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Abstract Background As an alternative to PCR methods, LAMP is increasingly being used in the field of molecular diagnostics. Under isothermal conditions at 65 °C, the entire procedure takes approximately 30 min to complete. In this study, we establish a sensitive and visualized LAMP method in a closed-tube system for the detection of Plasmodium knowlesi. Methods A total of 71 malaria microscopy positive blood samples collected in blood spots were obtained from the Sarawak State Health Department. Using 18s rRNA as the target gene, nested PCR and SYBR green I LAMP assay were performed following the DNA extraction. The colour changes of LAMP end products were observed by naked eyes. Results LAMP assay demonstrated a detection limit of 10 copies/µL in comparison with 100 copies/µL nested PCR. Of 71 P. knowlesi blood samples collected, LAMP detected 69 microscopy-positive samples. LAMP exhibited higher sensitivity than nested PCR assay. The SYBR green I LAMP assay was 97.1% sensitive (95% CI 90.2–99.7%) and 100% specific (95% CI 83.2–100%). Without opening the cap, incorporation of SYBR green I into the inner cap of the tube enabled the direct visualization of results upon completion of amplification. The positives instantaneously turned green while the negatives remained orange. Conclusions These results indicate that SYBR green I LAMP assay is a convenient diagnosis tool for the detection of P. knowlesi in remote settings.

Plasmodium knowlesi is an emerging species for malaria in Malaysia, particularly in East Malaysia. This infection contributes to almost half of all malaria cases and deaths in Malaysia and poses a challenge in eradicating malaria. The aim of this study was to develop a predictive model for P. knowlesi susceptibility areas in Sabah, Malaysia, using geospatial data and artificial neural networks (ANNs). Weekly malaria cases from 2013 to 2014 were used to identify the malaria hotspot areas. The association of malaria cases with environmental factors (elevation, water bodies, and population density, and satellite images providing rainfall, land surface temperature, and normalized difference vegetation indices) were statistically determined. The significant environmental factors were used as input for the ANN analysis to predict malaria cases. Finally, the malaria susceptibility index and zones were mapped out. The results suggested integrating geospatial data and ANNs to predict malaria cases, with overall correlation coefficient of 0.70 and overall accuracy of 91.04%. From the malaria susceptibility index and zoning analyses, it was found that areas located along the Crocker Range of Sabah and the East part of Sabah were highly susceptible to P. knowlesi infections. Following this analysis, targetted entomological mapping and malaria control programs can be initiated. [ABSTRACT FROM AUTHOR]

Abstract Objective Acute kidney injury (AKI) is a frequent presentation in malaria infections. Several cases of AKI that are accompanied by clinical symptoms of malaria infection, such as fever, nausea, respiratory distress, and anemia remain undiagnosed due to challenges in accurate diagnosis using peripheral blood microscopy and rapid diagnostic tests that are currently used in clinical settings. This is particularly true for P. vivax and P. knowlesi infections. As a result, these patients are not able to receive anti-malarial therapy in a timely manner. The objective of the present study was to investigate if patients presenting with AKI harbored any of the five human Plasmodium species (P. falciparum, P. vivax, P. knowlesi, P. malariae, and P. ovale) within their renal tissues. Results We found that renal biopsies from malaria associated AKI patients harbor the human malaria parasites P. falciparum, P. vivax and P. knowlesi as mono- and mixed species infections. Presence of microvascular injury in a majority of the malaria associated AKI cases suggested vascular involvement of P. vivax and P. knowlesi. This research note also highlights P. knowlesi as an emerging pathogen in the Indian subcontinent.

Osteoprotegerin (OPG) is a soluble decoy receptor for receptor activator of NF-ƙB ligand (RANKL) and TNF-related apoptosis-inducing ligand (TRAIL), and is increasingly recognised as a marker of poor prognosis in a number of diseases. Here we demonstrate that in Malaysian adults with falciparum and vivax malaria, OPG is increased, and its ligands TRAIL and RANKL decreased, in proportion to disease severity. In volunteers experimentally infected with P. falciparum and P. vivax , RANKL was suppressed, while TRAIL was unexpectedly increased, suggesting binding of OPG to RANKL prior to TRAIL. We also demonstrate that P. falciparum stimulates B cells to produce OPG in vitro , and that B cell OPG production is increased ex vivo in patients with falciparum, vivax and knowlesi malaria. Our findings provide further evidence of the importance of the OPG/RANKL/TRAIL pathway in pathogenesis of diseases involving systemic inflammation, and may have implications for adjunctive therapies. Further evaluation of the role of B cell production of OPG in host responses to malaria and other inflammatory diseases is warranted., Competing Interests: Declaration of Interests None of the authors have conflicts of interest to declare.

Background: As an alternative to PCR methods, LAMP is increasingly being used in the field of molecular diagnostics. Under isothermal conditions at 65 °C, the entire procedure takes approximately 30 min to complete. In this study, we establish a sensitive and visualized LAMP method in a closed-tube system for the detection of Plasmodium knowlesi. Methods: A total of 71 malaria microscopy positive blood samples collected in blood spots were obtained from the Sarawak State Health Department. Using 18s rRNA as the target gene, nested PCR and SYBR green I LAMP assay were performed following the DNA extraction. The colour changes of LAMP end products were observed by naked eyes. Results: LAMP assay demonstrated a detection limit of 10 copies/µL in comparison with 100 copies/µL nested PCR. Of 71 P. knowlesi blood samples collected, LAMP detected 69 microscopy-positive samples. LAMP exhibited higher sensitivity than nested PCR assay. The SYBR green I LAMP assay was 97.1% sensitive (95% CI 90.2–99.7%) and 100% specific (95% CI 83.2–100%). Without opening the cap, incorporation of SYBR green I into the inner cap of the tube enabled the direct visualization of results upon completion of amplification. The positives instantaneously turned green while the negatives remained orange. Conclusions: These results indicate that SYBR green I LAMP assay is a convenient diagnosis tool for the detection of P. knowlesi in remote settings. [ABSTRACT FROM AUTHOR]

Background Malaysia aims to eliminate malaria by 2020. However, while cases of Plasmodium falciparum and Plasmodium vivax have decreased substantially, the incidence of zoonotic malaria from Plasmodium knowlesi continues to increase, presenting a major challenge to regional malaria control efforts. Here we report incidence of all Plasmodium species in Sabah, including zoonotic P. knowlesi , during 2015–2017. Methods Microscopy-based malaria notification data and polymerase chain reaction (PCR) results were obtained from the Sabah Department of Health and State Public Health Laboratory, respectively, from January 2015 to December 2017. From January 2016 this was complemented by a statewide prospective hospital surveillance study. Databases were matched, and species was determined by PCR, or microscopy if PCR was not available. Results A total of 3867 malaria cases were recorded between 2015 and 2017, with PCR performed in 93%. Using PCR results, and microscopy if PCR was unavailable, P. knowlesi accounted for 817 (80%), 677 (88%), and 2030 (98%) malaria cases in 2015, 2016, and 2017, respectively. P. falciparum accounted for 110 (11%), 45 (6%), and 23 (1%) cases and P. vivax accounted for 61 (6%), 17 (2%), and 8 (0.4%) cases, respectively. Of those with P. knowlesi , the median age was 35 (interquartile range: 24–47) years, and 85% were male. Conclusions Malaysia is approaching elimination of the human-only Plasmodium species. However, the ongoing increase in P. knowlesi incidence presents a major challenge to malaria control and warrants increased focus on knowlesi-specific prevention activities. Wider molecular surveillance in surrounding countries is required. [ABSTRACT FROM AUTHOR]

Objective: Acute kidney injury (AKI) is a frequent presentation in malaria infections. Several cases of AKI that are accompanied by clinical symptoms of malaria infection, such as fever, nausea, respiratory distress, and anemia remain undiagnosed due to challenges in accurate diagnosis using peripheral blood microscopy and rapid diagnostic tests that are currently used in clinical settings. This is particularly true for P. vivax and P. knowlesi infections. As a result, these patients are not able to receive anti-malarial therapy in a timely manner. The objective of the present study was to investigate if patients presenting with AKI harbored any of the five human Plasmodium species (P. falciparum, P. vivax, P. knowlesi, P. malariae, and P. ovale) within their renal tissues. Results: We found that renal biopsies from malaria associated AKI patients harbor the human malaria parasites P. falciparum, P. vivax and P. knowlesi as mono- and mixed species infections. Presence of microvascular injury in a majority of the malaria associated AKI cases suggested vascular involvement of P. vivax and P. knowlesi. This research note also highlights P. knowlesi as an emerging pathogen in the Indian subcontinent. [ABSTRACT FROM AUTHOR]

Background Plasmodium knowlesi causes severe and fatal malaria, and incidence in Southeast Asia is increasing. Factors associated with death are not clearly defined. Methods All malaria deaths in Sabah, Malaysia, from 2015 to 2017 were identified from mandatory reporting to the Sabah Department of Health. Case notes were reviewed, and a systematic review of these and all previously reported fatal P. knowlesi cases was conducted. Case fatality rates (CFRs) during 2010–2017 were calculated using incidence data from the Sabah Department of Health. Results Six malaria deaths occurred in Sabah during 2015–2017, all from P. knowlesi. Median age was 40 (range, 23–58) years; 4 cases (67%) were male. Three (50%) had significant cardiovascular comorbidities and 1 was pregnant. Delays in administering appropriate therapy contributed to 3 (50%) deaths. An additional 26 fatal cases were included in the systematic review. Among all 32 cases, 18 (56%) were male; median age was 56 (range, 23–84) years. Cardiovascular-metabolic disease, microscopic misdiagnosis, and delay in commencing intravenous treatment were identified in 11 of 32 (34%), 26 of 29 (90%), and 11 of 31 (36%) cases, respectively. The overall CFR during 2010–2017 was 2.5/1000: 6.0/1000 for women and 1.7/1000 for men (P =.01). Independent risk factors for death included female sex (odds ratio, 2.6; P =.04), and age ≥45 years (odds ratio, 4.7; P <.01). Conclusions Earlier presentation, more rapid diagnosis, and administration of intravenous artesunate may avoid fatal outcomes, particularly in females, older adults, and patients with cardiovascular comorbidities. [ABSTRACT FROM AUTHOR]

In 2004, a large focus of human malaria infection in Malaysian Borneo alerted the world community about the emergence of Plasmodium knowlesi as a new threat to public health. It is a zoonotic disease transmitted primarily between nonhuman primate hosts by the Anopheles mosquitoes. At present it has become endemic in most of the South East Asian countries and human cases are being reported on a regular basis. The importance of this species lies in the fact that similar to P.falciparum, it can cause high parasitaemia and severe malaria which may require hospitalization. This review deals with the history, epidemiology, pathogenesis, clinical aspects, diagnosis, and treatment of P.knowlesi malaria, and the potential threat of this parasite for India.

In 2004, a large focus of human malaria infection in Malaysian Borneo alerted the world community about the emergence of Plasmodium knowlesi as a new threat to public health. It is a zoonotic disease transmitted primarily between nonhuman primate hosts by the Anopheles mosquitoes. At present it has become endemic in most of the South East Asian countries and human cases are being reported on a regular basis. The importance of this species lies in the fact that similar to P.falciparum, it can cause high parasitaemia and severe malaria which may require hospitalization. This review deals with the history, epidemiology, pathogenesis, clinical aspects, diagnosis, and treatment of P.knowlesi malaria, and the potential threat of this parasite for India. [ABSTRACT FROM AUTHOR]

The Duffy antigen receptor for chemokine (DARC) is a nonspecific receptor for several proinflammatory cytokines. It is homologous to the G-protein chemokine receptor superfamily, which is suggested to function as a scavenger in many inflammatory-and proinflammatory- related diseases. G-protein chemokine receptors are also known to play a critical role in infectious diseases; they are commonly used as entry vehicles by infectious agents. A typical example is the chemokine receptor CCR5 or CXCR4 used by HIV for infecting target cells. In malaria, DARC is considered an essential receptor that mediates the entry of the human and zoonotic malaria parasites Plasmodium vivax and Plasmodium knowlesi into human reticulocytes and erythrocytes, respectively. This process is mediated through interaction with the parasite ligand known as the Duffy binding protein (DBP). Most therapeutic strategies have been focused on blocking the interaction between DBP and DARC by targeting the parasite ligand, while strategies targeting the receptor, DARC, have not been intensively investigated. The rapid increase in drug resistance and the lack of new effective drugs or a vaccine for malaria constitute a major threat and a need for novel therapeutics to combat disease. This review explores strategies that can be used to target the receptor. Inhibitors of DARC, which block DBP-DARC interaction, can potentially provide an effective strategy for preventing malaria caused by P. vivax. [ABSTRACT FROM AUTHOR]

Ten years have passed since the publication of a large focus of Plasmodium knowlesi infections in the human population. The discovery was made during a molecular investigation of atypical P. malariae cases in the Kapit Health Division, Sarawak, Malaysian Borneo. Patients were more symptomatic with higher parasite counts than expected in P. malariae infections. The investigation found only P. knowlesi DNA present in patient blood samples. Morphological similarity had allowed P. knowlesi to masquerade as P. malariae during routine diagnostic microscopy for malaria. P. knowlesi, a malaria parasite of macaque monkeys, had entered the human population. The subsequent development of P. knowlesi species-specific PCR assays soon demonstrated that the entry was not confined to the Kapit Division but extended across island and mainland Southeast Asia. Relevant clinical descriptions and guidelines for the treatment and management of patents with P. knowlesi malaria were not available. Nor was it clear whether P. knowlesi had undergone a host switch event into the human population or if infections were zoonotic. The outputs of studies on P. knowlesi malaria during the past 10 years will be summarized, highlighting major findings within the context of pathophysiology, virulence, host switch events, treatment, control and importantly malaria elimination. [ABSTRACT FROM AUTHOR]

All Plasmodium species produce a brown birefringent crystal known as malarial pigment or hemozoin. This work compares the morphology of hemozoin from P. falciparum, P. vivax, P. ovale, P. malariae, P. knowlesi, P. brasilianum, P. yoelii and P. gallinaceum. The human, primate and mouse hemozoins have a regular, flat-faced cuboidal morphology with modest size differences in contrast to the larger, regularly irregular barrel shape with a waffle surface of the avian, P. gallinaceum, pigment. Hydrogen peroxide (H2O2), as a biochemical test reagent, can distinguish the hemozoins by different concentrations to degrade half of the crystals. A surface area to volume ratio explains both the appearance and susceptibility to H2O2 degradation. The hemozoin from each species is able to be a template for hemozoin extension inhibitable by the quinolines. P. gallinaceum hemozoin more closely resembles the hemozoin from another avian apicomplexan, Haemoproteus, rather than the hemozoin from the mammalian malaria species. These distinct morphological characteristics between mammalian and avian crystals suggest different biochemical environments that affect morphology. [Copyright &y& Elsevier]