Genetic diversity of Plasmodium falciparum in Bangladesh_MSA.pptx

In this presentation genetic diversity of falciparum malaria pasristes in Bangladesh has been reported.

1. Genetic diversity of Plasmodium falciparum in Bangladesh Mohammad Shafiul Alam Scientist Infectious Diseases Division icddr,b

2. Several  Plasmodium falciparum   genes show extensive genetic polymorphism High polymorphism has been shown in  several  genes in different geographical locations in malaria endemic areas Genetic polymorphism can be an essential tool for parasite population dynamics Comparing the genotypes of the different loci at baseline and at the time of parasite recurrence would be expected to discriminate between recrudescent and new infections Importance of genotyping:

3. 11 merozoite surface proteins (MSP) to date are described which are found on the surface of the merozoites , MSP1 to -11 MSP1 and MSP2 have been found to be polymorphic, while the others are mostly conserved The Glutamate Rich Protein (GLURP) is a  Plasmodium falciparum  antigen and expressed in all stages of the life cycle of the parasite GLURP polymorphisms mainly involve variations in the numbers of repeats of certain genomic sequences that therefore affect the size of the gene and its protein product. The presence of more than one allele represents a multiclonal infection.

4. Objectives Study the genetic diversity of P. falciparum clinical isolates in Bangladesh for: Merozoite surface protein-1 (MSP-1) Merozoite surface protein-2 (MSP-2) Glutamate rich protein (GLURP)

5. Materials and Methods 130 falciparum malaria positive samples from 7 districts (CHT: Bandarban , Rangamati , Khagrachari ; Non-CHT: Netrokona , Mymensingh , Sylhet and Cox’s Bazar ) were examined DNA was extracted using Qiagen blood mini kit Allelic frequency and diversity were investigated by PCR using WHO standard protocol PCR amplification of template DNA and analysis of region II of  glurp , central polymorphic region of  msp2  (3D7 and FC27 allelic families), and block 2 of  msp1  (K1, MAD20 and RO33 allelic families) was performed 

6. Table: List of primers for MSP1 , MSP2 and GLURP genotype PCR Materials and Methods Gene Primer Sequence Note MSP1 M1-OF 5 ′ - CTA GAA GCT TTA GAA GAT GCA GTA TTG - 3 ′ MSP1 Conserved- Nest 1 M1-OR 5 ′ -CTT AAA TAG TAT TCT AAT TCA AGT GGA TCA -3 ′ MSP1 Conserved- Nest 1 M1-KF 5 ′ -AAA TGA AGA AGA AAT TAC TAC AAA AGG TGC -3 ′ K1 family specific-Nest 2 M1-KR 5 ′ - GCT TGC ATC AGC TGG AGG GCT TGC ACC AGA -3 ′ K1 family specific-Nest 2 M1-MF 5 ′ ­- AAA TGA AGG AAC AAG TGG AAC AGC TGT TAC -3 ′ MAD20 family specific-Nest M1-MR 5 ′ - ATC TGA AGG ATT TGT ACG TCT TGA ATT ACC -3 ′ MAD20 family specific-Nest 2 M1-RF 5 ′ - TAA AGG ATG GAG CAA ATA CTC AAG TTG TTG -3 ′ RO33 family specific-Nest 2 ) M1-RR 5 ′ - CAT CTG AAG GAT TTG CAG CAC CTG GAG ATC -3 ′ RO33 family specific-Nest 2 MSP2 M2-OF 5 ′ - ATG AAG GTA ATT AAA ACA TTG TCT ATT ATA-3 ′ MSP2 Conserved- Nest 1 M2-OR 5 ′ - CTT TGT TAC CAT CGG TAC ATT CTT-3 ′ MSP2 Conserved- Nest 1 M2-FC27F1 5 ′ -AA T ACT AAG AGT GTA GGT GCA AAT GCT CCA-3 ′ FC27 family specific-Nest 2 M2- FC27F2 5 ′ -AAT ACT AAG AGT GTA GGT GCA GAT GCT CCA-3 FC27 family specific-Nest 2 M2- FC27R 5 ′ ­- TTT TAT TTG GTG CAT TGC CAG AAC TTG AAC-3 ′ FC27 family specific-Nest 2 M2-3D7/ICF 5 ′ - AGA AGT ATG GCA GAA AGT AAG CCT CCT ACT-3 ′ 3D7/IC family specific - Nest 2 M2-3D7/ICR 5 ′ - GAT TGT AAT TCG GGG GAT TCA GTT TGT TCG-3 ′ 3D7/IC family specific-Nest 2 GLURP G-OF 5 ′ - TGA ATT TGA AGA TGT TCA CAC TGA AC-3 ′ GLURP Conserved- Nest G-OR 5 ′ - GTG GAA TTG CTT TTT CTT CAA CAC TAA-3 ′ GLURP Conserved- Nest 1 and 2 G-NF 5 ′ - TGT TCA CAC TGA ACA ATT AGA TTT AGA TCA-3 ′ GLURP Conserved- Nest 2

7. Materials and Methods Gel photographs were visualized under UV illumunator and photographed Gel photographs were visually examined to identify sucessful amplified fragment The expected heterozygosity (H E ) was calculated by use of the formula H E  = [n/(n-1)] [(1-∑P i 2 )], where n = sample size, Pi = allele frequency The mean multiplicity of infection (MOI) was calculated as the quotient of the total number of  P. falciparum genotypes for each marker in a particular area and the number of positive PCR samples

8. Results Out of 130 samples : 125 were amplified in PCR for any of the MSP alleles 111 (85.4%) were amplified for MSP1 and 102 (78.5%) for MSP2 All 130 isolates were amplified for GLURP

9. 300 bp 175 bp 200 bp 250 bp Figure: Gel picture of the PCR product to detect K1 allelic family positive patients. Lane 1, 7, 14: 25 bp ladder, Lane 18: Positive control, Lane 6: Negative control, Lane 2-5, 8-13, 15-17, 19-20: PCR product. 250 bp 125 bp 150 bp 200 bp Figure: Gel picture of the PCR product to detect MAD20 allelic family positive patients. Lane 1, 6, 11, 16: 25 bp ladder, Lane 2: Positive control, Lane 3-5, 7-10, 12-15, 17-19: PCR product, Lane 20: Negative control. 250 bp 125 bp 150 bp 200 bp Fig: Gel picture of the PCR product to detect RO33 allelic family positive patients. Lane 1, 5, 11, 16: 25 bp ladder, Lane 2: Positive control, Lane 3-4, 6-10, 12-15, 17-19: PCR product, Lane 20: Negative control. 300 bp 175 bp 200 bp 250 bp

10. Results Table: DNA amplification success rate for each marker according to areas Area MSP1 MSP2 GLURP No K1 MAD20 RO33 Total (%) FC27 3D7 Total (%) Total (%) CHT 69 30 33 25 59 (85.5) 31 30 51 (73.9) 69 (100) Non-CHT 61 29 27 16 52 (85.2) 22 37 51 (83.6) 61 (100) BD 130 59 60 41 111 (85.4) 53 67 102 (78.5) 130 (100)

11. Results Area K1 MAD RO33     No of Clones No. Alleles Mol size ( bp ) No of Clones No. Alleles Mol size ( bp ) No of Clones No. Alleles Mol size ( bp ) CHT 33 7 126-300 35 5 126-250 25 1 160 Non-CHT 30 6 126-300 29 6 126-325 16 1 160 BD 63 7 126-300 64 6 126-325 41 1 160 Table: Number of MSP1 genotypes per allelic-family/locus in study areas

12. Results Area FC27 3D7 GLURP   No of Clones No. Alleles Mol size ( bp ) No of Clones No. Alleles Mol size ( bp ) No of Clones No. Alleles Mol size ( bp ) CHT 31 7 251-425 31 9 351-625 72 12 400-1000 Non-CHT 23 5 251-475 43 9 376-675 68 11 550-1050 BD 54 8 251-475 74 12 351-675 141 13 400-1050 Table: Number of MSP2 and GLURP genotypes per allelic-family/locus in study areas

13. MSP1 allelic frequency by region: Non-CHT CHT

14. MSP2 allelic frequency by region Non-CHT CHT

15. GLURP allelic frequency by area Non-CHT CHT

16. Area MSP1 MSP2 GLURP MOI H E MOI H E MOI H E CHT 1.35 0.76 0.90 0.93 1.04 0.86 Non-CHT 1.23 0.84 1.08 0.90 1.11 0.78 BD 1.29 0.80 0.98 0.93 1.08 0.83 Table: Mean multiplicity of infection (MOI) and genetic diversity of MSP1 , MSP2 and GLURP measured as expected heterozygosity ( H E ) in study areas

17. Topic summary There is no significant difference in genetic diversity of P. falciparum among CHT and non-CHT areas Overall P. falciparum genetical diversity in Bangladesh is less compare to India but similar to some African countries (Uganda, Malawi etc)