Advances in Infectious Diseases, 2013, 3, 55-59 Published Online March 2013 (
Estimates of Genetic Variability of Mycobacterium
tuberculosis Complex and Its Association with Drug
Resistance in Cameroon
Larissa Kamgue Sidze1,2, Emmanuel Mouafo Tekwu1,2, Christopher Kuaban3,
Jean-Paul Assam Assam1, Jean-Claude Tedom1, Stefan Niemann4, Matthias Frank2,
Véronique N. Penlap Beng1*
1Laboratory for Tuberculosis Research, Biotechnology Centre of Nkolbisson (BTC), University of Yaoundé I, Yaoundé, Cameroon;
2Institute for Tropical Medicine, University of Tübingen, Tübingen, Germany; 3Pneumology Unit, Jamot Hospital, Yaoundé, Cam-
eroon; 4Research Centre Borstel, Molecular Mycobacteriology, Borstel, Germany.
Email: *
Received November 5th, 2012; revised December 8th, 2012; accepted January 1st, 2013
The present study investigates the genetic diversity among Mycobacterium tuberculosis complex circulating in the Cen-
tre region of Cameroon and analyzes the relationship between genotypes and drug resistance patterns. Spoligotyping
was performed by PCR-amplification followed by the reverse hybridization of 298 cultured specimens. Spoligotypes
patterns were identified by comparison to reference strains in SPolDB4 database via the MIRU VNTR plus web appli-
cation. About 97.65% of all tuberculosis (TB) cases were attributed to M. tuberculosis. A total of 65 different profiles
were identified. Of these, 40 were represented as Shared Types (ST) while the others were orphans. LAM10_CAM and
Haarlem families were the most prevalent genetic families with 51.01% and 14.09% respectively. ST 61, a member of
the LAM10_ CAM family formed the largest cluster with 128 (42.95%) isolates. No association was found between
genotypes with regard to drug resistance and HIV sero-status. However, there was a significant association between
genotypes and age groups. Patients belonging to 15 - 24 and 35 - 44 age groups were more likely infected by
LAM10_CAM strains compared to others. The population structure of Mycobacterium tuberculosis complex strains
from the Centre region was found to be diverse and the spoligotype 61 of the LAM10_CAM family was highly pre-
dominant. Isolates of the LAM10_CAM seem to be not associated with drug resistance.
Keywords: M. tuberculosis; Spoligotyping; LAM10_CAM
1. Introduction
Tuberculosis (TB) is a cause of great mortality and suf-
fering, especially in poor and less-developed countries.
Its association with the HIV/AIDS pandemic forms a
lethal combination. In addition, multidrug resistant (MDR)
TB and extensively drug resistant (XDR) TB severely
complicate the management and control of the disease
worldwide [1]. More recently, the discovery of totally
drug resistant (TDR) TB, a deadly form of the disease
highlighted a crisis of mismanagement of the disease [2].
Elimination of TB by 2050 is a long-term goal of the
World Health Organization (WHO) and their strategy is
heavily based on the improvements in the current diag-
nostics, treatment and vaccination, as well as on the
development of new strategies to control and fight the
epidemic [3]. Any strategy for combating the epidemic
should be based on a thorough appreciation of the pro-
blem. Interventions driven by a poor understanding of the
pathogen in a specific geographical context will neces-
sarily entail a high risk of failure [4].
Our understanding of the transmission of tuberculosis
(TB) has been greatly enhanced since the introduction of
DNA fingerprinting techniques for Mycobacterium tu-
berculosis [5]. Spoligotyping is a very practical and re-
producible PCR-based method, which assays the pre-
sence or the absence of a set of target sequences in the
direct repeat (DR) locus [6]. The resulting genotype has a
simple binary format, which has recently leaded to the
construction of large databases, intended to facilitate
recognition of the origin of a particular clinical isolate [7].
Another advantage of spoligotyping is that it can be used
simultaneously for the detection and typing of the M.
tuberculosis complex bacteria in one assay.
As in most resource poor countries, TB epidemiology
in Cameroon has so far largely consisted of reporting the
*Corresponding author.
Copyright © 2013 SciRes. AID
Estimates of Genetic Variability of Mycobacterium tuberculosis Complex and
Its Association with Drug Resistance in Cameroon
number of cases detected and their demographic data.
Little is known about Mycobacterium tuberculosis strains
circulating in the Centre region of the country. The ob-
jective of the present study was to estimate the genetic
variability of Mycobacterium tuberculosis complex strains
circulating in the Centre region and to analyze the rela-
tionship between genotypes and drug resistance.
2. Materials and Methods
2.1. Mycobacterial Isolates
This study included 298 Mycobacterium tuberculosis
complex isolates. These isolates were selected from a co-
llection of Mycobacterium tuberculosis complex strains
isolated from smear positive pulmonary tuberculosis pa-
tients admitted in Jamot hospital and Mbalmayo District
hospital, whose age was ranged from 15 to 85 (mean age,
33.75 years). Among the selected isolates, 28 (10.64%)
were phenotypically drug resistant and 3 were multidrug
resistant. HIV serology was available for 296 (99.32%),
among which 86 (29.05%) were HIV positive.
2.2. DNA Extraction
Mycobacterium tuberculosis complex were scraped from
Lowestein-Jensen slopes, collected into Eppendorf tubes
containing Tris-EDTA (10 mM, 1 mM, pH 8) and heated
for 30 min at 90˚C. After centrifugation at 13,000 xg, the
supernatant was collected into a new tube and kept at
20˚C until further use.
2.3. Spoligotyping
All isolates were genotyped with a spoligotyping com-
mercial kit (Isogen Bioscience, BV Maarsen, The Neth-
erlands) according to the protocol previously described
by Kamerbeek et al. [6]. Briefly, the DR region of the
TB genome was amplified using primers DRa,
5’-GGTTTTGGGTCTGACGAC-3’ (biotinylated 5’ end)
PCR products were hybridized with a set of 43 spacer
oligonucleotides covalently linked to the spoligo-mem-
brane (Isogen Life Sciences, The Netherlands) according
to the manufacturer’s instructions. The hybridized PCR
products were then incubated with a streptavidin-per-
oxidase conjugate and the membrane exposed to chemi-
luminescence (Amersham ECL Direct™ nucleic acid
labeling and detection system, GE Healthcare Limited,
UK). The X-ray film was developed using standard pho-
tochemical procedures after 20 minutes exposure. DNA
extracts of M. tuberculosis H37Rv and M. bovis BCG
were used as controls.
2.4. Data Analysis
Spoligotype patterns in a binary format were entered in
an Excel sheet, and compared with the spoligotype data-
base SpolDB4 using MIRU VNTR plus [8]. The Hunter
Gaston Discriminatory Index (HGDI) was used to calcu-
late the discriminatory power of spoligotyping method
[9]. The Chi square or Exact Fisher test when necessary
were employed to evaluate difference in serology, age
group and drug resistance between LAM10_CAM and
non LAM10_CAM strains. Values of p of less than 0.05
were considered significant.
3. Results
Of the 298 isolates analyzed, 291 (97.65%) were classi-
fied as Mycobacterium tuberculosis and 6 (2.03%) as
Mycobacterium africanum species. The remaining one
isolate was identified as Mycobacterium bovis.
3.1. Distribution of Different Genetic Families
Among the 298 typed isolates, a total of 65 different pro-
files clustered into 17 genetic families were identified. Of
these, 152 (51.01%) isolates belong to the LAM10_CAM
family while 121 (42.7%) were non LAM10_CAM strains.
Strains classified into non LAM10_CAM family in-
cluded strains from the Haarlem family (14.09%), T fa-
mily (12.75%) and others (Table 1).
Table 1. Distribution of different genetic families identified
in a collection of 298 Mycobacterium tuberculosis complex
Genetic families No. isolates Frequency (%)
LAM10_CAM 152 51.01
H3 42 14.09
T2 22 7.38
U 14 4.70
T1 12 4.03
LAM1 6 2.01
U (likely H) 6 2.01
AFRI_2 3 1.01
H1 3 1.01
LAM9 3 1.01
U (likely H3) 2 0.67
AFRI 1 0.34
AFRI_1 1 0.34
AFRI_3 1 0.34
CAS1_DELHI 1 0.34
T1 (T4-CE1 ancestor0) 1 0.34
T2-T3 1 0.34
T5 1 0.34
T5_MAD2 1 0.34
Non identified 25 8.39
Total 298 100.00
Copyright © 2013 SciRes. AID
Estimates of Genetic Variability of Mycobacterium tuberculosis Complex and
Its Association with Drug Resistance in Cameroon
Copyright © 2013 SciRes. AID
3.2. Predominant Spoligotypes
Of the 65 spoligotypes identified, 40 were represented as
Shared Types (ST) according to SpolDB4 while the oth-
ers were reported for the first time. Among these Shared
Types, ST 61 member of the LAM10_CAM and ST 50
member of the Haarlem family respectively represented
42.95% and 11.41% (Table 2).
The HGDI value for spoligotyping was low (79.72%),
especially for the strains of the LAM10_CAM family
(96.71%). No correlation was found between the identi-
fied genotypes with regard to drug resistance, and HIV
sero-status (Table 3). However, a statistical association
was found between the LAM10_CAM isolates and age
groups. Patients belonging to 15 - 24 and 35 - 44 age
groups were more likely infected by LAM10_CAM
strains compared to others. In the LAM10_CAM family,
the distribution of different Share Types ST403, ST61,
ST838, ST850 and ST852 was not associated with HIV
Table 2. Distribution of Share Types (ST) identified in a collection of 298 Mycobacterium tuberculosis complex isolates.
Genotypes Share-Types (ST) Spoligo-patterns No. isolates Frequency (%)
AFRI 332 1 0.34
AFRI_1 715 1 0.34
AFRI_2 101 1 0.34
331 2 0.67
AFRI_3 856 1 0.34
1223 2 0.67
1324 2 0.67
144 1 0.34
53 7 2.35
T1 (T4-CE1 ancestor) 65 1 0.34
1056 2 0.67
125 1 0.34
317 2 0.67
52 8 2.68
848 1 0.34
853 8 2.68
T2-T3 73 1 0.34
T5 44 1 0.34
T5_MAD2 1227 1 0.34
LAM1 20 6 2.01
LAM9 42 3 1.01
403 3 1.01
61 128 42.95
838 14 4.70
850 5 1.68
852 2 0.67
H1 47 3 1.01
316 4 1.34
49 1 0.34
50 34 11.41
75 1 0.34
840 1 0.34
99 1 0.34
CAS1_DELHI 1 0.34
124 1 0.34
450 11 11.41
786 1 0.34
839 1 0.34
46 6 2.01
U (likely H) 237 2 0.67
Estimates of Genetic Variability of Mycobacterium tuberculosis Complex and
Its Association with Drug Resistance in Cameroon
Table 3. Distribution of LAM10_CAM and non LAM10_CAM genotype according to drug resistance, age group and HIV
N = 298LAM10_CAM
N = 152 % Non LAM10_CAM
N = 146 % p-value
DST results
Resistant 28 17 60.71 11 39.29
Susceptible 270 135 50.00 135 50.00
Age groups
15 - 24 72 46 63.87 26 36.13 0.01*
25 - 34 103 51 49.51 52 50.49 0.70
35 - 44 72 28 38.88 44 61.12 0.02*
45 - 54 28 18 64.28 10 35.72 0.14
55 - 64 23 9 39.13 14 60.87 0.23
HIV sero-status
Positive 86 50 58.14 36 41.86
Negative 274 98 35.76 111 64.23
*Statistically significant.
4. Discussion
It has been reported in some instances that, spoligotyping
can distinguish among members of the M. tuberculosis
complex based on the species-specific presence/absence
of spacers [10]. In our study, 3 different species were
identified among 298 M. tuberculosis complex isolates.
With more than 90% cases, M. tuberculosis was far the
most prevalent species. A similar observation was re-
ported among the M. tuberculosis complex isolates col-
lected from the West region of Cameroon [11].
The comparison of spoligotypes found in this study
with the International Spoligotyping Database SPolDB4,
showed that the most prevalent spoligotype was ST 61
followed by ST 50, which belong respectively to the
LAM10_CAM family and Haarlem family. A similar
predominance of the LAM10_CAM family was previ-
ously described among Mycobacterium tuberculosis iso-
lates from Burkina faso [12] and Benin [13]. Although
this genotype was described to be prevalent in some
countries of the West African coast [11], a study con-
ducted in Sierra Leone revealed only 4% of strains be-
longing to the LAM10_CAM [14]. The factors that might
contribute to the adaptability of M. tuberculosis strains or
lineages to a particular population or zone are poorly un-
derstood. As hypothesized for the Tunisian family [15],
mass BCG vaccination strictly applied for decades might
have profoundly shaped the population structure of M.
tuberculosis by concurrently favoring the selection and
accommodation of particular genotypes, as the LAM10_
CAM family in our setting.
W-Beijing family strains were not identified in our
isolates. It has been reported that this genotype is very
rare in some West African coast countries [12]. A pro-
portion of 10.3% was reported among M. tuberculosis
from Cotonou (Benin) [13]. As expected, the HGDI va-
lue for spoligotyping was low (79.72%). To increase the
discriminatory power and for a better understanding of
the molecular diversity of the studied population, a more
discriminatory technique such as MIRU-VNTR typing is
recommended [18].
As it was reported in some instances that, genotypes
like W-Beijing family are associated with drug resistance
[17], we analyzed the relationship between genotypes
circulating in our setting and drug resistance. However,
we did not find any statistical association between geno-
types and drug resistance (p = 0.47) even HIV serostatus
(p = 0.07) in our study, but only with age groups (p =
0.01). The distribution of the LAM10_CAM strains ac-
cording to age groups followed the trend of HIV infec-
tion in our study population. It has been reported that
strains well adapted to a specific population like the
LAM10_CAM family are more likely to transmit com-
pared to others [18]. Since HIV infection, a known risk
factor for tuberculosis was associated with age groups;
this could explain the association of LAM10_CAM strains
with age groups. Five different Share Types ST403, ST61,
ST838, ST850 and ST852 were identified in the LAM10_
CAM family. The distribution of these genotypes did not
correlate with HIV sero-status. This observation was pre-
viously reported in isolates from the West region of the
country [11].
Copyright © 2013 SciRes. AID
Estimates of Genetic Variability of Mycobacterium tuberculosis Complex and
Its Association with Drug Resistance in Cameroon
The population structure of Mycobacterium tuberculo-
sis complex strains from the Centre region was diverse
and included 65 different genotypes. The majority of
strains belonged to the LAM10_CAM which can be sub-
divided in 5 spoligotypes. The consequence of this diver-
sity for the TB epidemic are not yet clear and need to be
addressed in further studies.
5. Acknowledgements
This study was financially supported by EDCTP grant
through the CANTAM-TB “Central Africa Network for
Tuberculosis, AIDS/HIV and Malaria” project. Larissa
Kamgue Sidze and Emmanuel Mouafo Tekwu were re-
search fellow students at the Institute for Tropical Medi-
cine in Tübingen (Germany). We thank Mrs. Augusta
Tsasse (Centre Pasteur of Cameroon) and Tanja Ubben
(Research Centre Borstel, Germany) for their technical
[1] C. Dye, “Global Epidezmiology of Tuberculosis,” Lancet,
Vol. 367, No. 9514, 2006, pp. 938-940.
[2] K. Rowland, “Totally Drug-Resistant TB Emerges in
India,” Nature News and Comment, 13 January 2012.
[3] World Health Organization, “Global Tuberculosis Control:
WHO Report 2011,” Geneva, 2011.
[4] G. D. Van der Spuy, R. M. Warren and P. D. Van Helden,
“The Role of Molecular Epidemiology in Low-Income,
High-Burden Countries,” International Journal of Tuber-
cle and Lung Diseases, Vol. 13, No. 4, 2009, pp. 419-
[5] D. Van Soolingen, P. W. Hermans, P. E. De Haas, D. R.
Soll and J. D. Van Embden, “Occurrence and Stability of
Insertion Sequences in Mycobacterium tuberculosis Com-
plex Strains: Evaluation of an Insertion Sequence-De-
pendent DNA Polymorphism as a Tool in the Epidemiol-
ogy of Tuberculosis,” Journal of Clinical Microbiology,
Vol. 29, No. 11, 1991, pp. 2578-2586.
[6] J. Kamerbeek, L. Schouls, A. Kolk, M. Van Agterveld, D.
Van Soolingen, S. Kuijper, A. Bunschoten, H. Molhuizen,
R. Shaw, M. Goyal, et al., “Simultaneous Detection and
Strain Differentiation of Mycobacterium tuberculosis for
Diagnosis and Epidemiology,” Journal of Clinical Mi-
crobiology, Vol. 35, No. 4, 1997, pp. 907-914.
[7] I. Filliol, J. R. Driscoll, D. Van Soolingen, B. N. Kreis-
wirth, K. Kremer, G. Valetudie, D. D. Anh, R. Barlow, D.
Banerjee, P. J. Bifani, et al., “Global Distribution of My-
cobacterium tuberculosis Spoligotypes,” Emerging Infec-
tious Diseases, Vol. 8, No. 11, 2002, pp. 1347-1349.
[8] T. Weniger, J. Krawczyk, P. Supply, S. Niemann and D.
Harmsen, “MIRU-VNTR plus: A Web Tool for Polypha-
sic Genotyping of Mycobacterium tuberculosis Complex
Bacteria,” Nucleic Acids Research, Vol. 38, 2010, pp.
[9] P. R. Hunter and M. A. Gaston, “Numerical Index of the
Discriminatory Ability of Typing Sysems: An Applica-
tion of Simpson’s Index of Diversity,” Journal of Clinical
Microbiology, Vol. 26, No. 1, 1988, pp. 2465-2466.
[10] B. Mathema, N. E. Kurepina, P. J. Bifani and B. N. Krei-
swirth, “Molecular Epidemiology of Tuberculosis: Cur-
rent Insights,” Clinical Microbiology Review, Vol. 19, No.
4, 2006, pp. 658-685. doi:10.1128/CMR.00061-05
[11] S. N. Niobe-Eyangoh, C. Kuaban, P. Sorlin, P. Cunin, J.
Thonnon, C. Sola, et al., “Genetic Biodiversity of Myco-
bacterium tuberculosis Complex Strains from Patients
with Pulmonary Tuberculosis in Cameroon,” Journal of
Clinical Microbiology, Vol. 41, No. 6, 2003, pp. 2547-
2553. doi:10.1128/JCM.41.6.2547-2553.2003
[12] S. Godreuil, G. Torrea, D. Terru, F. Chevenet, S. Diag-
bouga, P. Supply, et al., “First Molecular Epidemiology
Study of Mycobacterium tuberculosis in Burkina Faso,”
Journal of Clinical Microbiology, Vol. 45, No. 3, 2007,
pp. 921-927. doi:10.1128/JCM.01918-06
[13] D. Affolabi, G. Anyo, F. Faihun, N. Sanoussi, I. C. Sham-
puta, L. Rigouts, et al., “First Molecular Epidemiological
Study of Tuberculosis in Benin,” International Journal of
Tubercle and Lung Diseases, Vol. 13, No. 13, 2009, pp.
[14] S. Homolka, E. Post, B. Oberhauser, A. G. George, L.
Westman, F. Dafae, S. Rusch-Gerdes and S. Niemann,
“High Genetic Diversity among Mycobacterium tubercu-
losis Complex Strains from Sierra Leone,” BMC Micro-
biology, Vol. 8, No. 103, 2008, pp. 489-495.
[15] A. Namouchi, A. Karboul, B. Mhenni, N. Khabouchi, R.
Haltiti, R. Ben Hassine, et al., “Genetic Profiling of My-
cobacterium tuberculosis in Tunisia: Predominance and
Evidence for the Establishment of a Few Genotypes,”
Journal of Medical Microbiology, Vol. 57, No. 7, 2008,
pp. 864-872. doi:10.1099/jmm.0.47483-0
[16] P. Supply, E. Mazars, S. Lesjean, V. Vincent, B. Gicquel
and C. Locht, “Variable Human Minisatellite-Like Re-
gions in the Mycobacterium tuberculosis Genome,” Mo-
lecular Microbiology, Vol. 36, No. 3, 2000, pp. 762-771.
[17] W. A. Githui, A. M. Jordaan, E. S. Juma, P. Kinyanjui, F.
G. Karimi, J. Kimwomi, et al., “Identification of MDR-
TB Beijing/W and Other Mycobacterium tuberculosis
Genotypes in Nairobi, Kenya,” International Journal of
Tubercle and Lung Diseases, Vol. 8, No. 3, 2004, pp.
[18] S. Gagneux and P. M. Small, “Global Phylogeography of
Mycobacterium tuberculosis and Implications for Tuber-
culosis Product Development,” Lancet Infectious Dis-
eases, Vol. 7, No. 5, 2007, pp. 328-337.
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