Fidelis Cho-Ngwa1,3, Jean-Paul Assam-Assam1,2,3, Irene Ane-Anyangwe1, Robert A. Skilton3, Roger Pelle3, Mercy Kitavi3, Inosters Nzuki3, J. Nyonka1,3,
J.C. Tedom4, V. Penlap Beng1, Akoachere Jane-Francis1, Mbome Njie4, Vincent P. K. Titanji1*
(1) Laboratory of Molecular Biology, Biotechnology Unit Faculty of Science, University of Buea Cameroon (2) Laboratory of Tuberculosis Research, Biotechnology center of Nkolbisson Faculty of Science, University of
Biology Unit, Science Buea, Cameroon; Research Nkolbisson, Science
Yaoundé I, Cameroon; (3) Biosciences eastern and central Africa (BecA) at Hub ILRI, Nairobi, Kenya; (4) Buea District Hospital, Buea, SW Region, Cameroon; *Group Leader
Tuberculosis (TB), is an ancient disease predominantly of the lungs caused by airborne germs of the Mycobacterium tuberculosis complex (MTBC). It has continued to be a
public health hazard world‐wide, and a major cause of suffering and death to humans in sub‐Saharan Africa. The number of new human TB cases in most African countries
has more than quadrupled since 1990, with roughly 735 thousand deaths occurring annually on the continent. Single and multi‐drug resistant (MDR) forms are already
widespread, while a form that is resistant to both first‐line and some second‐line drugs, referred to as extensively drug resistant TB (XDR‐TB) is now gaining grounds. While
many countries globally have made great progress in controlling the disease, the problem has persisted in Africa because the correct tools are not being deployed or
adapted/adopted adequately for use on the continent In the present study we determined the best published PCR assay/combination for use in Cameroon; determined
continent. study,
the pattern of resistance of TB bacteria to first line TB drugs in parts of the country; determined the strains of MTBC circulating in the country and developed capacity for
tackling the problem on a regional or continent‐wide scale.
 Overall objective: To contribute to the control of TB •This was a cross‐sectional and hospital‐based molecular epidemiological survey
in Cameroon through the development / validation •A combination of bacteriological, biochemical, molecular biology and
of efficient tools and strategies for use. bioinformatics approaches were used
 Specific objectives were to: •Sputa and urine were collected at selected hospitals and health centers in 7
1. Determine the best PCR assay or combination of
southern regions of Cameroon from February to July 2009
assays for use in the rapid diagnosis of
y p g
•Drug sensitivity t t were carried out b th i di t proportion method
D iti it tests i d t by the indirect ti th d
pulmonary TB in Cameroon
•DNA was extracted from heat killed bacteria by the CTAB method
2. Determine the phenotypic drug resistance
•Diagnostic PCR assays were based on purified DNA and bacterial thermolysate.
pattern of isolated Mycobacteria to first line TB
drugs in Cameroon •Genotyping was done by MIRU‐VNTR and Spoligotyping at the BecA Hub, ILRI,
3. Determine the genotypes of MTBC circulating in
Nairobi
Cameroon and predict possible routes of spread Map of TB incidence rates (Source: WHO, 2009 report)
•Binary data for spoligotyping, and number of repeats for MIRU‐VNTR were
4. To strengthen the TB research capacity of the
analysed using online bioinformatics programmes at www.miru‐vntrplus.org
NEPAD/BecA Node in Buea, Cameroon
•The combined targeting of the IS6110 and rpoB genes should yield the most sensitive PCR procedure for the diagnosis of TB in Cameroon
•TB in Cameroon appears to be caused largely by M. tuberculosis and to a much smaller extent by M. africanum.
• Following the re‐organisation of the Cameroon National TB control Programme, 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, with ethambutol now showing no resistance in both Regions.
•The prevalence of the Cameroon family TB genotype has dropped from 42% in 2003 to 34.25% in the present study (2009). The Ghana strain seems to be spreading in Cameroon,
with Douala, the economic capital, being the epicenter.
•Only three M. africanum isolates were detected in 265 isolates. This represents a significant drop in the relative prevalence of this species in Cameroon from 56% in the early 1970s
to 9% in early 2000 to 1.2% in the present study.
NO. of NO +VE by No. No. With Drug Retained
1 2 3 4 5 6 7 8 9
REGION samples microscopy Cultured Growth sensitivity for DNA
collected (%) (% of No Done typing
cultured)
SOUTH 1,183 254 (21.5) 129 100 25 59 DNA
WEST (77.5)
LITTORAL 678 133 (19.6) 67 44 (65.7) 10 26 Resistance to: Drug(s) Region Number of resistant % in patients of Total % in
NORTH 544 178 (32.7) 173 151 5 37
cases in Region Region: N= 74 for patients of both
Figure: Purified DNA Centre and 75 for West Regions
WEST (87.3)
CENTER 313 75 (23.9) 74 74 (100) 74 71 1 2 3 4 5 6 7 8 9 One drug INH Centre 02 2.70 2.68
WEST 443 79 (17.8) 75 75 (100) 75 72 West 02 2.66
SOUTH 215 70 (32.6) 70 70 (100) - - RIF Centre 01 1.35 0.67
EAST 102 30 (29.4) 30 30 (100) - - West 0 0
SM Centre 01 1.35 2.68
TOTAL 3,478 819 618 544 189 265 West 03 4.0
(23.5) (88.0)
Figure: DNA from bacterial thermolysate EMB Centre 00 0 0
West 0 0
Two drugs Centre 2 2.70 1.34
INH, SM West 0 0
Centre 0 0 1.34
INH, RIF West 2 2.66
Three drugs Centre 0 0 2.01
INH, RIF, West 3 4.0
Test All culture positives (n Culture Culture SM
= 125) positive/Biochem positive/Biochem Totals to at least 16 8.1
8 1 (Centre); 10.73
10 73
positive (n = 110) negative (n = 15) one drug 13.3 (West)
Freq % Sens Freq % Sens Freq % Sens
IS6110 124 99.2 109 99.1 15 100 Resistance Drug(s) Region Acquired % among old Initial % among new
to: resistance cases; N= 6 for resistance cases ; N= 68
hupB 117 93.6 104 94.5 13 86.7 C am eroon G hana H a a r le m U g a n d a W est LAM D e h li/ TUR m u ltip le U nknow
Centre and 13 for for Centre and I A fr ic a CAS m a tc h e s n
rpoB 123 98.4 109 99.1 14 93.3 West 62 for West
oxyR 119 95.2 106 96.4 13 86.7
One drug INH Centre 00 / 02 2.94 3 4 .2 5 % 3 1 .5 1 % 1 3 .7 % 4 .1 1 % 2 .7 4 % 2 .7 4 % 0 0 4 .1 1 % 4 .1 1 %
IS1081 118 94.4 104 94.5 14 93.3
West 00 / 02 3.22
RIF Centre 00 / 01 1.47
Table below: PCR assay sensitivity based on the combined targeting of the West 00 / 02 3.22
IS6110 and the other genes where at least one of the genes gave a positive SM Centre 01 16.66 00 0.00
West 00 / 01 1.61
PCR result (n = 125). EMB Centre 00 / 00 0.00
Gene IS6110 IS6110+rpo IS6110+hup IS6110+oxy IS6110+IS10 West 00 / 00 0.00 ‐Three MSc theses on TB defended
Two drugs Centre 00 / 02 2.94
or gene alone B B R 81 INH, SM West 00 / 00 0.00 ‐One PhD thesis on TB submitted and awaiting
combination Centre 00 / 00 0.00
Number 124 125 125 125 125
INH, RIF West 00 / 02 3.22 defence
Three drugs Centre 00 / 00 0.00
p
positive INH, RIF, West 03 23.07 00 0.00 ‐A TB culture laboratory established and hands‐on
y
Sensitivity 99.2 100 100 100 100 SM TB genotyping techniques acquired
% Totals for both Regions 04 21.05 12 9.20
‐Three Post‐Docs received further training in area
Acknowledgements: Biosciences Eastern and Central Africa‐ Network (BecANet), a network of the New Partnership for Africa’s Development (NEPAD) for financial and training support; The local, regional and district hospitals for interviewing patients and collecting samples; The
Centre Pasteur, Yaounde, Cameroon staff for their technical assistance during the bacteriological studies; The BecA Hub at ILRI, Nairobi, Kenya where the genetic characterizations were undertaken.