White blood cell differential fluorescence abnormal scattergram: A useful indicator for early detection of malarial parasite

Background & Objective: Undiagnosed malarial infectionis associated with significant mortality and morbidity. Laboratory investigations leading to rapid, accurate and timely diagnosis of malaria is still a challenge. This study was done to assess the utility of abnormal White blood cell differential fluorescence (WDF) scattergram for diagnosis of malaria. Our aim was to study the utility of WDF scattergram for early detection of malarial parasite. Methods: All EDTA anti-coagulated blood samples received in laboratory during a period from Dec 2019 to May 2020 were analyzed on anautomated hematology analyzer, Sysmex XN 1000. All abnormal WDF scattergrams pertaining to plasmodium specie were critically evaluated and recorded. Review of Leishman-stained peripheral smears as well as immune-chromatographic assay by rapid test devices (RTD) was done. Accordingly, sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) for detection of malaria by abnormal scattergram were calculated. Results: Out of total 1, 26,000of samples analyzed, abnormal WDF scattergrams were detected in 96 cases. Amongst these, 95.8% (92) were positive for MP on Leishman-stained peripheral smear as well as on ICT with a p-value of 0.05. WDF scattergram abnormalities typical of malaria showed a sensitivity of 80% and specificity of 93.26%. Positive predictive value of 95.8% whereas negative predictive value of 99.9% was detected. Significant findings of hemolysis, platelet clumps, nucleated RBCS (NRBCs) and RBC agglutination were noted in cases (n=4) with abnormal WDF scattergram negative for malaria on peripheral smear. Conclusion: Interpretation of abnormal WDF scattergram not only increases the early detection rate for malarial parasite but isa strong indicator for presence of hemolysis, RBC agglutination, platelet clumps and leucoerythroblastic blood picture as well. doi: https://doi.org/10.12669/pjms.38.3.4702 How to cite this:Rehan M, Khalid A, Nasreen F. White blood cell differential fluorescence abnormal scattergram: A useful indicator for early detection of malarial parasite. Pak J Med Sci. 2022;38(3):---------. doi: https://doi.org/10.12669/pjms.38.3.4702 This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Pseudoeosinophilia with Abnormal WBC Scattergram: an Important Diagnostic Clue on Hematology Analyzer

Malaria is one of the most common parasitic diseases causing a significant burden on health care, especially in India. Light microscopy, the gold standard for malaria diagnosis is time-consuming especially in a setup where the caseload is high and parasite index is low. Knowledge of abnormal scattergrams by the malarial parasite is very essential to suspect malaria and screen those cases thoroughly on a peripheral smear for accurate diagnosis. A timely and accurate diagnosis is crucial to the prognosis of this disease. Herein, we report a case of a 40-year-old male from Punjab who presented to the emergency with fever and an altered sensorium. Analyzer showed characteristic pseudo eosinophilia with greying of neutrophil eosinophil zone with double eosinophil cluster. According to flagging rules because of eosinophilia, peripheral blood film (PBF) was made and examined, which revealed Plasmodium falciparum gametocytes.

Absence of PEXEL-Dependent Protein Export in Plasmodium Liver Stages Cannot Be Restored by Gain of the HSP101 Protein Translocon ATPase

Host cell remodeling is critical for successful Plasmodium replication inside erythrocytes and achieved by targeted export of parasite-encoded proteins. In contrast, during liver infection the malarial parasite appears to avoid protein export, perhaps to limit exposure of parasite antigens by infected liver cells. HSP101, the force-generating ATPase of the protein translocon of exported proteins (PTEX) is the only component that is switched off during early liver infection. Here, we generated transgenic Plasmodium berghei parasite lines that restore liver stage expression of HSP101. HSP101 expression in infected hepatocytes was achieved by swapping the endogenous promoter with the ptex150 promoter and by inserting an additional copy under the control of the elongation one alpha (ef1α) promoter. Both promoters drive constitutive and, hence, also pre-erythrocytic expression. Transgenic parasites were able to complete the life cycle, but failed to export PEXEL-proteins in early liver stages. Our results suggest that PTEX-dependent early liver stage export cannot be restored by addition of HSP101, indicative of alternative export complexes or other functions of the PTEX core complex during liver infection.

Hematological changes in patients with malaria in a tertiary care hospital, Multan, Punjab, Pakistan

Background: Malaria is one of the most common human infections and continues to cause significant morbidity and mortality all over the world. To assess and compare the hematological changes in common types of malaria in our patients.Methods: This observational study included 100 diagnosed malaria patients of Multan Medical and Dental college, Multan both from out patient department (OPD) and in-patient department, between March 2020 and March 2021. The diagnosis of malaria was confirmed by thick and thin film stained with Geimsa’s staining for malaria parasite and plasmodium species and the parasite index (MPI) in some cases. Complete blood counts (CBCs) were performed and WBC differential was done on all cases.Results: The most common type of malarial parasite was Plasmodium vivax followed by Plasmodium falciparum and 89% of the patient had thrombocytopenia, 70 % anemia, 23% had leukopenia and 10% had raised WBC count. The mean parasite load was 1.275±0.629%, 20 % of the cases showed neutropenia, 40 % had lymphopenia and 40 % showed monocytopenia. Of all the cases 15 % had neutrophilia, 8 % had lymphocytosis and 5 % had eosinophilia. Thrombocytopenia was slightly more in P. falciparum (58.69%) than P. vivax (30.18%) cases, p>0.05, whereas there was no significant difference in the incidence of anemia in two groups (34.68% vs 33.82%) with p>0.05.Conclusions: P. vivax is the common malarial parasite in our population. Both P. vivax and P. falciparum can cause marked hematological changes including thrombocytopenia, anemia, lymphopenia and monocytopenia.

Insights into the Relation between Oxidative Stress and Malaria: A Mechanistic and Therapeutic Approach

The mortality rate due to malaria has increased tremendously in the last decade. Even though the causative agent of this disease is known, the preventive measures are not potent enough to control the spread of this disease. Malarial infection involves a strong interrelationship between oxidative stress and pathogenesis. This review addresses the various oxidative stress-related mechanisms associated with vector defense, host immunity, plasmodial pathogenesis, and corresponding therapeutic strategies. The mechanisms involving host and vector defense show both similarity and contradiction to the processes involving plasmodial pathogenesis under different circumstances. Therefore, corresponding ameliorative peculiarities are observed in the therapeutic mechanisms adopted by the anti-malarial drugs. The malarial parasite augments oxidative stress to weaken the host and exerts antioxidant effects against host defense mechanisms. However, the anti-malarial drugs induce oxidative insult to reduce parasitic load and exert antioxidant effects against parasite infection-induced oxidative stress in host. Thus, the anti-malarial drugs exhibit antioxidant activity in hosts and/or pro-oxidant activity in parasites.

Global diversity of the gene encoding the Pfs25 protein—a Plasmodium falciparum transmission-blocking vaccine candidate

Background Vaccines against the sexual stages of the malarial parasite Plasmodium falciparum are indispensable for controlling malaria and abrogating the spread of drug-resistant parasites. Pfs25, a surface antigen of the sexual stage of P. falciparum, is a leading candidate for transmission-blocking vaccine development. While clinical trials have reported that Pfs25-based vaccines are safe and effective in inducing transmission-blocking antibodies, the extent of the genetic diversity of Pfs25 in malaria endemic populations has rarely been studied. Thus, this study aimed to investigate the global diversity of Pfs25 in P. falciparum populations. Methods A database of 307 Pfs25 sequences of P. falciparum was established. Population genetic analyses were performed to evaluate haplotype and nucleotide diversity, analyze haplotypic distribution patterns of Pfs25 in different geographical populations, and construct a haplotype network. Neutrality tests were conducted to determine evidence of natural selection. Homology models of the Pfs25 haplotypes were constructed, subjected to molecular dynamics (MD), and analyzed in terms of flexibility and percentages of secondary structures. Results The Pfs25 gene of P. falciparum was found to have 11 unique haplotypes. Of these, haplotype 1 (H1) and H2, the major haplotypes, represented 70% and 22% of the population, respectively, and were dominant in Asia, whereas only H1 was dominant in Africa, Central America, and South America. Other haplotypes were rare and region-specific, resulting in unique distribution patterns in different geographical populations. The diversity in Pfs25 originated from ten single-nucleotide polymorphism (SNP) loci located in the epidermal growth factor (EGF)-like domains and anchor domain. Of these, an SNP at position 392 (GGA/GCA), resulting in amino acid substitution 131 (Gly/Ala), defined the two major haplotypes. The MD results showed that the structures of H1 and H2 variants were relatively similar. Limited polymorphism in Pfs25 could likely be due to negative selection. Conclusions The study successfully established a Pfs25 sequence database that can become an essential tool for monitoring vaccine efficacy, designing assays for detecting malaria carriers, and conducting epidemiological studies of P. falciparum. The discovery of the two major haplotypes, H1 and H2, and their conserved structures suggests that the current Pfs25-based vaccines could be used globally for malaria control. Graphical Abstract

Clinico-Epidemiological Studies of Plasmodium Falciparum and Salmonella Typhi Co-Infection among Patients Attending Selected General Hospital in Northern Nigeria

Study on the prevalence of co-infection between Plasmodium falciparum and Salmonella typhi among patients in Northern Nigeria was carried out. The study is cross-sectional designed to determine the socio-demographic characteristics as well as the risk factors for malaria and typhoid. A total of 100 consented patients of age group of 21-40 years were recruited for the study. A structured questionnaire was administered, and venous blood samples were collected and analyzed using standard microbiological methods. The isolated salmonella species were biochemically characterized, and subjected to antimicrobial susceptibility test using Kirby-Bauer disc diffusion method. The prevalence of malaria and typhoid was found to be 56% and 68% respectively. The prevalence of malarial parasite and Salmonella typhi infections was 40%. Females recorded low malarial infection of 56.9% compared to their male counterparts 43.1% (P= 0.510). The age group, educational levels and occupations of the study participants were not associated with the likelihood of having malarial parasite infection (P= 0.297, 0.15 and 0.503 respectively). Participants who did not sleep under the insecticide treated nets were more likely to have malaria than those who did (P ≤ 0.0001). The educational levels of the study participants were statistically associated with Salmonella typhi infection (P= 0.026). Water sources, use of pit latrine, hand washing before and after meal were significantly associated with Salmonella typhi infections (P= <0.0001 and P= 0.003 respectively). The isolates of Salmonella typhi and Salmonella paratyphi were found to be sensitive to chloramphenicol (86.8%), ciprofloxacin (80.9%) and amoxicillin (79.4%), but relatively resistant to penicillin and augmentin that recorded sensitivities of 19.1% and 35.3% respectively. The prevalence of malaria and typhoid infections as well as malarial parasite and Salmonella typhi co-infections is high among the study population. Fortunately, the isolated bacteria are highly sensitive to chloramphenicol and ciprofloxacin.

Computational and experimental studies of plasmodium falciparum protein PfAMA1 domain-II loop dynamics: implications in PfAMA1-PfRON2 binding event

Protein-protein interactions are interesting targets for various drug discovery campaigns. One such promising and therapeutically pertinent protein-protein complex is PfAMA1-PfRON2, which is involved in malarial parasite invasion into human red blood cells. A thorough understanding of the interactions between these macromolecular binding partners is absolutely necessary to design better therapeutics to fight against the age-old disease affecting mostly under-developed nations. Although crystal structures of several PfAMA1-PfRON2 complexes have been solved to understand the molecular interactions between these two proteins, the mechanistic aspects of the domain II loop-PfRON2 association is far from clear. The current work investigates a crucial part of the recognition event; i.e., how the domain II loop of PfAMA1 exerts its effect on the alpha helix of the PfRON2, thus influencing the overall kinetics of this intricate recognition phenomenon. To this end, we have conducted thorough computational investigation of the dynamics and free energetics of domain II loop closing processes using molecular dynamics simulation. The computational results are validated by systematic alanine substitutions of the PfRON2 peptide helix. The subsequent evaluation of the binding affinity of Ala-substituted PfRON2 peptide ligands by surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC) provides a rank of the relative importance of the residues in context. Our combined (computational and experimental) investigation has revealed that the domain II loop of PfAMA1 is in fact responsible for arresting the PfRON2 molecule from egress, K2027 and D2028 of PfRON2 being the determinant residues for the capturing event. Our study provides a comprehensive understanding of the molecular recognition event between PfAMA1 and PfRON2, specifically in the post binding stage, which potentially can be utilized for drug discovery against malaria.

Structures of plasmepsin X from P. falciparum reveal a novel inactivation mechanism of the zymogen and molecular basis for binding of inhibitors in mature enzyme

Plasmodium falciparum plasmepsin X (PfPMX), involved in the invasion and egress of this deadliest malarial parasite, is essential for its survival and hence considered as an important drug target. We report the first crystal structure of PfPMX zymogen containing a novel fold of its prosegment. A unique twisted loop from the prosegment and arginine 244 from the mature enzyme are involved in zymogen inactivation; such mechanism, not previously reported, might be common for apicomplexan proteases similar to PfPMX. The maturation of PfPMX zymogen occurs through cleavage of its prosegment at multiple sites. Our data provide thorough insights into the mode of binding of a substrate and a potent inhibitor 49c to PfPMX. We present molecular details of inactivation, maturation, and inhibition of PfPMX that should aid in the development of potent inhibitors against pepsin-like aspartic proteases from apicomplexan parasites.

Hemozoin (beta-hematin) formation inhibitors; A promising target for the development of new antimalarials: Current update and A future prospect

Background: Malaria is responsible for a social and an economic burden in most low-income malaria-affected countries. Thus, newer antimalarials are needed to tackle morbidities and mortalities associated with the drug-resistant malarial strains. Haemoglobin digestion inside the food vacuole of malarial parasite would lead to producing redox-active and toxic-free heme. The detoxification process adopted by Plasmodium sp. would give rise to hemozoin (Hz) (beta-hematin) formation. Targeting the pathway of hemozoin formation is considered as a validated target for the discovery of newer antimalarials. Objective: This study aims to collect detailed information about aspects of hemozoin (Hz) (beta-hematin) inhibitors. Methods: A systemic search has been carried out using PubMed, Google Scholar, CNKI, etc., for relevant studies having the keyword, ' hemozoin or beta-hematin' for almost the last 2 decades (2000-2021). Results: This mini-review tries to summarize all the recent advancements made for the developments of synthetic, natural isolated phytoconstituents and plant extracts inhibiting the hemozoin (beta-hematin) formation. Conclusion: thus, would act as promising antimalarial candidates in near future.