Title

Genetic diversity, drug resistance patterns and indicators of disease severity in Plasmodium vivax clinical isolates from Southern Pakistan

Date of Award

2013

Document Type

Thesis

Degree Name

Doctor of Philosophy in Health Science (PhD)

Department

Biological and Biomedical Sciences

Abstract

Pakistan is one of seven countries within the WHO Eastem Mediterranean region with nearly all of the population living 'at-risk' of malaria. Approximately,4.5 million people annually are infected by malaria in Pakistan. Both. Plasmodiumfalciparum and Plasmodium vivax co-exist in Pakistan in ratio of 30 andTOoh respectively Although Plasmodium vivac is the prevalent species in Pakistan, however, epidemiological data on population structure, drug resistance and disease severity on this species is lacking. Therefore, the aim of this study was to determine the extent of genetic diversity, map patterns of drug resistance and determine the mechanism of pathogenesis/indicators of disease severity in clinical isolates of P.vivm from southem Pakistan. The study areas included Karachi,'Balochistan and rural Sindh. Genetic diversity win assessed in two single copy polymorphic genes of P.vivac; the circumsporozoite protein(csp) and the merozoite surface protein-I (mspl).Extensive diversity wrr observed in both genes. A total of 250 microscopically confirmed P.vivax cases were enrolled in the study. Amongst these, 227 isolates (91%) were included in the analysis on the basis of PCR results. Findings on cspvariants showed that both VK210 (85.5o/o,194/227) and VK247 type (I4.5%,33/227) were found circulating in southern Pakistan. With respect to study areas, VK210 type was found to be the predominant variant in all the three study areas (Karachi : 87.4%o, Balochistan : 60.8Yo and Rural Sindh : 95.2%) whereas percent positivity of YK247 type was found to be l2.5Yo in Karachi, 39Yo inBalochistan and, 4.8%o in Rural Sindh. Mixed genetic infection (VK 210+VK247) was observed in Karachi isolates only (2.2Yd. In msp l, atotal of 87 distinct genotypes (Karachi : 36, Balochistan:28 and Rural Sindh = 23) were observed. Furthermore, twenty one, four and five isolates in Karachi, Balochistan and Sindh respectively showed mixed genetic infection, thus indicating a high transmission rate of P.vivax \n the study areas. The secondary aim of the study was to report on the prevalence of drug resistance associated mutations in southern Pakistan. Sulphadoxine-pyrimethamine (SP) is an anti-malarial drug used against uncomplicated P.falciparum malaria. However, due to inappropriate diagnosis, mixed infection and sub-therapeutic 'dosage P.vivm is inadvertently exposed to SP. The target genes for SP arc dihydrofolate reductase (dhfr) and dihydropteroate synthase (dhpl. Both genes produce enzymes required for de novo tetrahydrofolate synthesis and subsequent DNA replication in P.vivox. However, accumulation of mutations via single nucleotide polymorphisms (SNPs) in dhfr and dhps gene allow the parasite to disturb SP binding and accommodation, thus making SP defunct for use. Dhfr and dhps sequences were obtained from 131 P.vivax clinical isolates from the study areas. Direct sequencing of purified amplified product was performed and sequencing data was compared to wild type reference sequence s. In dhfr, wild type isolates were observed in 33.6Yo of the total samples. Of these, 25.2o/o were from Karach|l.5yo from Balochistan and 6.9%o from Rural Sindh. Single mutations were observed at codon position 58& 117N/T, l14R and 119K while double/mixed mutation was observed in combinations of 57Ll58& 58R/117N/T, 50Vl l7N, 50Vl19K 114R/119K and llgWIl7N. Predominant double mutation in all the sampling sites was found to be 58R/l l7N and 50V1l7N.The frequency distribution of these mutations were 28o/o and 5.lYo in Karachi, 30.7% and 7.7%o in Balochistan and 30Yo and ll%inSindh respectively. ln dhps, wild type isolates were observed in 61.8% of the total isolates. Frequency distribution of wild types with respect to study areas were 46.5yo,6.1% and9.2Yo in Karachi, Balochistan and Sindh respectively. 383G mutant allele was observed most frequently as single, double, triple and quadruple mutation. Double mutation in combination of 383G/553Gwas observed in Balochistan only. Moreover, nonsynonymous mutations were observed at codon positipns S373T, E3 80KP3 84L,N3 89T,V 392D,T393P, D459A,M60 I I,A65 lD and 4.66 1 V.These results indicate that the extent of SP pressure on P.vivm is almost equivalent to that observed in high transmission areas worldwide thus serving as an indicator for drug resistance in other areas of Pakistan. With reports of complicated P.viv6 cases being documented consistently from Pakistan, another objective focused on determining the mechanism of pathogenesis and indicators of disease severity in P.vivm. Cytokine-mediated endothelial activation pathway is a mechanism of pathogenesis employed by Plasmodiumfalciparum to induce severe disease symptoms in human host. Therefore, it was hypothesized thatP.vivmutilizes similar mechanism of pathogenesis for manifestations of severe malaria as that used byP.falciparum.The role of cytokines and endothelial activation markers were thus characterized for complicated P.vivmclinical isolates from southem Pakistan. Plasma samples were tested from well-characterized groups infected with P.vivax infection including uncomplicated cases (n:100), complicated cases (n:82) and healthy controls (n:1O0).Levels of cytokines and endothelial adhesion molecules, Tumor necrosis factor-o (TNF-o), Interleukin-6 (IL-6), lnterleukin-I0 (IL-10), Intracellular adhesion molecule-1 (ICAM-1), Vascular adhesion molecule- I(VCAM-I) and E-selectin were measured by Enzyme Linked Immunosorbant Assay (ELISA). Results showed that TNF-c, IL-10, ICAM-land VCAM-lwere 3- fold, 3.7-fold and 2-fold increased between uncomplicated and complicated cases while IL-6 and E-selectin was 1.8 and 1.2 fold decreased between the two groups. Comparison of healthy controls with uncomplicated cases showed .no significant difference in TNF-a concentrations while IL-6, IL-10, ICAM-I, VCAM-I and Eselectin were found to be 3.5-fold,20-fold,3-fold,4-fold andlO-fold elevated respectively. To determine complex correlations between cytokines and endothelial adhesion molecules, Spearman's correlation matrix was generated. Significant positive correlation was observed between TNF-o and ICAM-I (r: 0.314, p-value : 0.004), TNF-o and IL-10 (r: 0.287, p-value : 0.009), IL-10 and IL-6 (r :0.345, p-value:0.002), ICAM-I and VCAM-I (r: 0.237, p-value < 0.03). In addition, the usefulness of these markers as indicators of disease severity was assessed using the Receiver operating curve (ROC). TNF-c, IL-10, ICAM-I and VCAM-I were observed to be the best individual predictors of complicated P.vivm malaria. The results from this thesis provides useful regional baseline information on genetic diversity, extent of SP resistance, possible role of cytokines and endothelial adhesion molecules in pathogenesis and indicators of disease severity which may be useful in planning malaria surveillance and implementation of control strategies within Pakistan and in the region.

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