Estimates of Genetic Variability of <i>Mycobacterium tuberculosis</i> Complex and Its Association with Drug Resistance in Cameroon

doi:10.4236/aid.2013.31007

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Advances in Infectious Diseases, 2013, 3, 55-59

http://dx.doi.org/10.4236/aid.2013.31007 Published Online March 2013 (http://www.scirp.org/journal/aid)

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: *[email protected]

Received November 5th, 2012; revised December 8th, 2012; accepted January 1st, 2013

ABSTRACT

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.

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)

and DRb, -CCGA-GAGGGGACGGAAAC-3’.

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

isolates.

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

Estimates of Genetic Variability of Mycobacterium tuberculosis Complex and

Its Association with Drug Resistance in Cameroon

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

sero-status.

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

LAM10_CAM

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

sero-status.

Total

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

0.28

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

0.07

*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].

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

assistance.

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