Fidelis Cho-Ngwa1,3, Jean-Paul Assam-Assam1,2,3, Irene Ane-Anyangwe1, Robert A. Skilton3, Roger Pelle3, Mercy Kitavi3, Ino...
Upcoming SlideShare
Loading in …5
×

Molecular and phenotypic characterisation of mycobacterium tuberculosis complex

1,014 views

Published on

Poster by Fidelis Cho-Ngwa, Jean-Paul Assam-Assam, Irene Ane-Anyangwe, Robert A. Skilton, Roger Pelle, Mercy Kitavi, Inosters Nzuki, J. Nyonka, J.C. Tedom, V. Penlap Beng, Jane-Francis Akoachere, Mbome Njie, Vincent P. K. Titanji for the BecA Opening, Nairobi, 5 November 2010

Published in: Technology, Health & Medicine
0 Comments
0 Likes
Statistics
Notes
  • Be the first to comment

  • Be the first to like this

No Downloads
Views
Total views
1,014
On SlideShare
0
From Embeds
0
Number of Embeds
1
Actions
Shares
0
Downloads
19
Comments
0
Likes
0
Embeds 0
No embeds

No notes for slide

Molecular and phenotypic characterisation of mycobacterium tuberculosis complex

  1. 1. 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.

×