Mycobacterium tuberculosis complex drug resistance pattern and identification of species causing tuberculosis in the West and Centre regions of Cameroon.

Abstract

Data on the levels of resistance of Mycobacterium tuberculosis complex (MTBC) strains to first line anti-tuberculosis drugs in Cameroon, and on the species of MTBC circulating in the country are obsolete. The picture about 10 years after the last studies, and 6 years after the re-organisation of the National Tuberculosis (TB) Control Programme (NTBCP) is not known.
The study was conducted from February to July 2009 in the West and Centre regions of Cameroon. A total of 756 suspected patients were studied. MTBC species were detected by the standard Ziehl-Neelsen staining method. Bacterial susceptibility to the first line drugs [isoniazid (INH), rifampicin (RIF), ethambutol (EMB) and streptomycin (SM)] were performed on cultures using the indirect proportion method. MTBC species were identified by standard biochemical and culture methods.
Of the 756 suspected patients, 154 (20.37%) were positive by smear microscopy. Of these, 20.77% were HIV patients. The growth of Mycobacterium was observed with the sputa from 149 (96.75%) subjects. All the isolates were identified as either M. tuberculosis or M. africanum. Among these, 16 (10.73%) were resistant to at least one drug (13.3% for the West region and 8.1% for the Centre). The initial resistance rates were 7.35% for the Centre region and 11.29% for the West region, while the acquired resistance rates were 16.66% (1/6) for the Centre region and 23.07% (3/13) for the West. Within the two regions, the highest total resistance to one drug was obtained with INH and SM (2.68% each). Multidrug-resistance (MDR) was observed only in the West region at a rate of 6.67%. No resistance was recorded for EMB.
M. tuberculosis and M. africanum remain the MTBC species causing pulmonary TB in the West and Centre regions of Cameroon. Following the re-organisation of the NTBCP, resistance to all first line anti-TB drugs has declined significantly (p < 0.05 for West; and p < 0.01 for Centre) in comparison to previous studies. However, the general rates of anti-TB drug resistance remain high in the country, underscoring the need for greater enforcement of control strategies.

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RESEARCH ARTICLEOpen Access
Mycobacterium tuberculosis complex drug
resistance pattern and identification of species
causing tuberculosis in the West and Centre
regions of Cameroon
Jean-Paul Assam-Assam1,2, Veronique B Penlap2, Fidelis Cho-Ngwa1, Jean-Claude Tedom2, Irene Ane-Anyangwe1
and Vincent P Titanji1*
Abstract
Background: Data on the levels of resistance of Mycobacterium tuberculosis complex (MTBC) strains to first line
anti-tuberculosis drugs in Cameroon, and on the species of MTBC circulating in the country are obsolete. The
picture about 10 years after the last studies, and 6 years after the re-organisation of the National Tuberculosis (TB)
Control Programme (NTBCP) is not known.
Methods: The study was conducted from February to July 2009 in the West and Centre regions of Cameroon. A
total of 756 suspected patients were studied. MTBC species were detected by the standard Ziehl-Neelsen staining
method. Bacterial susceptibility to the first line drugs [isoniazid (INH), rifampicin (RIF), ethambutol (EMB) and
streptomycin (SM)] were performed on cultures using the indirect proportion method. MTBC species were
identified by standard biochemical and culture methods.
Results: Of the 756 suspected patients, 154 (20.37%) were positive by smear microscopy. Of these, 20.77% were
HIV patients. The growth of Mycobacterium was observed with the sputa from 149 (96.75%) subjects. All the
isolates were identified as either M. tuberculosis or M. africanum. Among these, 16 (10.73%) were resistant to at
least one drug (13.3% for the West region and 8.1% for the Centre). The initial resistance rates were 7.35% for the
Centre region and 11.29% for the West region, while the acquired resistance rates were 16.66% (1/6) for the Centre
region and 23.07% (3/13) for the West. Within the two regions, the highest total resistance to one drug was
obtained with INH and SM (2.68% each). Multidrug-resistance (MDR) was observed only in the West region at a
rate of 6.67%. No resistance was recorded for EMB.
Conclusions: M. tuberculosis and M. africanum remain the MTBC species causing pulmonary TB in the West and
Centre regions of Cameroon. Following the re-organisation of the NTBCP, resistance to all first line anti-TB drugs
has declined significantly (p < 0.05 for West; and p < 0.01 for Centre) in comparison to previous studies. However,
the general rates of anti-TB drug resistance remain high in the country, underscoring the need for greater
enforcement of control strategies.
* Correspondence: vpk.titanji@yahoo.com
1Biotechnology Unit, Faculty of Science, University of Buea, P.O. Box 63 Buea,
Cameroon
Full list of author information is available at the end of the article
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© 2011 Assam-Assam et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly cited.

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Background
Every year approximately 9 million people contract
tuberculosis (TB) and close to 2 million die from the dis-
ease [1]. The success of propagation of TB remains
directly linked to the socio-economic and hygienic condi-
tions of human populations [1]. While most TB cases are
in Asia, the highest incidence rates are in Africa where
high rates of HIV and malnutrition weaken immune sys-
tems and accelerate the spread of the disease [2]. Conse-
quently, the number of new TB cases in most African
countries has more than quadrupled since 1990, with 2.8
million new TB cases and roughly 735 thousand deaths
annually [3]. The emergence of anti-tuberculosis drug
resistance is a serious problem for TB control pro-
grammes in industrialized and developing countries alike.
A global project on anti-tuberculosis drug resistance sur-
veillance by the WHO and the International Union
Against Tuberculosis and Lung Disease (IUATLD) [4]
reported a prevalence of initial drug resistance of more
than 10% in over 30 countries; it also identified 14 coun-
tries in which the prevalence of initial multidrug resistant
TB strains (MDR-TB) (defined as resistance to at least
isoniazid [INH] and rifampicin [RMP]) was more than
3%. In a 2000 WHO report on Africa, the overall level of
initial resistance varied from 6.3% to 24.8%, and the level
of MDR-TB from 1% to 5.3% [4]. However, the data
available on the rates of drug resistance in Africa are not
extensive and up to date.
In Cameroon, a country with over 18 million inhabi-
tants distributed in 10 regions (previously provinces),
the incidence of tuberculosis is relatively high, with
more than 300 new cases per 100,000 inhabitants yearly
[5]. Further contribution to the increased death rate due
to TB in the country has been the emergence of drug-
resistant strains to nearly all first line drugs [6]. RIF,
INH, SM, and EMB are components of first-line multi-
drug therapy in Cameroon [7]. Between 1997 and 1998
in the West region of Cameroon, the overall resistance
rate to at least one anti-TB drug was 26.9%, with initial
resistance being 19.7% and acquired resistance 51.1%
[6]. In the Centre region of the country, the rate of
initial resistance to at least one drug in 1995 was 31.8%,
and in 1998 it was 35.2% [8,9]. In 2000 the rate of
acquired resistance was determined in the Centre region
to be 58.2% [10]. Since then, no other report on the
rates of resistance to these drugs in the country has
been carried out. The rising prevalence of MDR strains
has resulted in outbreaks and cases that are not only
marginally treatable, but also often fatal. Following the
awareness generated by the previous studies and the re-
organsiation of the National TB Control Programme
with a focus on proper treatment methods, it has been
necessary to reassess the levels of resistance to the main
anti-TB drugs in the country. Thus, in the present
paper, we report on the current levels of resistance to
these first line drugs in the West and Centre regions of
Cameroon and identify the species of MTBC causing
TB in the regions.
Methods
Study population and classification of samples
The study was conducted from February - July 2009 in
six centres for the detection and treatment of TB (CDT)
of the Centre region (Jamot Hospital, Mbalmayo District
Hospital and Catholic Health Centre of Mvolye) and of
the West region (Regional Hospital of Bafoussam, Dis-
trict Hospital of Baleng and District Hospital of Dje-
leng). Ethical permit was obtained from the Cameroon
National Ethics Committee in Yaounde. In addition,
each subject had to sign or thumbprint a patient con-
sent form before they could be admitted into the study.
A patient was confirmed sputum smear positive if at
least two of three sputum samples collected over a 48
hour period were positive for acid fast bacilli (AFB). All
sputum smear positive patients were included in the
study. Patients confirmed as having pulmonary tubercu-
losis for the first time and without any history of pre-
vious anti-tuberculosis treatment were considered as
new (or initial) cases and any drug resistance associated
to them were reported as initial resistance. Patients with
previous anti-tuberculosis treatment lasting a month or
more were considered as old cases and any drug resis-
tance based on them were reported as acquired resis-
tance. Other pieces of information (notably age,
occupation, gender, HIV status) were taken from the
patients using a questionnaire. The sputum samples
which were smear positive were kept temporarily at +4°
C and transported within 48 hours to the Mycobacter-
ium Laboratory of the Centre Pasteur du Cameroun in
Yaoundé for bacteriological procedures.
Bacteriological procedures
Each sputum sample was examined for acid-fast bacilli
(AFB) by the standard Ziehl Neelsen staining method,
and if positive, was cultured on Lőwenstein-Jensen (LJ)
agar slants in tubes and in triplicate. One of the tubes
was supplemented with sodium pyruvate at 0.4% to
encourage the growth of any M. bovis present. The cul-
tures were incubated at 37°C and examined weekly for a
maximum duration of 10 weeks. Strain identification
was based on the following criteria: growth rate, colony
morphology, growth affinity for pyruvate, niacin produc-
tion, reduction of nitrates and catalase activity [11].
Drug susceptibility testing was performed using the
indirect proportion method on LJ medium as described
by Canetti and collaborators [11]. The first line anti-
tuberculosis drugs were tested as follows: rifampicin at
40 mg/l, isoniazid at 0.1 mg/l, isoniazid at 0.2 mg/l,
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streptomycin at 4 mg/l, and ethambutol at 2 mg/l. Drug
resistance was defined as growth on a drug containing
medium greater than or equal to 1% of that recorded on
the drug-free control medium of the same experiment.
Statistical analysis
Data were analyzed using Epi info 2000 version 12 or,
when appropriate, by Fischer’s Exact test, the Student`s
t-test or the z-test for comparison of proportions. A dif-
ference was considered significant if p was < 0.05.
Results
From a total of 756 subjects who presented clinical
symptoms of tuberculosis at the hospital study sites, 154
(20.37%) were diagnosed positive by smear microscopy.
They were 100 men (64.93%) and 54 women (35.07%)
(Table 1). Patients’ age distribution showed a steady
decline in age-specific prevalence from 30.58% in age
group 15-29 years to 15.51% in the age group above 60
years (Table 2). Of the 154 patients, 32 (20.77%) were
HIV positive (Table 3); while 135 (87.66%) were new
cases and 19 (12.33%) were old cases.
The growth of Mycobacterium tuberculosis complex
strains was observed with sputa from 149 (96.75%) of
the 154 patients. The 5 samples without growth were
thus excluded from further analysis. All the 149 isolates
were identified as either M. tuberculosis or M. africa-
num species. Among these, 133 (89.26%) were suscepti-
ble to all the drugs tested, while 16 (10.73%) were
resistant to at least one drug. This overall resistance rate
was higher in the West region (13.3%) than in the Cen-
tre region (8.1%). Within the two regions, the highest
resistance rate to one drug was obtained with INH and
SM (2.68% each). We did not find any resistance to
EMB in this study. Resistance to more than one drug
stood at 4.69% (7 isolates) for the two regions (Table 4).
A total of 4 isolates (2.68%) were resistant to two drugs
only, and 3 (2.01%) to three drugs. We did not find any
case of resistance to all four drugs (Table 4).
The pattern of initial and acquired drug resistance is
shown in Table 5. Initial resistance to at least one drug
amongst the new cases of both regions (n = 130) was
9.23%, with resistance to INH being the most common
(3.08%). The initial resistance rate was 7.35% (5/68) for
the Centre region and 11.29% (7/62) for the West
region. Acquired resistance to one drug amongst the old
cases (n = 19) of both regions was 21.05%, with the
resistance to SM being the most common (4/19 or
21.05%). The acquired resistance to at least one drug
was 16.66% (1/6) for the Centre region and 23.07% (3/
13) for the West region. Multidrug-resistant M. tubercu-
losis (MDR-TB) complex strains according to the defini-
tion were found in 5 (6.67%) of all the 75 isolates from
the West region. By contrast, MDR-TB was not detected
in any of the 74 isolates from the Centre region. MDR-
TB was more frequently observed in the old cases than
in the new cases (p < 0.05).
Discussion and conclusions
Only a relatively small number of studies on anti-tuber-
culosis drug resistance have so far been carried out in
Cameroon. The last of such studies carried out in the
West (pilot region) and Centre (capital city being
Yaounde) regions of the country were done over 10
years ago and before the re-organisation of the Camer-
oon National TB Control Programme [6,8,9]. The objec-
tive of the present study was to identify the
mycobacterial species responsible for the spread of pul-
monary TB in these two regions and to study the pat-
tern of resistance of isolates to common first line anti-
tuberculosis drugs in the regions. These regions are
amongst the most socio-economically active in the
country.
The growth of MTBC strains was observed with the
sputa of 149 (96.75%) of the 756 suspected patients. The
identification of the isolates by biochemical tests
revealed the presence of only M. tuberculosis or M. afri-
canum species. These results are in line with those of
Table 1 Distribution of sputum samples collected
RegionSites Number of clinically
suspected cases
Number of positive
sputum smears
Centre
Jamot Hospital
District Hospital of Mbalmayo
Catholic health Centre of Mvolyé
75
125
113
313
130
209
104
443
39 (52%)
27 (21.60%)
9 (7.96%)
75 (23.96%)
35 (26.92%)
22 (10.52%)
22 (21.15%)
79 (17.83%)
Subtotal
West
District Hospital of Djeleng
Regional Hospital of Bafoussam
District Hospital of Baleng
Subtotal
Total756154 (20.37%)
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previous studies in the West and Centre regions of
Cameroon where in 1995 and 1998, only these species
were detected [8,9].
The majority of the patients were men (63.93%) and
this was found to be in agreement with previous find-
ings [12]. The prevalence of TB decreased steadily with
age, with the youths being at greater risk of becoming
active patients. The youths are socio-economically more
active, and have the highest HIV infection rates in
Cameroon. At least 32 (20.77%) of the 154 patients were
HIV positive based on analysis of the questionnaires.
The official prevalence of HIV infection in Cameroon is
about 5.5%, indicating a greater than 4-fold increase in
the prevalence of HIV infection amongst TB patients.
On the global scale in 2007, 15% of all new cases of TB
were infected with HIV [2]. Global figures suggest that
HIV-positive people are between 6 to 37 times more
likely than HIV-negative people to develop TB [2].
However, all the drug resistant MTBC isolates in the
present study were from people who indicated they
were HIV negative.
The overall resistance rates (one or more drugs) were
8.1% and 13.3% for the Centre and West regions respec-
tively. The initial resistance rates were 7.35% and
11.29% for the Centre and West regions respectively,
while the acquired resistance rates were 16.66% and
23.07% for the Centre and West regions respectively.
Within the two regions, the highest resistance rates to
one drug were obtained with INH and SM, while no
resistance was recorded for EMB. A previous study car-
ried out in the Centre region (capital city, Yaounde) of
Cameroon in 1998 indicated an overall resistance rate of
35.2%, with initial resistance being 31.8% (164/516) and
acquired resistance, 55.8% (48/86). In the study, resis-
tance to streptomycin (20.5%), and isoniazid (12.4%)
were the highest, while ethambutol showed the least
(0.4%) [13,14]. A similar study carried out in the West
region from 1997-1998 indicated the overall resistance
rate to be 26.9%, with initial resistance being 19.7% (86/
437) and acquired resistance, 51.1% (66/129). Again, the
resistance to streptomycin (11.7%) and isoniazid (12.1%)
were the highest, while the rates for ethambutol (2.5%)
and rifampicin (2.1%) were low [6]. Our observations
therefore follow the trends of these previous results
obtained for the same regions, albeit a significant drop
(p < 0.05 for West; and p < 0.01 for Centre) in resis-
tance to all the drugs tested. This drop could be the
fall-out of the reorganization of the National TB Control
Programme, which emphasises the implementation of the
directly observed treatment strategy (DOTs). The lower
resistance rates for the Centre region in the present study
could be accounted for, at least in part, by the fact that
the region is the seat of the capital city of Cameroon,
Yaounde where better health care facilities may be found.
Both SM and INH have been in use in Cameroon for
longer periods than RIF and EMB. However, a compila-
tion of 63 surveys on resistance to anti-tuberculosis
drugs carried out between 1985 and 1994 in some coun-
tries showed the rate of acquired EMB resistance to be as
high as 13.7% [15]. The rate of initial resistance in the
present study was found to be higher (9.2% for the two
regions) than that reported from other African countries
such as Zimbabwe (3.3%) and Bostwana (3.7%) [16], but
lower than those reported in similar studies from Uganda
[17], Ivory Coast [16], and Sierra Leone [16] (27.4%,
13.4%, and 28.1% respectively). In some African countries
the rates of acquired drug resistance are reported to be
lower or similar (e.g. Zimbabwe, 13.9%; Botswana, 14.9%;
and Swaziland, 20.5%) [16].
A more serious aspect of the TB drug problem is
when the infecting organism is resistant to both INH
and RIF, referred to as multi-drug resistant TB (MDR-
TB) [18]. Under this condition, the duration of treat-
ment is prolonged from 6 to 18-24 months, and the
cure rate could decrease from nearly 100% to less than
60%. This makes the treatment of multi-drug-resistant
cases particularly challenging [19]. MDR-TB strains
were found in 5 (6.67%) of the 75 isolates from the
West region. No MDR-TB strain was detected in any of
the 74 isolates from the Centre region. A similar work
done in the West region in 2000 indicated an MDR-TB
rate of 4.1% [6]. This indicates a slight increase in the
rate of MDR-TB. MDR-TB is an important problem
globally [20]. It results from deficiencies in TB case
Table 3 Distribution of patients according to their HIV
serological status
HIV
Positive
HIV
Negative
Unknown HIV
status
Number of
patients
32 (20.77%)103 (66.88%) 19 (12.33%)
Table 2 Age distribution of study subjects and patients
Age range (years) 15- 29 30-4445 - 60> 60
Number of suspected cases in age range (%)
Number positive by smear microscopy (% of positive cases)
206
63
(41.00%)
216
53
(34.41%)
122
29
(18.83%)
58
9
(5.84%)
% Smear positives in age group 30.58% 24.53%23.77%15.51%
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management and control programmes, and there is
growing evidence now of an overlap between this epi-
demic and HIV infection [21]. In 2004, it was estimated
that 4.3% of all new and previously treated tuberculosis
cases world-wide were multidrug resistant [22]. The
results of a worldwide survey showed that in 2006, 2%
of M. tuberculosis isolates were extensively multidrug
resistant (XDR-TB, caused by MDR strains also resistant
to quinolones plus one of the injectable second line
agents) [23].
We conclude that M. tuberculosis and M. africanum
remain the MTBC species causing pulmonary TB in the
West and Centre regions of Cameroon, and that follow-
ing the re-organisation of the NTBCP, resistance to all
first line anti-TB drugs has declined significantly in
comparison to previous studies, with ethambutol now
Table 5 Pattern of acquired and initial resistance to first line anti-tuberculosis drugs in the Centre and West regions of
Cameroon
Resistance
to:resistance
Drug(s) RegionAcquired % among old
cases; N = 6
for Centre and 13 for
West
Initial
resistance
% among
new cases;
N = 68 for
Centre and
62 for West
One drug
INH Centre
West
0
0
/
/
2
2
2.94
3.22
RIF Centre
West
0
0
/
/
1
2
1.47
3.22
SM Centre
West
1
0
16.66
/
0
1
0
1.61
EMB Centre
West
0
0
/
/
0
0
0
0
Two drugs
INH, SM Centre
West
0
0
/
/
2
0
2.94
0
INH, RIFCentre
West
0
0
/
/
0
2
0
3.22
Three drugs
INH, RIF, SM Centre
West
0
3
/
23.07
0
0
0
0
Totals for both regions421.05 129.20
(INH) = Isoniazid; (RIF) = Rifampicin; (SM) = Streptomycin; (EMB) = Ethambutol
Table 4 First line anti-tuberculosis drug resistance pattern in the Centre and West regions of Cameroon
Resistance
to:
Drug(s)Region Number of
resistant
cases in
region
% in patients of
region: N = 74 for
Centre and 75 for
West
Total % in
patients of
both regions
One drug
INH Centre
West
Centre
West
Centre
West
Centre
West
Centre
West
Centre
West
Centre
West
2
2
1
0
1
3
0
0
2
0
0
2
0
3
2.70
2.66
1.35
0
1.35
4.00
0
0
2.70
0
0
2.66
0
4.00
2.68
RIF0.67
SM2.68
EMB0
Two drugs
INH, SM1.34
INH, RIF1.34
Three drugs
INH, RIF, SM2.01
Totals to at least one drug
16 8.10 (Centre); 13.30 (West)10.73
(INH) = Isoniazid; (RIF) = Rifampicin; (SM) = Streptomycin; (EMB) = Ethambutol
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