BackgroundThe genetic diversity of Plasmodium falciparum has been extensively studied in various parts of the world. However, limited data are available from Pakistan. This study aimed to establish molecular characterization of P. falciparum field isolates in Pakistan measured with two highly polymorphic genetic markers, i.e. the merozoite surface protein 1 (msp-1)and 2 (msp-2).MethodsBetween October 2005 and October 2007, 244 blood samples from patients with symptomatic blood-slide confirmed P. falciparum mono-infections attending the Aga Khan University Hospital, Karachi, or its collection units located in Sindh and Baluchistan provinces, Pakistan were collected. The genetic diversity of P. falciparum was analysed by length polymorphism following gel electrophoresis of DNA products from nested polymerase chain reactions (PCR) targeting block 2 of msp-1 and block 3 of msp-2, including their respective allelic families KI, MAD 20, RO33, and FC27, 3D7/IC.ResultsA total of 238/244 (98%) patients had a positive PCR outcome in at least one genetic marker; the remaining six were excluded from analysis. A majority of patients had monoclonal infections. Only 56/231 (24%) and 51/236 (22%) carried multiple P. falciparum genotypes in msp-1 and msp-2, respectively. The estimated total number of genotypes was 25 msp-1 (12 KI; 8 MAD20; 5 RO33) and 33 msp-2 (14 FC27; 19 3D7/IC).ConclusionsThis is the first report on molecular characterization of P. falciparum field isolates in Pakistan with regards to multiplicity of infection. The genetic diversity and allelic distribution found in this study is similar to previous reports from India and Southeast Asian countries with low malaria endemicity.

To compare the severity of Plasmodium vivax malaria with that of P. falciparum malaria, we conducted a retrospective cross-sectional study of 356 adults hospitalized with malaria (2009–2011) in Pakistan. P. vivax and P. falciparum accounted for 83% and 13% of cases, respectively; 79.9% of patients with severe malaria were infected with P. vivax.

BackgroundPlasmodium vivax is the prevalent malarial species accounting for 70% of malaria burden in Pakistan; however, there is no baseline data on the circulating genotypes. Studies have shown that polymorphic loci of gene encoding antigens pvcsp and pvmsp1 can be used reliably for conducting molecular epidemiological studies. Therefore, this study aimed to bridge the existing knowledge gap on population structure on P. vivax from Pakistan using these two polymorphic genes.MethodsDuring the period January 2008 to May 2009, a total of 250 blood samples were collected from patients tested slide positive for P. vivax, at the Aga Khan University Hospital, Karachi, or its collection units located in Baluchistan and Sindh Province. Nested PCR/RFLP was performed, using pvcsp and pvmsp1 markers to detect the extent of genetic diversity in clinical isolates of P. vivax from southern Pakistan.ResultsA total of 227/250 (91%) isolates were included in the analysis while the remainder were excluded due to negative PCR outcome for P.vivax. Pvcsp analysis showed that both VK 210 (85.5%, 194/227) and VK 247 type (14.5%, 33/227) were found to be circulating in P. vivax isolates from southern Pakistan. A total of sixteen and eighty-seven genotypes of pvcsp and pvmsp-1 were detected respectively.ConclusionThis is the first report from southern Pakistan on characterization of P. vivax isolates confirming that extensively diverse pvcsp and pvmsp1 variants are present within this region. Results from this study provide valuable data on genetic diversity of P. vivax that will be helpful for further epidemiological studies.

Understanding key host protective mechanisms against SARS-CoV-2 infection can help improve treatment modalities for COVID-19. We used a blood transcriptome approach to study biomarkers associated with differing severity of COVID-19, comparing severe and mild Symptomatic disease with Asymptomatic COVID-19 and uninfected Controls. There was suppression of antigen presentation but upregulation of inflammatory and viral mRNA translation associated pathways in Symptomatic as compared with Asymptomatic cases. In severe COVID-19, CD177 a neutrophil marker, was upregulated while interferon stimulated genes (ISGs) were downregulated. Asymptomatic COVID-19 cases displayed upregulation of ISGs and humoral response genes with downregulation of ICAM3 and TLR8. Compared across the COVID-19 disease spectrum, we found type I interferon (IFN) responses to be significantly upregulated (IFNAR2, IRF2BP1, IRF4, MAVS, SAMHD1, TRIM1), or downregulated (SOCS3, IRF2BP2, IRF2BPL) in Asymptomatic as compared with mild and severe COVID-19, with the dysregulation of an increasing number of ISGs associated with progressive disease. These data suggest that initial early responses against SARS-CoV-2 may be effectively controlled by ISGs. Therefore, we hypothesize that treatment with type I interferons in the early stage of COVID-19 may limit disease progression by limiting SARS-CoV-2 in the host.

BackgroundScarce data are available on Plasmodium falciparum anti-malarial drug resistance in Pakistan. The aim of this study was, therefore, to determine the prevalence of P. falciparum resistance associated polymorphisms in field isolates from southern Pakistan.MethodsBlood samples from 244 patients with blood-slide confirmed P. falciparum mono-infections were collected between 2005-2007. Single nucleotide polymorphisms in the P. falciparum chloroquine resistance transporter (pfcrt K76T), multi drug resistance (pfmdr1 N86Y), dihydrofolate reductase (pfdhfr A16V, N51I, C59R, S108N, I164L) and dihydropteroate synthetase (pfdhps A436S, G437A and E540K) genes and pfmdr1 gene copy numbers were determined using PCR based methods.ResultsThe prevalence of pfcrt 76T and pfmdr1 86Y was 93% and 57%, respectively. The prevalence of pfdhfr double mutations 59R + 108N/51R + 108N was 92%. The pfdhfr triple mutation (51I, 59R, 108N) occurred in 3% of samples. The pfdhfr (51I, 59R, 108N) and pfdhps (437G, 540E) quintuple mutation was found in one isolate. Pfdhps 437G was observed in 51% and 540E in 1% of the isolates. One isolate had two pfmdr1 copies and carried the pfmdr1 86Y and pfcrt 76T alleles.ConclusionsThe results indicate high prevalence of in vivo resistance to chloroquine, whereas high grade resistance to sulphadoxine-pyrimethamine does not appear to be widespread among P. falciparum in southern Pakistan.

BackgroundCytokine-mediated endothelial activation pathway is a known mechanism of pathogenesis employed by Plasmodium falciparum to induce severe disease symptoms in human host. Though considered benign, complicated cases of Plasmodium vivax are being reported worldwide and from Pakistan. It has been hypothesized that P.vivax utilizes similar mechanism of pathogenesis, as that of P.falciparum for manifestations of severe malaria. Therefore, the main objective of this study was to characterize the role of cytokines and endothelial activation markers in complicated Plasmodium vivax isolates from Pakistan.Methods and Principle FindingsA case control study using plasma samples from well-characterized groups suffering from P.vivax infection including uncomplicated cases (n=100), complicated cases (n=82) and healthy controls (n=100) were investigated. Base line levels of Tumor necrosis factor-α (TNF-α), Interleukin-6 (IL-6), Interleukin-10 (IL-10), Intercellular adhesion molecule-1 (ICAM-1), Vascular adhesion molecule-1(VCAM-1) and E-selectin were measured by ELISA. Correlation of cytokines and endothelial activation markers was done using Spearman’s correlation analysis. Furthermore, significance of these biomarkers as indicators of disease severity was also analyzed. The results showed that TNF-α, IL-10, ICAM-1and VCAM-1 were 3-fold, 3.7 fold and 2 fold increased between uncomplicated and complicated cases. Comparison of healthy controls with uncomplicated cases showed no significant difference in TNF-α concentrations while IL-6, IL-10, ICAM-1, VCAM-1 and E-selectin were found to be elevated respectively. In addition, significant positive correlation was observed between TNF-α and IL-10/ ICAM-1, IL-6 and IL-10, ICAM-1 and VCAM-1.A Receiver operating curve (ROC) was generated which showed that TNF-α, IL-10, ICAM-1 and VCAM-1 were the best individual predictors of complicated P.vivax malaria.ConclusionThe results suggest that though endothelial adhesion molecules are inducible by pro-inflammatory cytokine TNF-α, however, cytokine-mediated endothelial activation pathway is not clearly demonstrated as a mechanism of pathogenesis in complicated P.vivax malaria cases from Pakistan.

The Asia–Pacific region faces formidable challenges in achieving malaria elimination by the proposed target in 2030. Molecular surveillance of Plasmodium parasites can provide important information on malaria transmission and adaptation, which can inform national malaria control programmes (NMCPs) in decision-making processes. In November 2019 a parasite genotyping workshop was held in Jakarta, Indonesia, to review molecular approaches for parasite surveillance and explore ways in which these tools can be integrated into public health systems and inform policy. The meeting was attended by 70 participants from 8 malaria-endemic countries and partners of the Asia Pacific Malaria Elimination Network. The participants acknowledged the utility of multiple use cases for parasite genotyping including: quantifying the prevalence of drug resistant parasites, predicting risks of treatment failure, identifying major routes and reservoirs of infection, monitoring imported malaria and its contribution to local transmission, characterizing the origins and dynamics of malaria outbreaks, and estimating the frequency of Plasmodium vivax relapses. However, the priority of each use case varies with different endemic settings. Although a one-size-fits-all approach to molecular surveillance is unlikely to be applicable across the Asia–Pacific region, consensus on the spectrum of added-value activities will help support data sharing across national boundaries. Knowledge exchange is needed to establish local expertise in different laboratory-based methodologies and bioinformatics processes. Collaborative research involving local and international teams will help maximize the impact of analytical outputs on the operational needs of NMCPs. Research is also needed to explore the cost-effectiveness of genetic epidemiology for different use cases to help to leverage funding for wide-scale implementation. Engagement between NMCPs and local researchers will be critical throughout this process.

Background We investigated the genome diversity of SARS-CoV-2 associated with the early COVID-19 period to investigate evolution of the virus in Pakistan. Materials and methods We studied ninety SARS-CoV-2 strains isolated between March and October 2020. Whole genome sequences from our laboratory and available genomes were used to investigate phylogeny, genetic variantion and mutation rates of SARS-CoV-2 strains in Pakistan. Site specific entropy analysis compared mutation rates between strains isolated before and after June 2020. Results In March, strains belonging to L, S, V and GH clades were observed but by October, only L and GH strains were present. The highest diversity of clades was present in Sindh and Islamabad Capital Territory and the least in Punjab province. Initial introductions of SARS-CoV-2 GH (B.1.255, B.1) and S (A) clades were associated with overseas travelers. Additionally, GH (B.1.255, B.1, B.1.160, B.1.36), L (B, B.6, B.4), V (B.4) and S (A) clades were transmitted locally. SARS-CoV-2 genomes clustered with global strains except for ten which matched Pakistani isolates. RNA substitution rates were estimated at 5.86 x10−4. The most frequent mutations were 5’ UTR 241C > T, Spike glycoprotein D614G, RNA dependent RNA polymerase (RdRp) P4715L and Orf3a Q57H. Strains up until June 2020 exhibited an overall higher mean and site-specific entropy as compared with sequences after June. Relative entropy was higher across GH as compared with GR and L clades. More sites were under selection pressure in GH strains but this was not significant for any particular site. Conclusions The higher entropy and diversity observed in early pandemic as compared with later strains suggests increasing stability of the genomes in subsequent COVID-19 waves. This would likely lead to the selection of site-specific changes that are advantageous to the virus, as has been currently observed through the pandemic.