QUB4165
4
11
2–4
6.6
0.639
0.999
7
3690
Mtu39
3
6
2
2.8
0.66
0.999
8
2401
Mtu30
3
6
2
2.8
0.64
0.999
9
2996
MIRU26
2
4
2
1.8
0.628
0.999
10
424
Mtu04
2
4
2
1.8
0.613
0.999
11
577
ETRC
2
4
2
1.8
0.553
0.999
12
1995
Mtub21
2
4
2
1.8
0.579
0.999
13
2531
VNTR23
2
4
2
1.8
0.555
0.999
14
1644
MIRU16
2
4
2
1.8
0.452
0.999
15
3192
MIRU31
1
2
2
0.9
0.37
0.999
16
580
MIRU4
1
2
2
0.9
0.092
0.999
(N = 106).
doi:10.1371/journal.pone.0092675.t002
Evaluation of Customised MIRU-VNTR Loci in Ghana
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Table 3. Cumulative HGI and clustering rate for Lineage 5 with successive addition of individual MIRU-VNTR Loci.
4
Locus
VNTR Loci
Alias
No. of Clusters
No. of clustered isolates
No. of isolates in individual cluster
Clustering Rate (%)
Individual HGI
Cumulative HGI
1
2163B
QUB11B
6
52
2–13
88.5
0.797
0.798
2
4156
QUB4156
12
45
2–9
63.5
0.731
0.935
3
802
MIRU 40
2
4
2
3.8
0.7
0.971
4
2996
MIRU 26
7
16
2–3
15.9
0.7
0.992
5
4052
QUB26
10
31
2–5
40.4
0.69
0.997
6
1644
MIRU 16
3
6
2
5.7
0.689
0.998
7
3007
MIRU 27
2
4
2
3.8
0.6
0.998
8
2531
MIRU 23
2
4
2
3.8
0.58
0.998
9
424
Mtub04
2
4
2
3.8
0.572
0.998
10
960
MIRU 10
2
4
2
3.8
0.526
0.998
11
1955
Mtub21
2
4
2
3.8
0.489
0.998
12
577
ETRC
2
4
2
3.8
0.487
0.998
13
2401
Mtub30
1
2
2
1.9
0.473
0.998
14
580
MIRU04
1
2
2
1.9
0.472
0.999
15
3690
Mtub39
0
0
0
0
0.428
0.999
16
2165
ETRA
0
0
0
0
0.387
0.999
17
3192
MIRU31
0
0
0
0
0.352
0.999
N = 52.
doi:10.1371/journal.pone.0092675.t003
Evaluation of Customised MIRU-VNTR Loci in Ghana
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Evaluation of Customised MIRU-VNTR Loci in Ghana
Figure 1. Individual and Cumulative HGI of analysed Loci. Individual and cumulative HGI was calculated for each locus and after successive
loci addition respectively for all analysed loci using the mathematical formula as proposed by Hunter and Gaston (36). The black bars are the
cumulative HGI values while crossed bars are for the individual locus values. Fig. 1a and 1b show values for lineage 4 and 5 respectively.
doi:10.1371/journal.pone.0092675.g001
standard MIRU-15 as described by Supply et al. [34] (Table 1).
Each PCR mixture contained 10X PCR buffer, 1.5 mM MgCl2,
200 mM concentrations of deoxyribonuclueotide triphosphate,
5 mM concentration of each primer, 1 ml of HotstarTaq DNA
polymerase enzyme, 5 ml Q solution and 10 ng of DNA template
in a total volume of 25 ml. Negative (sterile water) and positive
controls (H37Rv) were added to each PCR reaction to validate the
assay. Locus amplification was carried out under the following
conditions: initial denaturation at 95uC for 15 minutes, and then
40 cycles of 95uC for 1 minute, 59uC for1 minute and 72uC for 3
minutes, followed by a final extension at 72uC for 7 minutes. Gel
electrophoresis was done in 2% agarose for 5 hours at 80 constant
Voltage. The amplicons were sized using a 100 bp marker and the
obtained size compared with allelic table as published by Supply
et al. [34].
17 months (from October 2007 to March 2009) from sputum
AFB-positive pulmonary TB cases attending four main government health centres covering three different regions: Central,
Greater Accra and Western regions of Ghana respectively before
commencement of anti-TB drug. DNA was extracted as described
previously [33]. MTBC was confirmed by IS6110 PCR [40]. The
isolates were then classified into lineages by analyses of various
regions of difference (RDs) as previously described [31]. Specifically, all isolates were first screened for RD9. RD9-deleted strains
were screened for RD4. Isolates identified as RD9 deleted and
RD4 undeleted were further sub-typed for Lineage 5 and 6 (M.
africanum West Africa I and II) using RD711 and RD702 flanking
primers, respectively. TaqMan real time PCR was performed
according to standard procedures using probes designed by Stucki
et al for the confirmation of Lineages [35]. Although Lineage 6
strains (M. africanum West Africa II) are present in Ghana [27,28],
they were removed from further analysis due to limited number (6
isolates) identified.
SNP Typing
TaqMan real time PCR was performed as published by Stucki
et al. [35]. Briefly, in a 200 ml sterile PCR tube, 2 ml of DNA was
added to a 5 ml sterile water containing 0.21 mM each reverse and
forward primers for the targeted regions, 0.83 mM each probe A
for ancestral allele and probe B for mutant allele (each labelled
with different dyes); and 5 ml Taqman Universal MasterMix II
MIRU-VNTR Analysis
Two sets of PCRs were performed for each isolate. The first set
was performed using the 12 lineage-specific MIRU-VNTR loci
proposed by Comas et al. [30], while the second set consisted of the
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Figure 2. Clustering rate for lineages 4 and 5 calculated using after successive addition of analysed loci using the formula (nc - c)/n,
where nc is the total number of clustered cases, c is the number of clusters, and n is the total number of cases in the sample a was
calculated after successive addition of individual locus. Fig. 2a and b shows clustering rate values for lineages 4 and 5 respectively.
doi:10.1371/journal.pone.0092675.g002
(Applied Biosystem). The reaction was performed in Applied
Biosystems 7300 real time PCR system under the following
conditions: 60uC for 30 seconds, 95uC 10 minutess, 95uC 15
seconds and 60uC 1 minute for 40 cycles; 60uC for 30 seconds.
The fluorescence intensity in the dyes (VIC and FAM) channels
were measured at the end of each cycle.
under investigation. Twelve loci were shared between the
customised Lineage 4 set and standard MIRU-15, addition of
the remaining 4 non-shared loci from standard MIRU-15 gave a
total of 16 loci for analysis. For Lineage 5, addition of 6 nonshared loci to the 9 shared loci gave a total of 17 loci.
Subsequently, we calculated individual locus HGDI. The results
obtained were arranged in a descending order. Starting with the
highest HGDI, cumulative HGDI was then calculated by
successively adding one locus after the other. Finally, the clustering
rate was calculated in a similar manner by successively adding one
locus after the other. The result (cumulative HGDI and
percentage clustering) obtained for each lineage was plotted on a
graph and the cut-off point for selection of the minimal set of loci
was set at where graph plateaued meaning further addition of loci
resulted in the same cumulative HGDI. The customized minimal
loci-set was then extracted from the graph.
Data Analysis
The number of repeats for each locus was determined based on
the allelic table by Supply et al. [34] and clustering analysis was
done using the online tool at http://www.MIRU-VNTRplus.org.
MIRU-VNTR clusters were defined as isolates sharing identical
patterns. The clustering rate was defined as (nc - c)/n, where nc is
the total number of clustered cases, c is the number of clusters, and
n is the total number of cases in the sample [29].
The Hunter-Gaston Discriminatory Index (HGDI) was used to
calculate the discriminatory power of each locus as well as that of
each method [36].
Results
Determination of a Minimal Set of MIRU-VNTR Loci
MTBC Isolates and Lineage Determination
All 178 isolates included in this study were classified into
Lineage 4 (N = 126) or Lineage 5 (N = 52) based on the RD and
SNP typing analysis [31,35]. Discordant samples were excluded
Stepwise analysis was performed to identify a set of loci needed
to achieve maximum discrimination. Firstly, we combined loci
from the customised sets and standard MIRU-15 for each lineage
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Evaluation of Customised MIRU-VNTR Loci in Ghana
from the study. A full set of MIRU allelic data was obtained for
158/178 (88.8%), comprising 106 Lineage 4 and 52 Lineage 5
isolates, respectively. The remaining 20 of the 178 (11.2%) isolates
were excluded from the analysis for various reasons. 90% (18/20)
of excluded isolates had no PCR amplicon at one or several loci
whilst the remaining 10% (2/20) had double alleles at one or more
MIRU-VNTR loci, indicative of the possible presence of two
independent strains [32].
Discussion and Conclusion
Different combinations of MIRU and other VNTR loci have
been proposed to complement the standard MIRU-15 scheme to
achieve higher discrimination. Results accumulated from such
studies clearly revealed that due to the strong phylogeographic
structure exhibited by MTBC, the most relevant MIRU-VNTR
typing schemes will likely differ depending on the specific
geographical setting. For example, Shamputa et al. [18] successfully identified a reduced set of 8 loci from standard MIRU-24,
which could be used to discriminate, isolates from the Republic of
Korea. Similarly, Musare et al. [37], Dong et al. [38] and Zhou
et al. [39] successfully defined a minimal set of 12 loci for
genotyping Beijing strains which made up more than 90% of the
isolates investigated from Asia. Most of the studies have been
focused on Lineage 2 including the clinically important Beijing
family based on its association with drug resistance [48]. However,
no study has been carried out in most resource-limited settings like
Ghana, where M. africanum is an important pathogen [27–28,49].
If customized lineage-specific sets of MIRU-VNTR loci could be
implemented in such settings, this will have an impact in terms of
reducing work load and saving resources. In the present study, we
evaluated such an approach for genotyping MTBC strains from
Ghana, [27,28] and compared our results with the current gold
standard typing method; standard MIRU 15 as proposed by
Supply et al. [34].
Although standard MIRU-15 showed higher discrimination in
its ability to accurately identify clusters among these two lineages
in our study when compared to customised lineage-specific
MIRU-12 proposed previously [30], we found that not all the
15 loci were as informative for typing MTBC strains in Ghana.
Even with the customized MIRU-12, based on our data, not all 12
loci were needed to achieve maximum discrimination (Figure 1).
Specifically, our analysis showed that 10 of a total of 16 loci tested
for Lineage 4 strains added no or only limited additional
information in terms of discriminatory power. Similarly, 11 of a
total of 17 loci screened for Lineage 5 strains showed limited
discriminatory power. We thus explored the possibility of a
minimal set of loci HGDI selected by combining the standard
MIRU-15 and the customized MIRU-12 data set. Based on
individual and cumulative HGDIs, and clustering rate, we defined
the six top discriminatory loci for Lineage 4 (4052, 2163B, 40,
2165, 10 and 4165) (Figure 1a and 2a) and similarly for Lineage 5
(2163B, 4165, 40, 26, 4052 and 16) (Figure 1b and 2b) with 4 loci
shared among the two sets (4052, 2163B, 40 and 4165). A
combination of loci from Lineage 4 and 5 gave a unique
customized set of 8 loci with HGDIs similar to that of standard
MIRU-15.
We now plan to apply these minimal MIRU-VNTR set for
molecular epidemiological investigation of MTBC transmission in
population based study in Ghana. We anticipate that this
approach will save a significant amount of time. In addition we
perform cost analysis on the different VNTR schemes analysed in
this study. Cost was calculated based on the direct cost of reagents,
materials and equipment. We compared the cost of genotyping
using standard MIRU-15 and our proposed customized set of
MIRU-8. With a unit cost of $11.24, the cost of performing
standard MIRU-15 on one sample was $168.60, with the total
material costs of analyses using our proposed customized MIRU-8
set for one sample being $89.2. Hence, by screening for only the
relevant loci, we not only maximize discriminatory power but also
minimize genotyping costs.
Currently, human-associated MTBC is known to comprise a
total of seven main phylogenetic lineages [23,46–47]. We propose
Evaluation of Customized MIRU-12 for Lineage 4
One hundred and four distinct patterns (Table S1) comprising
102 singleton and 2 clusters (2 isolate per cluster) was obtained
using customized MIRU-12 (clustering rate: 1.8%). Discriminatory power was calculated separately for each locus and classified
into highly (HGDI $0.6), moderately (0.3 to 0.6), and poorly (,
0.3) discriminatory based on the HGDI scores as previously
reported [21]. Based on this definition, the discriminatory power
of 10 loci (MIRU-VNTR loci 10, 40, 2163b, 2165, 3690, 4052,
4165 2401, 0424, and 0577) was higher than 0.6, supporting their
designation as highly discriminatory loci with the remaining 2
MIRU/VNTR loci (VNTR 1955, and 23) being ‘‘moderately
discriminatory’’(DI: 0.3–0.59) (Table 2). Using the same set of
isolates, standard MIRU-15 identified 105 distinct patterns with
only one cluster of two isolates (clustering rate: 0.9%). Ten loci
(66.6%; MIRU-VNTR loci 4052, 2163b, 40, 2165, 10, 4165,
3690, 2401, 26 and 0424) were highly discriminatory, 4 (26.6%)
(VNTR 1955, 0577 and 23) moderately discriminatory and the
remaining 1 (MIRU 4: 6.7%) poorly discriminated among the
isolates.
Evaluation of Customized MIRU-12 for Lineage 5
Among 52 isolates analyzed, we obtained 51 unique patterns
with 1 cluster (Table S1) comprising 2 isolates and a clustering rate
of 0.9%. Five MIRU-VNTR loci (2163B, 4156, 4052, 40, 27) were
highly discriminatory and the remaining 7 (23, 0577, 2165, 10,
0424, 31, 1955) being moderately discriminatory (Table 3).
Standard MIRU-15 on the other hand identified 52 unique
patterns. Six of the 15 loci (VNTR 2163B, 4156, 4052, 26, 16 and
40) had HGDI above 0.6 with the remaining nine MIRU-VNTR
loci (0424, 10, 1955, 0577, 4, 2401, 3690, 2165 and 31) showing
moderate discrimination (HGDI: 0.3–0.59).
Determination of a Minimal Set of MIRU-VNTR Loci for
Genotyping Main MTBC Lineages from Ghana
Customized MIRU-12 for Lineage 4 shared 11 loci with
standard MIRU-15 whilst 9 loci were shared between customized
MIRU-12 for Lineage 5 and standard MIRU-15. A union of both
sets of typing schemes gave a total of 16 and 17 loci for Lineage 4
and 5, respectively (Table S1). For Lineage 4, we identified six top
most discriminatory loci (4052, 2163B, 40, 2165, 10 and 4165)
with a cumulative HGDI of 0.99 (Table 2). Similarly, for Lineage
5, six loci: 2163B, 4165, 40, 26, 4052 and 16 (Table 3) with a
cumulative HGDI of 0.99 were identified. Further addition of loci
gave no significant change in cumulative HGDI values as shown in
Figure 1. Note that 4 loci (4052, 2163B, 4162 and 40) were among
the 6 most discriminatory in both lineage-specific sets. Hence,
based on this, we propose the usage of a new set of customised
typing system comprising 8 loci showing the highest discriminatory
power for genotyping strains from the two most common lineages
circulating in Ghana.
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that additional lineage-specific sets of MIRU-VNTR could be
identified for molecular epidemiological investigation of TB
transmission in resource-limited settings. Moreover, each MTBC
lineage consists of a number of sub-lineages, some of which also
show strong geographical associations [22, 24, and 45]. For
example, the ‘‘Uganda’’ sub-lineage of Lineage 4 causes up to
60% of TB in Kampala, Uganda [41], suggesting that a similar
customized Lineage 4 set for Uganda could be developed,which
possibly would include other loci considering that most of Lineage
4 in Ghana consists of the ‘‘Cameroon’’ sub-lineage [42–44].
This study set out to define a set of loci for genotyping MTBC
strains from Ghana. We acknowledge the high prevalence of M.
africanum strains in Ghana, however, this prevalence is driven by
Lineage 5 (M. africanum West Africa I) with limited number of
Lineage 6 (M. africanum West Africa II). We acknowledge the fact
that this makes our proposed customized MIRU-8 countryspecific, and thus suggest that countries within West African where
the high prevalence of M. africanum is driven by Lineage 6 (M.
africanum West Africa II) could equally determine the minimal set
of loci which gives the highest discrimination. Nevertheless, the
strength of our study is the ability to genotype an unknown strain
in Ghana with the proposed customized MIRU-8 loci in the most
cost-efficient way.
In conclusion, this study identified a reduced set which can be
applied for strain differentiation of the main MTBC lineages from
Ghana.
Supporting Information
Table S1 MIRU-VNTR Profiles of the Mycobacterial Isolates
Analysed.
(XLS)
Author Contributions
Conceived and designed the experiments: DYM SG. Performed the
experiments: AAP SB MSN. Analyzed the data: AAP DYM SB MSN IC.
Wrote the paper: AAP DYM SG.
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