Prevalence and distribution of human Plasmodium infection in Pakistan.

BACKGROUND: Both Plasmodium vivax and Plasmodium falciparum are prevalent in Pakistan, yet up-to-date data on the epidemiology of malaria in Pakistan are not available. This study was undertaken to determine the current prevalence and distribution of Plasmodium species across the country. METHODS: A malariometric population survey was conducted in 2011 using blood samples collected from 801 febrile patients of all ages in four provinces and the capital city of Islamabad. Microscopically confirmed Plasmodium-positive blood samples were reconfirmed by polymerase chain reaction (PCR). Confirmed parasite-positive samples were subjected to species-specific PCR capable of detecting four species of human malaria. RESULTS: Of the 707 PCR-positive samples, 128 (18%) were P. falciparum, 536 (76%) were P. vivax, and 43 (6%) were mixed P. falciparum and P. vivax. Ninety-four microscopy-positive samples were PCR-negative, and Plasmodium malariae and Plasmodium ovale were not detected. Prevalence of P. vivax ranged from 2.4% in Punjab Province to 10.8% in Sindh Province and prevalence of P. falciparum ranged from 0.1% in Islamabad to 3.8% in Balochistan. CONCLUSIONS: Plasmodium infections in Pakistan are largely attributed to P. vivax but P. falciparum and mixed species infections are also prevalent. In addition, regional variation in the prevalence and species composition of malaria is high.

Prevalence and patterns of antifolate and chloroquine drug resistance markers in Plasmodium vivax across Pakistan.

BACKGROUND: Plasmodium vivax is the most prevalent malaria species in Pakistan, with a distribution that coincides with Plasmodium falciparum in many parts of the country. Both species are likely exposed to drug pressure from a number of anti-malarials including chloroquine, sulphadoxine-pyrimethamine (SP), and artemisinin combination therapy, yet little is known regarding the effects of drug pressure on parasite genes associated with drug resistance. The aims of this study were to determine the prevalence of polymorphisms in the SP resistance-associated genes pvdhfr, pvdhps and chloroquine resistance-associated gene pvmdr1 in P. vivax isolates collected from across the country. METHODS: In 2011, 801 microscopically confirmed malaria-parasite positive filter paper blood samples were collected at 14 sites representing four provinces and the capital city of Islamabad. Species-specific polymerase chain reaction (PCR) was used to identify human Plasmodium species infection. PCR-positive P. vivax isolates were subjected to sequencing of pvdhfr, pvdhps and pvmdr1 and to real-time PCR analysis to assess pvmdr1 copy number variation. RESULTS: Of the 801 samples, 536 were determined to be P. vivax, 128 were P. falciparum, 43 were mixed vivax/falciparum infections and 94 were PCR-negative for Plasmodium infection. Of PCR-positive P. vivax samples, 372 were selected for sequence analysis. Seventy-six of the isolates (23%) were double mutant at positions S58R and S117N in pvdhfr. Additionally, two mutations at positions N50I and S93H were observed in 55 (15%) and 24 (7%) of samples, respectively. Three 18 base pair insertion-deletions (indels) were observed in pvdhfr, with two insertions at different nucleotide positions in 36 isolates and deletions in 10. Ninety-two percent of samples contained the pvdhps (S382/A383G/K512/A553/V585) SAKAV wild type haplotype. For pvmdr1, all isolates were wild type at position Y976F and 335 (98%) carried the mutation at codon F1076L. All isolates harboured single copies of the pvmdr1 gene. CONCLUSIONS: The prevalence of mutations associated with SP resistance in P. vivax is low in Pakistan. The high prevalence of P. vivax mutant pvmdr1 codon F1076L indicates that efficacy of chloroquine plus primaquine could be in danger of being compromised, but further studies are required to assess the clinical relevance of this observation. These findings will serve as a baseline for further monitoring of drug-resistant P. vivax malaria in Pakistan.

Genetic structure of Plasmodium vivax and Plasmodium falciparum in the Bannu district of Pakistan.

BACKGROUND: Plasmodium vivax and Plasmodium falciparum are the major causative agents of malaria. While knowledge of the genetic structure of malaria parasites is useful for understanding the evolution of parasite virulence, designing anti-malarial vaccines and assessing the impact of malaria control measures, there is a paucity of information on genetic diversity of these two malaria parasites in Pakistan. This study sought to shed some light on the genetic structure of P. vivax and P. falciparum in this understudied region. METHODS: The genetic diversities of P. vivax and P. falciparum populations from the densely populated, malaria-endemic Bannu district of Pakistan were evaluated by analysis of their merozoite surface protein (msp) genes by PCR-RFLP. Specifically, the Pvmsp-3alpha and Pvmsp-3beta genes of P. vivax and the Pfmsp-1 and Pfmsp-2 genes of P. falciparum were analysed. RESULTS: In P. vivax, genotyping of Pvmsp-3alpha and Pvmsp-3beta genes showed a high level of diversity at these loci. Four distinct allele groups: A (1.9 kb), B (1.5 kb), C (1.2 kb), and D (0.3 kb) were detected for Pvmsp-3alpha, type A being the most prevalent (82%). Conversely, amplification of the P. vivax msp-3beta locus produced two allele groups: A (1.7-2.2 kb, 62%) and B (1.4-1.5 kb, 33%), with 5% mixed-strain infections. Restriction analysis of Pvmsp-3alpha and Pvmsp-3beta yielded 12 and 8 distinct alleles, respectively, with a combined mixed genotype prevalence of 20%. In P. falciparum, all three known genotypes of Pfmsp-1 and two of Pfmsp-2 were observed, with MAD20 occurring in 67% and 3D7/IC in 65% of the isolates, respectively. Overall, 24% P. falciparum samples exhibited mixed-strain infections. CONCLUSION: These results indicate that both P. vivax and P. falciparum populations in Pakistan are highly diverse.

LC-MS-MS Characterization of Forced Degradation Products of Fidarestat, a Novel Aldose Reductase Inhibitor: Development and Validation of a Stability-Indicating RP-HPLC Method.

An accurate, precise, robust and selective stability-indicating liquid chromatographic (LC) method has been developed for the monitoring of fidarestat in the presence of its forced degradants. The drug was subjected to hydrolysis (acid, alkali and neutral degradation), oxidation, photolysis and thermal stress conditions. The drug degraded significantly under hydrolytic (basic, acidic and neutral) and oxidative stress conditions, whereas it was found to be stable in photolytic and thermal conditions. The chromatographic separation was achieved on a Grace C18, (250 mm × 4.6 mm × 5 µm) column using gradient mobile phase system consisting of 10 mM of ammonium acetate buffer at pH 4 and acetonitrile at a flow rate of 1 mL/min with UV detection at 283 nm. The developed method was extended to liquid chromatography quadrupole time-of-flight tandem mass spectrometry (LC-QTOF-MS-MS) for characterization of all the degradation products. A total of five new degradation products were identified and characterized by LC-QTOF-MS-MS. The developed LC method was validated as per ICH guideline Q2 (R1). The proposed method was found to be successively applied for the quality control of fidarestat in bulk drug analysis.

Synthesis of gallinamide A analogues as potent falcipain inhibitors and antimalarials.

Analogues of the natural product gallinamide A were prepared to elucidate novel inhibitors of the falcipain cysteine proteases. Analogues exhibited potent inhibition of falcipain-2 (FP-2) and falcipain-3 (FP-3) and of the development of Plasmodium falciparum in vitro. Several compounds were equipotent to chloroquine as inhibitors of the 3D7 strain of P. falciparum and maintained potent activity against the chloroquine-resistant Dd2 parasite. These compounds serve as promising leads for the development of novel antimalarial agents.

Sequence analysis of genes associated with resistance to chloroquine and sulphadoxine pyrimethamine in P. falciparum and P. vivax isolates from the Bannu district of Pakistan.

Plasmodium vivax and Plasmodium falciparum are becoming resistant to drugs including antifolates, sulphonamides and chloroquine. This study was focused at sequence analysis of resistant genes of these parasites against sulphadoxine-pyrimethamine and chloroquine, from Bannu, Pakistan. Known mutations were detected at codons 57, 58 and 117 of pvdhfr gene of P. vivax, while none of the isolates had any pvdhps mutation. Similarly P. falciparum isolates exhibited double 59R+108N mutations in pfdhfr, and single 437G in pfdhps thus demonstrating the existance of triple mutant 59R+108N+437G haplotype in this region. The key chloroquine resistance mutation, 76T in pfcrt was observed in 100% of the P. falciparum isolates, with haplotype SVMNT which is also associated with resistance to amodiaquine. Some novel mutations were also observed in pvdhfr and pfdhfr genes.

Genetic diversity in human malarial parasites of Khyber Agency Pakistan.

Malaria is wide spread in poor world and its burden has been assessed by the enumeration of malarial parasites in blood of patients. This study was designed to find a relationship between social structure, and spread of malaria in Khyber agency. The average parasite density was 2050 parasite/µl in Khyber Agency. Due to economic and social setup most of the people have habit of sleeping in open air thus playing role in high malaria prevalence and Plasmodium vivax remains the prevalent species. Genetic study performed on 110 Blood samples showed less genetic diversity for both Plasmodium vivax and Plasmodium falciparum. Eight alleles were distinguished both for Pvmsp 3α and Pvmsp 3ß in total of 20 and 39 amplified samples of P. vivax respectively. Out of 17 samples amplified for P. falciparum 11 showed genotype K1 and 10 for MAD at Pfmsp-1 while 14 alleles were identified for 3D7/1C and two for FC27 of corresponding families of Pfmsp-2 gene. This shows that Plasmodium parasites are not genetically diverse in Khyber agency.

Genetic diversity of Plasmodium vivax and Plasmodium falciparum in Kohat District, Pakistan.

Malaria is one of the serious diseases threatening human health in Pakistan and contributes to a large proportion of the total malaria deaths in South Asia. However, little is known about the nature and extent of genetic diversity of the malarial parasites circulating in Pakistan. This study was designed to assess the infection status of Plasmodium and the genetic diversity of Plasmodium vivax and Plasmodium falciparum by analyzing msp-3α, msp-3ß and msp-1, msp-2 genes respectively using allele specific nested PCR and RFLP assays. For this purpose, 130 field isolates were collected from the individuals who exhibited clinical symptoms associated with malaria in the Kohat region of Khyber Pakhtoonkhwa (KPK), Pakistan. Among 130 blood samples collected, P. vivax was detected in 105/130 (80.8%) and P. falciparum in 21/130 (16.2%). Mixed infections with both parasites were detected in 4/130 (3%) of the isolates. A large number of distinguishable alleles were found for msp genetic markers: 10 alleles for msp-3α and seven for msp-3ß with one mixed infection in case of msp-3ß. The genotyping of P. falciparum showed that K1+MAD20 mixed genotype was dominant in msp-1 and FC27 in msp-2. The results collectively suggest that P. vivax and P. falciparum populations in this region are highly polymorphic and mixed infections are prevalent.

Prevalence of antimalarial drug resistance mutations in Plasmodium vivax and P. falciparum from a malaria-endemic area of Pakistan.

To study drug resistance in Bannu district, a malaria-endemic area in Pakistan, molecular-based analyses were undertaken. In Plasmodium vivax, antifolate resistance mutations were detected in pvdhfr gene codons 57, 58, and 117, with a 117N mutation frequency of 93.5%. All P. falciparum isolates exhibited double 59R + 108N mutations in pfdhfr, whereas the triple mutant 59R + 108N + 437G haplotype was found in 31.8% isolates. Furthermore, all (100%) P. falciparum isolates exhibited the key chloroquine resistance mutation, pfcrt 76T, which is also associated with resistance to amodiaquine. Additionally, pfmdr1 86Y and D1042Y mutations were, respectively, detected in 32% and 9% isolates. These results indicate an emerging multi-drug resistance problem in P. vivax and P. falciparum malaria parasites in Pakistan.