Change in mutation patterns of Plasmodium vivax dihydrofolate reductase (Pvdhfr) and dihydropteroate synthase (Pvdhps) in P. vivax isolates from malaria endemic areas of Thailand

Malaria is the most important public health problem in several countries. In Thailand, co-infections of Plasmodium vivax and Plasmodium falciparum are common. We examined the prevalence and patterns of mutations in P. vivax dihydrofolate reductase (Pvdhfr) and P. vivax dihydropteroate synthase (Pvdhps) in 103 blood samples collected from patients with P. vivax infection who had attended the malaria clinic in Mae Sot, Tak Province during 2009 and 2010. Using nested polymerase chain reaction-restriction fragment length polymorfism, we examined single nucleotide polymorphisms-haplotypes at amino acid positions 13, 33, 57, 58, 61, 117 and 173 of Pvdhfr and 383 and 553 of Pvdhps. All parasite isolates carried mutant Pvdhfr alleles, of which the most common alleles were triple mutants (99%). Eight different types of Pvdhfr and combination alleles were found, as follows: 57I/58R/117T, 57I/58R/117T, 57I/58R/117T/N, 57L/58R/117T, 57L/58R/117T, 58R/61M/117N, 58R/61M/117N and 13L/57L/58R/117T. The most common Pvdhfr alleles were 57I/58R/117T (77.7%), 57I/58R/117T/N (1%), 57L/58R/117T (5.8%) and 58R/61M/117N (14.5%). The most common Pvdhfr alleles were 57I/58R/117T (77.7%), 57I/58R/117T/N (1%), 57L/58R/117T (5.8%) and 58R/61M/117N (14.5%). Additionally, we recovered one isolate of a carrying a quadruple mutant allele, 13L/57L/58R/117T. The most prevalent Pvdhps allele was a single mutation in amino acid 383 (82.5%), followed by the wild-type A383/A553 (17.5%) allele. Results suggest that all P. vivax isolates in Thailand carry some combination of mutations in Pvdhfr and Pvdhps. Our findings demonstrate that development of new antifolate drugs effective against sulfadoxine-pyrimethamine-resistant P. vivax is required.

PCR - based diagnosis to evaluate the performance of malaria reference centers

Although the Giemsa-stained thick blood smear (GTS) remains the gold standard for the diagnosis of malaria, molecular methods are more sensitive and specific to detect parasites and can be used at reference centers to evaluate the performance of microscopy. The description of the Plasmodium falciparum, P. vivax, P. malariae and P. ovale ssrRNA gene sequences allowed the development of a polymerase chain reaction (PCR) that had been used to differentiate the four species. The objective of this study was to determine Plasmodium species through PCR in 190 positive smears from patients in order to verify the quality of diagnosis at SUCEN's Malaria Laboratory. Considering only the 131 positive results in both techniques, GTS detected 4.6% of mixed and 3.1% of P. malariae infections whereas PCR identified 19.1% and 13.8%, respectively.

Molecular screening of Plasmodium sp. asymptomatic carriers among transfusion centers from Brazilian Amazon region

The transmission of malaria in Brazil is heterogeneous throughout endemic areas and the presence of asymptomatic Plasmodium sp. carriers (APCs) in the Brazilian Amazon has already been demonstrated. Malaria screening in blood banks is based on the selection of donors in respect to possible risks associated with travel or residence, clinical evidence and/or inaccurate diagnostic methods thereby increasing the probability of transfusion-transmitted infection. We evaluated the frequency of APCs in four blood services in distinct areas of the Brazilian Amazon region. DNA was obtained from 400 human blood samples for testing using the phenol-chloroform method followed by a nested-PCR protocol with species-specific primers. The positivity rate varied from 1 to 3% of blood donors from the four areas with an average of 2.3%. All positive individuals had mixed infections for Plasmodium vivax and Plasmodium falciparum. No significant differences in the results were detected among these areas; the majority of cases originated from the transfusion centres of Porto Velho, Rondônia State and Macapá, Amapá State. Although it is still unclear whether APC individuals may act as reservoirs of the parasite, efficient screening of APCs and malaria patients in Brazilian blood services from endemic areas needs to be improved.

Relationship between plasma and red blood cell concentrations of quinine in Brazilian children with uncomplicated Plasmodium falciparummalaria on oral therapy

We determined the relationship between plasma and red blood cell concentrations of quinine in children with uncomplicated falciparum malaria from an endemic area of Amazonian region. Quinine was determined by high performance liquid chromatography with ultraviolet detection. In the steady state the ratio between plasma and red blood cell quinine concentration was 1.89 ± 1.25 ranging from 1.05 to 2.34. This result demonstrated that quinine do not concentrate in red blood cell of Brazilian children and characterize the absence of interracial difference in this relationship.

Epidemiological profile of malaria in the state of Tocantins, Brazil, from 2003 to 2008

This study describes the epidemiological profile of malaria in the State of Tocantins, in the period 2003-2008, investigates the association between the frequency of malaria and population growth, classifies the cases by 'autochthonous' and 'imported', reports the indices of the disease and analyses the distribution of the cases by Plasmodium species, age and gender. The retrospective study was based on secondary data, stored in SIVEP-malaria and analyzed using the software Epi-Info 3.5.1. and Bioestat 5.0. 19,004 samples were investigated for malaria, 19% of them were positive, 73.32% with Plasmodium vivax, 21.80% with Plasmodium falciparum, 4.79% with mixed infections and only 0.08% with Plasmodium malariae. Male individuals accounted for 76.95% and predominated in all years and age groups, especially in the 15 to 49 years old group. From the overall cases, 34.27% were autochthonous and 65.73% were imported (χ2 = 356.8, p = 0.0001). The frequency of malaria decreased significantly during the entire series (rp = 0.96, p = 0.002) and the number of municipalities with autochthonous transmission also diminished. It was found that malaria is predominantly imported, related to land activities, which confirms the need for effective measures to maintain vigilance throughout the state and enhance educational activities in order to guide the population towards early treatment-seeking.

MALARIA DIAGNOSIS BY LOOP-MEDIATED ISOTHERMAL AMPLIFICATION (LAMP) IN THAILAND

The loop-mediated isothermal amplification method (LAMP) is a recently developed molecular technique that amplifies nucleic acid under isothermal conditions. For malaria diagnosis, 150 blood samples from consecutive febrile malaria patients, and healthy subjects were screened in Thailand. Each sample was diagnosed by LAMP, microscopy and nested polymerase chain reaction (nPCR), using nPCR as the gold standard. Malaria LAMP was performed using Plasmodiumgenus and Plasmodium falciparum specific assays in parallel. For the genus Plasmodium, microscopy showed a sensitivity and specificity of 100%, while LAMP presented 99% of sensitivity and 93% of specificity. For P. falciparum, microscopy had a sensitivity of 95%, and LAMP of 90%, regarding the specificity; and microscopy presented 93% and LAMP 97% of specificity. The results of the genus-specific LAMP technique were highly consistent with those of nPCR and the sensitivity of P. falciparum detection was only marginally lower.

Infrequency of asymptomatic malaria in an endemic area in Amazonas, Brazil

A malaria survey was conducted in an area of high transmission (Costa Marques, Rondonia, Brazil) to determine the prevalence of asymptomatic parasitemia and its clinical significance. Most of the people surveyed were immigrants who had lived in the endemic area < 5 years. The people had easy access to free diagnostic and treatment services at the Malaria Clinic in the town of Costa Marques. The prevalence of plasmodial parasitemia in 344 people was 22%. There were 36 individuals with asymptomatic infections among the 77 parasitemic patients. During the two days following the initial examination, 19 ofthe 36 individuals: with asymptomatic infections developed malaria. Among the 17 patients who remained asymptomatic for > 2 days, 4 had only gametocytes, 1 had taken inadequate anti-malarial treatment, 3 were under treatment and 2 moved. Six asymptomatic patients denied the use of anti-malarial drugs and they developed malaria 3-6 days after the initial parasitological diagnosis. The final patient remained asymptomatic during the 7 day observation period. He had a history of > 40 malaria attacks and denied the use of antimalarial treatment. With the exception of the latter all of the other asymptomatic patients, were either in the incubation period or had been treated It is concluded that asymptomatic malaria is rare in the Costa Marques area and that it is necessary to treat all individuals with plasmodial parasitemia.

Dynamics and kinetics of natural killer cell cytotoxicity in human malaria as evaluated by a novel stepwise cytotoxicity assay

Malaria causes important functional alterations of the immune system, but several of them are poorly defined. To evaluate thoroughly the natural killer cell cytotoxicity in patients with malaria, we developed a technique capable to assess both the dynamics and the kinetics of the process. For the kinetics assay, human peripheral blood mononuclear cells were previously incubated with K562 cells and kept in agarose medium, while for the dynamics assay both cells were maintained in suspension. NK activity from patients with vivax malaria presented a kinetics profile faster than those with falciparum malaria. NK cytotoxicity positively correlated with parasitemia in falciparum malaria. The dynamics of NK cytotoxicity of healthy individuals was elevated at the beginning of the process and then significantly decreased. In contrast, malaria patients presented successive peaks of NK activity. Our results confirmed the occurrence of alteration in NK cell function during malaria, and added new data about the NK cytotoxicity process.

Malaria outbreaks in a non-endemic area of Brazil, 2005

In March 2005, a resident of the municipality of Monte Alegre de Minas, State of Minas Gerais, without any history of traveling to endemic areas for malaria, was diagnosed with Plasmodium vivax infection and local mosquito-borne transmission was suspected. The epidemiological investigation identified another 10 cases with local transmission and all of them were related to the imported malaria case that was detected in this region. The potential exposure site was the banks of the river Tejuco, an area frequented by mineral prospectors. Some of these prospectors were known to have come from states with malaria transmission. In the autochthonous cases, Plasmodium vivax and Plasmodium falciparum were diagnosed. Entomological investigation identified Anopheles (Nyssorhynchus) darlingi, Anopheles (Nyssorhynchus) albitarsis, Anopheles (Nyssorhynchus) triannulatus and Anopheles (Nyssorhynchus) parvus. After the first outbreak, another three autochthonous cases were notified in municipality of Monte Alegre de Minas, in the same year. The occurrence of these outbreaks highlights the importance of surveillance systems in areas that are nonendemic for malaria.

An unusual case of heart failure due to Plasmodium vivax infection with a favorable outcome

Although malaria is one of the oldest types of parasitic infection, we have recently witnessed substantial changes in the outcome of malarial infections. Severe Plasmodium vivax infections have recently become more frequent, and are occasionally associated with fatal outcomes. Cardiac arrhythmia and myocardial failure have also been reported, typically in association with Plasmodium falciparum infections. We report a case of myocarditis and heart failure, due to Plasmodium vivaxinfection, along with the favorable outcome.

Basic sanitation, socioeconomic conditions, and degree of risk for the presence and maintenance of malaria in a low-transmission area in the Brazilian Amazon

ABSTRACTINTRODUCTION:This study aimed to evaluate basic sanitation and socioeconomic indicators, reported cases of malaria, and risk of contracting malaria in the Ananindeua municipality, State of Pará.METHODS:Data on basic sanitation and socioeconomic dimensions were taken from the Brazilian Institute of Geography and Statistics [ Instituto Brasileiro de Geografia e Estatística (IBGE)] 2010 census. Epidemiological malaria information was taken from the Epidemiological Malaria Surveillance Information System [ Sistema de Informação de Vigilância Epidemiológica de Malária (SIVEP/Malaria)], between 2003 and 2013 of the Ministry of Health and from the SIVEP/Malaria forms of the municipality's Endemic Diseases Unit for 2,013 cases.RESULTS:Our data do not confirm the correlation among indicators of basic sanitation, socioeconomic conditions, and water supply with malaria cases. Of the 1,557 cases evaluated, most were caused by Plasmodium vivax , with rare cases of Plasmodium falciparum and mixed infections. There were 756 notifications in 2003. The number of reported cases was sharply reduced between 2006 and 2012, but a 142-case outbreak occurred in 2013. Ananindeua municipality's Annual Parasite Index indicated low risk in 2003 and no risk in other years, and the 2,013 cases were predominantly male individuals aged ≥40 years.CONCLUSIONS:Our data confirm the non-endemicity of malaria in the Ananindeua municipality, as the Annual Parasite Indices described for the years 2004-2013 classify it as a risk-free area. However, the 2013 outbreak indicates the need to strengthen prevention, surveillance, and control activities to reduce the risk of new outbreaks and consequent economic and social impacts on the population.

Malaria seroepidemiology: comparison between indirect fluorescent antibody test and enzyme immunoassay using bloodspot eluates

Blood sampling on filter paper is a current practice seroepidemiological studies by indirect fluorescent antibody test (IFAT). There is, however, scant comparative information about the use of bloodspot eluates for detection of malarial IgG antibodies simultaneously by IFAT and enzyme immunoassay (ELISA). Here we report data obtained by both serological methods done on 219 bloodspot eluate samples collected in a rural community in Brazilian Amazon Basin (Alto Paraíso, Ariquemes municipality) where malaria is endemic. Plasmodium falciparum and P. vivax thick smear antigens were used in the IFAT; a detergent-soluble P. falciparum antigen was prepared for ELISA. Substantial agreement of results (Kappa coefficient k = 0.686) was observed when P. falciparum antigen was used in both tests, and IFAT titers were found to be strongly correlated ELISA antibody units (Spearman correlation coeficient rs = 0.818, p < 0.0001). Only moderate agreement (k = 0.467) between IFAT with P. vivax antigen and ELISA with P. falciparum antigen was observed. Spearman correlation coefficient value between quantitative results (IFAT titers and ELISA antibody units) in this case was numerically lowe (rs = 0.540, p < 0.0001). Our results suggest that, with P. falciparum antigen, both IFAT and ELISA performed on bloodspot eluates are equivalent for seropidemiological purposes.

A malaria merozoite surface protein (MSP1)-structure, processing and function

Merozoite surface protein-1 (MSP-1, also referred to as P195, PMMSA or MSA 1) is one of the most studied of all malaria proteins. The proteins. The protein is found in all malaria species investigated and structural studies on the gene indicate that parts of the molecule are well-conserved. Studies on Plasmodium falciparum have shown that the protein is in a processed form on the merozoite surface, a result of proteolytic cleavage of the large percursor molecule. Recent studies have identified some of these cleavage sites. During invasion of the new red cell most of the MSP1 molecule is shed from the parasite surface except for a small C-terminal fragment which can be detected in ring stages. Analysis of the structure of this fragment suggests that it contains two growth factor-like domains that may have a functional role.

Mefloquine resistant malaria in cameroon and correlation with resistance quinine

Based on the results of in vitro sensitivity of Plasmodium falciparum to chloroquine, quinine and mefloquine, and evaluation of drug consumption conducted in 1987-1988 in four areas in the noth and south-west of Cameron, two opposite situations were encountered in this country. In northern Cameron where mefloquine resistance is prevalent a close correlation was found between the responses of P. falciparum to mefloquine and to quinine, but not between mefloquine and chloroquine. In the south, where chloroquine resistance is highly prevalent, no correlation was found neither between mefloquine and chloroquine nor mefloquine and quinine, but the responses to quinine and chloroquine appear partly correlated. These lead to formulate the hypothesis of a "southern" type of P. falciparum submitted to a high chloroquine drug pressure inducing a secondary cross resistance, whilst a "northern"type submitted to a relatively high and abortive quinine drug pressure inducing a primary quinine resistance and a secondary cross resistance with mefloquine.

Variation in the cytoadherence characteristics of malaria parasites: is this a true virulence factor?

The sequestration of Plasmodium falciparum-infected erythrocytes to the endothelial cells of brain capillaries is believed to represent one of the determining factors in the pathogenesis of cerebral malaria. In vitro studies of cytoadherence provide an experimental approach to understand the mechanism of sequestration and the respective roles played by parasite and host components in this interaction. This paper critically reviews current studies on cytoadherence, with particular emphasis on the nature of the information provided by such studies and their limitations. The paper also describes how cytoadherence studies using the patient's own monocytes can provide original information on the level of receptor up-regulation in the course of malarial infection.