Plasmodium vivax drug resistance markers: Genetic polymorphisms and mutation patterns in isolates from Malaysia.

Transmission of Plasmodium vivax still persist in Malaysia despite the government's aim to eliminate malaria in 2020. High treatment failure rate of chloroquine monotherapy was reported recently. Hence, parasite drug susceptibility should be kept under close monitoring. Mutation analysis of the drug resistance markers is useful for reconnaissance of anti-malarial drug resistance. Hitherto, information on P. vivax drug resistance marker in Malaysia are limited. This study aims to evaluate the mutations in four P. vivax drug resistance markers pvcrt-o (putative), pvmdr1 (putative), pvdhfr and pvdhps in 44 isolates from Malaysia. Finding indicates that 27.3%, 100%, 47.7%, and 27.3% of the isolates were carrying mutant allele in pvcrt-o, pvmdr1, pvdhfr and pvdhps genes, respectively. Most of the mutant isolates had multiple point mutations rather than single point mutation in pvmdr1 (41/44) and pvdhfr (19/21). One novel point mutation V111I was detected in pvdhfr. Allelic combination analysis shows significant strong association between mutations in pvcrt-o and pvmdr1 (X2 = 9.521, P < 0.05). In the present study, 65.9% of the patients are non-Malaysians, with few of them arrived in Malaysia 1-2 weeks before the onset of clinical manifestations, or had previous history of malaria infection. Besides, few Malaysian patients had travel history to vivax-endemic countries, suggesting that these patients might have acquired the infections during their travel. All these possible imported cases could have placed Malaysia in a risk to have local transmission or outbreak of malaria. Six isolates were found to have mutations in all four drug resistance markers, suggesting that the multiple-drugs resistant P. vivax strains are circulating in Malaysia.

Prevalence and associated risk factors of malaria among adults in East Shewa Zone of Oromia Regional State, Ethiopia: a cross-sectional study.

BACKGROUND: Malaria is one of the most important causes of morbidity and mortality in sub-Saharan Africa. The disease is prevalent in over 75% of the country's area making it the leading public health problems in the country. Information on the prevalence of malaria and its associated factors is vital to focus and improve malaria interventions. METHODS: A cross-sectional study was carried out from October to November 2012 in East Shewa zone of Oromia Regional State, Ethiopia. Adults aged 16 or more years with suspected malaria attending five health centers were eligible for the study. Logistic regression models were used to examine the effect of each independent variable on risk of subsequent diagnosis of malaria. RESULTS: Of 810 suspected adult malaria patients who participated in the study, 204 (25%) had microscopically confirmed malaria parasites. The dominant Plasmodium species were P. vivax (54%) and P. falciparum (45%), with mixed infection of both species in one patient. A positive microscopic result was significantly associated with being in the age group of 16 to 24 years [Adjusted Odds Ratio aOR 6.7; 95% CI: 2.3 to 19.5], 25 to 34 years [aOR 4.2; 95% CI: 1.4 to 12.4], and 35 to 44 years [aOR 3.7; 95% CI: 1.2-11.4] compared to 45 years or older; being treated at Meki health center [aOR 4.1; 95% CI: 2.4 to 7.1], being in Shashemene health center [aOR = 2.3; 95% CI: 1.5 to 4.5], and living in a rural area compared to an urban area [aOR 1.7; 95% CI: 1.1 to 2.6)]. CONCLUSION: Malaria is an important public health problem among adults in the study area with a predominance of P. vivax and P. falciparum infection. Thus, appropriate health interventions should be implemented to prevent and control the disease.

The Aotus nancymaae erythrocyte proteome and its importance for biomedical research.

The Aotus nancymaae species has been of great importance in researching the biology and pathogenesis of malaria, particularly for studying Plasmodium molecules for including them in effective vaccines against such microorganism. In spite of the forgoing, there has been no report to date describing the biology of parasite target cells in primates or their biomedical importance. This study was thus designed to analyse A. nancymaae erythrocyte protein composition using MS data collected during a previous study aimed at characterising the Plasmodium vivax proteome and published in the pertinent literature. Most peptides identified were similar to those belonging to 1189 Homo sapiens molecules; >95% of them had orthologues in New World primates. GO terms revealed a correlation between categories having the greatest amount of proteins and vital cell function. Integral membrane molecules were also identified which could be possible receptors facilitating interaction with Plasmodium species. The A. nancymaae erythrocyte proteome is described here for the first time, as a starting point for more in-depth/extensive studies. The data reported represents a source of invaluable information for laboratories interested in carrying out basic and applied biomedical investigation studies which involve using this primate. SIGNIFICANCE: An understanding of the proteomics characteristics of A. nancymaae erythrocytes represents a fascinating area for research regarding the study of the pathogenesis of malaria since these are the main target for Plasmodium invasion. However, and even though Aotus is one of the non-human primate models considered most appropriate for biomedical research, knowledge of its proteome, particularly its erythrocytes, remains unknown. According to the above and bearing in mind the lack of information about the A. nancymaae species genome and transcriptome, this study involved a search for primate proteins for comparing their MS/MS spectra with the available information for Homo sapiens. The great similarity found between the primate's molecules and those for humans supported the use of the monkeys or their cells for continuing assays involved in studying malaria. Integral membrane receptors used by Plasmodium for invading cells were also found; this required timely characterisation for evaluating their therapeutic role. The list of erythrocyte protein composition reported here represents a useful source of basic knowledge for advancing biomedical investigation in this field.

Malaria Prevalence and Its Associated Risk Factors among Patients Attending Chichu and Wonago Health Centres, South Ethiopia.

BACKGROUND: There were about of 124 to 283 million cases of malaria with 367,000 to 755,000 deaths annually. This study aimed to assess the prevalence of malaria cases and associated risk factors among attendants at Chichu and Wonago health centers, South Ethiopia. METHODS: In this health institution based cross sectional study, 324 subjects, attendants from outpatient department who came for any kind of medical services, were included during May to June 2016. A blood film examination format and structured questionnaire were used for data collection. Peripheral blood samples were collected and the presence of malaria cases was observed microscopically. The collected data were analyzed by SPSS version 20.0. RESULTS: Malaria cases were detected in 91 (28.1%) of the participants with higher infection rate amongst (56.04%). The predominant Plasmodium species detected was P. vivax (52.75%) followed by P. falciparum (35.16%) and mixed malaria infection by both of the species (12.09%). Housing construction and not using of insecticide treated bed nets for the last 6 months were significantly associated with the risk of getting malaria. Individuals who had stagnant water in their compound were more likely to get malaria than those who did not (OR=1.87, 95% CI: 1.20, 2.76). Houses that had been sprayed with insecticide in the past 6 months were protected against malaria infection (OR=0.33, 95% CI: 0.11, 0.92). Moreover, bed net utilization was associated with a significantly lower risk of infection (OR=0.19, 95% CI: 0.09, 0.37). CONCLUSIONS: Type of housing construction, not using bed net, insecticide spraying and residing near stagnant water were associated risk factors with malaria positivity in the study area.

Vivax malaria in a blood donor in Spain, relapse or a new infection in a malaria non-endemic country?

Malaria is a vectorborne disease caused by protozoan of the genus Plasmodium, which can also be transmitted by the transfusion of infected red blood cells. One year after return from a travel to Honduras, a Spanish traveller developed vivax malaria. Prior to the onset of symptoms, the donor made a donation that tested non-reactive using an immunological test for malaria. Samples from the donor taken before donation and tested by serological and molecular methods were negative but positive at the time of hospital admission. The possible sources of the donors' infection, imported versus locally acquired, are discussed.

Characteristics of Travel-Related Severe Plasmodium vivax and Plasmodium falciparum Malaria in Individuals Hospitalized at a Tertiary Referral Center in Lima, Peru.

Severe Plasmodium falciparum malaria is uncommon in South America. Lima, Peru, while not endemic for malaria, is home to specialized centers for infectious diseases that admit and manage patients with severe malaria (SM), all of whom contracted infection during travel. This retrospective study describes severe travel-related malaria in individuals admitted to one tertiary care referral hospital in Lima, Peru; severity was classified based on criteria published by the World Health Organization in 2000. Data were abstracted from medical records of patients with SM admitted to Hospital Nacional Cayetano Heredia from 2006 to 2011. Of 33 SM cases with complete clinical data, the mean age was 39 years and the male/female ratio was 2.8. Most cases were contracted in known endemic regions within Peru: Amazonia (47%), the central jungle (18%), and the northern coast (12%); cases were also found in five (15%) travelers returning from Africa. Plasmodium vivax was most commonly identified (71%) among the severe infections, followed by P. falciparum (18%); mixed infections composed 11% of the group. Among the criteria of severity, jaundice was most common (58%), followed by severe thrombocytopenia (47%), hyperpyrexia (32%), and shock (15%). Plasmodium vivax mono-infection predominated as the etiology of SM in cases acquired in Peru.

Investigating the Pathogenesis of Severe Malaria: A Multidisciplinary and Cross-Geographical Approach.

More than a century after the discovery of Plasmodium spp. parasites, the pathogenesis of severe malaria is still not well understood. The majority of malaria cases are caused by Plasmodium falciparum and Plasmodium vivax, which differ in virulence, red blood cell tropism, cytoadhesion of infected erythrocytes, and dormant liver hypnozoite stages. Cerebral malaria coma is one of the most severe manifestations of P. falciparum infection. Insights into its complex pathophysiology are emerging through a combination of autopsy, neuroimaging, parasite binding, and endothelial characterizations. Nevertheless, important questions remain regarding why some patients develop life-threatening conditions while the majority of P. falciparum-infected individuals do not, and why clinical presentations differ between children and adults. For P. vivax, there is renewed recognition of severe malaria, but an understanding of the factors influencing disease severity is limited and remains an important research topic. Shedding light on the underlying disease mechanisms will be necessary to implement effective diagnostic tools for identifying and classifying severe malaria syndromes and developing new therapeutic approaches for severe disease. This review highlights progress and outstanding questions in severe malaria pathophysiology and summarizes key areas of pathogenesis research within the International Centers of Excellence for Malaria Research program.

Clinical, geographical, and temporal risk factors associated with presentation and outcome of vivax malaria imported into the United Kingdom over 27 years: observational study.

OBJECTIVE: To examine temporal and geographical trends, risk factors, and seasonality of imported vivax malaria in the United Kingdom to inform clinical advice and policy. DESIGN: Observational study. SETTING: National surveillance data from the UK Public Health England Malaria Reference Laboratory, data from the International Passenger Survey, and international climactic data. PARTICIPANTS: All confirmed and notified cases of malaria in the UK (n=50,187) from 1987 to 2013, focusing on 12,769 cases of vivax malaria. MAIN OUTCOME MEASURES: Mortality, sociodemographic details (age, UK region, country of birth and residence, and purpose of travel), destination, and latency (time between arrival in the UK and onset of symptoms). RESULTS: Of the malaria cases notified, 25.4% (n=12,769) were due to Plasmodium vivax, of which 78.6% were imported from India and Pakistan. Most affected patients (53.5%) had travelled to visit friends and relatives, and 11.1% occurred in tourists. Imported P vivax is concentrated in areas with large communities of south Asian heritage. Overall mortality was 7/12,725 (0.05%), but with no deaths in 9927 patients aged under 50 years. Restricting the analysis to those aged more than 50 years, mortality was 7/2798 (0.25%), increasing to 4/526 (0.76%) (adjusted odds ratio 32.0, 95% confidence interval 7.1 to 144.0, P<0.001) in those aged 70 years or older. Annual notifications decreased sharply over the period, while traveller numbers between the UK and South Asia increased. The risk of acquiring P vivax from South Asia was year round but was twice as high from June to September (40 per 100,000 trips) compared with the rest of the year. There was strong seasonality in the latency from arrival in the UK to presentation, significantly longer in those arriving in the UK from South Asia from October to March (median 143 days) versus those arriving from April to September (37 days, P<0.001). CONCLUSIONS: Travellers visiting friends and family in India and Pakistan are most at risk of acquiring P vivax, and older patients (especially those >70 years) are most at risk of dying; these groups should be targeted for advice before travelling. The risk of acquiring vivax malaria is year round but higher during summer monsoons, masked by latency. The latency of time to clinical presentation of imported vivax malaria in the UK is highly seasonal; seasonal latency has implications for pretravel advice but also for the control of malaria in India and Pakistan. A reduced incidence of vivax malaria in travellers may mean further areas of South Asia can be considered not to need malaria chemoprophylaxis.

Spatial-temporal analysis of malaria and the effect of environmental factors on its incidence in Yongcheng, China, 2006-2010.

BACKGROUND: In 2003, Plasmodium vivax malaria has re-emerged in central eastern China including Yongcheng prefecture, Henan Province, where no case has been reported for eleven years. Our goals were to detect the space-time distribution pattern of malaria and to determine significant environmental variables contributing to malaria incidence in Yongcheng from 2006 to 2010, thus providing scientific basis for further optimizing current malaria surveillance and control programs. METHODS: This study examined the spatial and temporal heterogeneities in the risk of malaria and the influencing factors on malaria incidence using geographical information system (GIS) and time series analysis. Univariate analysis was conducted to estimate the crude correlations between malaria incidence and environmental variables, such as mosquito abundance and climatic factors. Multivariate analysis was implemented to construct predictive models to explore the principal environmental determinants on malaria epidemic using a Generalized Estimating Equation (GEE) approach. RESULTS: Annual malaria incidence at town-level decreased from the north to south, and monthly incidence at prefecture-level demonstrated a strong seasonal pattern with a peak from July to November. Yearly malaria incidence had a visual spatial association with yearly average temperature. Moreover, the best-fit temporal model (model 2) (QIC = 16.934, P<0.001, R2 = 0.818) indicated that significant factors contributing to malaria incidence were maximum temperature at one month lag, average humidity at one month lag, and malaria incidence of the previous month. CONCLUSIONS: Findings supported the effects of environment factors on malaria incidence and indicated that malaria control targets should vary with intensity of malaria incidence, with more public resource allocated to control the source of infections instead of large scale An. sinensis control when malaria incidence was at a low level, which would benefit for optimizing the malaria surveillance project in China and some other countries with unstable or low malaria transmission.

Plasmodium vivax adherence to placental glycosaminoglycans.

BACKGROUND: Plasmodium vivax infections seldom kill directly but do cause indirect mortality by reducing birth weight and causing abortion. Cytoadherence and sequestration in the microvasculature are central to the pathogenesis of severe Plasmodium falciparum malaria, but the contribution of cytoadherence to pathology in other human malarias is less clear. METHODOLOGY: The adherence properties of P. vivax infected red blood cells (PvIRBC) were evaluated under static and flow conditions. PRINCIPAL FINDINGS: P. vivax isolates from 33 patients were studied. None adhered to immobilized CD36, ICAM-1, or thrombospondin, putative ligands for P. falciparum vascular cytoadherence, or umbilical vein endothelial cells, but all adhered to immobilized chondroitin sulphate A (CSA) and hyaluronic acid (HA), the receptors for adhesion of P. falciparum in the placenta. PvIRBC also adhered to fresh placental cells (N = 5). Pre-incubation with chondroitinase prevented PvIRBC adherence to CSA, and reduced binding to HA, whereas preincubation with hyaluronidase prevented adherence to HA, but did not reduce binding to CSA significantly. Pre-incubation of PvIRBC with soluble CSA and HA reduced binding to the immobilized receptors and prevented placental binding. PvIRBC adhesion was prevented by pre-incubation with trypsin, inhibited by heparin, and reduced by EGTA. Under laminar flow conditions the mean (SD) shear stress reducing maximum attachment by 50% was 0.06 (0.02) Pa but, having adhered, the PvIRBC could then resist detachment by stresses up to 5 Pa. At 37 °C adherence began approximately 16 hours after red cell invasion with maximal adherence at 30 hours. At 39 °C adherence began earlier and peaked at 24 hours. SIGNIFICANCE: Adherence of P. vivax-infected erythrocytes to glycosaminoglycans may contribute to the pathogenesis of vivax malaria and lead to intrauterine growth retardation.

Two cases of imported malaria in Western Romania, 2010-2011.

Malaria is a major problem for European travelers to endemic regions. In Romania during 1980-2007 approximately 20 imported cases were detected annually. The aim of our short communication is to present 2 interesting cases of imported malaria detected in Western Romania. The first patient was a 20-year female who traveled to India and acquired an infection with Plasmodium vivax (P. vivax). The second patient, a 60-year female, contracted an infection with Plasmodium falciparum (P. falciparum) during a trip to Ghana; the evolution of the disease was severe with many complications and the patient finally died. The cases presented revealed the difficulties in establishing a correct diagnosis of malaria in a non-endemic country, consequences of an incomplete taken anamnesis. Travel history should always represent a mandatory part of a well conducted investigation. At the same time, we must underline the importance of a correct and complete prophylaxis prior to every departure to tropical countries.

Serum proteome analysis of vivax malaria: An insight into the disease pathogenesis and host immune response.

Vivax malaria is the most widely distributed human malaria resulting in 80-300 million clinical cases every year. It causes severe infection and mortality but is generally regarded as a benign disease and has not been investigated in detail. The present study aimed to perform human serum proteome analysis in a malaria endemic area in India to identify potential serum biomarkers for vivax malaria and understand host response. The proteomic analysis was performed on 16 age and gender matched subjects (vivax patients and control) in duplicate. Protein extraction protocols were optimized for large coverage of the serum proteome and to obtain high-resolution data. Identification of 67 differentially expressed and statistically significant (Student's t-test; p<0.05) protein spots was established by MALDI-TOF/TOF mass spectrometry. Many of the identified proteins such as apolipoprotein A and E, serum amyloid A and P, haptoglobin, ceruloplasmin, and hemopexin are interesting from a diagnostic point of view and could further be studied as potential serum biomarkers. The differentially expressed serum proteins in vivax malaria identified in this study were subjected to functional pathway analysis using multiple software, including Ingenuity Pathway Analysis (IPA), Protein ANalysis THrough Evolutionary Relationships (PANTHER) and Database for Annotation, Visualization and Integrated Discovery (DAVID) functional annotation tool for better understanding of the biological context of the identified proteins, their involvement in various physiological pathways and association with disease pathogenesis. Functional pathway analysis of the differentially expressed proteins suggested the modulation of multiple vital physiological pathways, including acute phase response signaling, complement and coagulation cascades, hemostasis and vitamin D metabolism pathway due to this parasitic infection. This article is part of a Special Issue entitled: Proteomics: The clinical link.

Southeast Asian ovalocytosis is associated with increased expression of Duffy antigen receptor for chemokines (DARC).

The Duffy antigen receptor for chemokines (DARC or Fy glycoprotein) carries antigens that are important in blood transfusion and is the main receptor used by Plasmodium vivax to invade reticulocytes. Southeast Asian ovalocytosis (SAO) results from an alteration in RBC membrane protein band 3 and is thought to mitigate susceptibility to falciparum malaria. Expression of some RBC antigens is suppressed by SAO, and we hypothesized that SAO may also reduce Fy expression, potentiallyleading to reduced susceptibility to vivax malaria. Blood samples were collected from individuals living in the Madang Province of Papua New Guinea. Samples were assayed using a flow cytometry assay for expression of Fy on the surface of RBC and reticulocytes by measuring the attachment of a phycoerythrin-labeled Fy6 antibody. Reticulocytes were detected using thiazole orange. The presence of the SAO mutation was confirmed by PCR. There was a small (approximately 10%) but statistically significant (p=0.049, Mann-Whitney U test) increase in Fy expression on SAO RBC compared with RBC from individuals without this polymorphism: mean Fy expression (mean fluorescence intensity [MFI]) was 10.12 +/- 1.22 for SAO heterozygotes versus an MFI of 8.95 +/- 1.1 for individuals without SAO. For reticulocytes the MFI values were 27.61 +/- 19.12 for SAO heterozygotes and 16.47 +/- 3.81 for controls. SAO is associated with increased and not decreased Fy6 expression so that susceptibility to P. vivax infection is unlikely to be affected.

Nucleotide sequence polymorphism at the apical membrane antigen-1 locus reveals population history of Plasmodium vivax in Thailand.

Apical membrane antigen-1 is a candidate for inclusion in a vaccine for the human malaria parasite Plasmodium vivax. We collected 231 complete sequences of the gene encoding this antigen (pvama-1) from three regions of Thailand, the most extensive collection to date of sequences at this locus. The domain II loop (previously mentioned as a potential vaccine component) was almost completely conserved, with a single amino acid variant (I313R) observed in a single sequence. The 3' portion of the gene (domain II through the stop codon) showed significantly lower nucleotide diversity than the 5' portion (start codon through domain I); and a given domain I sequence might be found in a haplotype with more than one domain II sequence. These results imply a hotspot of recombination between domains I and II. We found significant geographic subdivision among the three regions of Thailand (NW, East, and South) in which collections were made in 2007. Numbers of P. vivax infections have experienced overall declines since 1990 in all three regions; but the decline has been most recent in the NW, and there has been a rebound in numbers of infections in the South since 2000. Consistent with population history, amino acid sequence diversity was greatest in the NW. The South, which had by far the lowest sequence diversity of the three regions, showed signs of a population that has expanded from a small number of founders after a bottleneck.

Pregnancy outcomes associated with Plasmodium vivax malaria in northeastern Venezuela.

Although Plasmodium vivax is increasingly recognized as an important cause of morbidity in pregnancy in low malaria-transmission areas of Asia, little is know about the epidemiologic and clinical profiles of P. vivax in pregnant women in Latin America. We describe the clinical features and pregnancy outcomes in a series of 12 cases of P. vivax malaria in pregnant women complicated in some by miscarriage or preterm deliveries and in others with significant degrees of anemia and thrombocytopenia in a population where P. vivax is endemic in northeastern Venezuela.

Chloroquine resistant malaria in neonates.

OBJECTIVE: To analyze the clinical presentation, treatment given, and outcome of patients suffering from congenital and acquired malaria in neonatal period. DESIGN: Analytical study. PLACE AND DURATION OF STUDY: Paediatrics Ward-2, QAMC/BVH, Bahawalpur for 02 years from October 2001 to October 2003. PATIENTS AND METHODS: The study included 45 cases of neonatal malaria. Thirty cases of malaria, admitted during first ten days of life, diagnosed as congenital malaria, were kept in group A, while 15 cases admitted in the ward from the age of 11 to 28 days, labeled as acquired malaria, were named group B. The clinical features at the time of presentation were noted in each group from the charts having positive malarial parasite (M.P.) on thick and thin slides. The diagnosed cases were treated with the standard dose of chloroquine sulphate. Those patients who improved clinically as well as revealed no parasite on follow-up were labeled as chloroquine sensitive. On the other hand, patients showing poor clinical response with persistence of the parasites in the blood or initially disappearing but later again having a clinical disease with positive M.P. on follow-up, were labeled as chloroquine resistant. They were treated with quinine sulphate. Outcome was compared in both the groups regarding the pattern of chloroquine resistance and death/ survival. Data was collected on which Fischer's exact test of significance was performed to know the level of significance. P-value of < 0.05 was taken as statistically significant. RESULTS: Low birth weight, severe hemolytic anemia with history of fever in the mother were main features in group A while in group B fever, anaemia and history of blood transfusion were marked features. In group A 76.66% were caused by Plasmodium (P.) falciparum. While in group B 60% were caused by Plasmodium vivax. In group A 26.66% were chloroquine resistant while 33.65% were chloroquine resistant in group B. The mortality was 16.66% in group A and 13.33% in group B. CONCLUSION: Intrauterine growth retardation, hemolytic jaundice and history of fever in the mother in the last trimester of pregnancy in the congenital while fever, history of blood transfusion in the neonates in acquired malaria but pallor in both the groups, were important clinical features. Pattern of chloroquine resistance and mortality in both the groups was not statistically different.