SIMPLIFIED DIAGNOSIS OF MALARIA INFECTION: GFM/PCR/ELISA A SIMPLIFIED NUCLEIC ACID AMPLIFICATION TECHNIQUE BY PCR/ELISA

We report an adaptation of a technique for the blood sample collection (GFM) as well as for the extraction and amplification of Plasmodium DNA for the diagnosis of malaria infection by the PCR/ELISA. The method of blood sample collection requires less expertise and saves both time and money, thus reducing the cost by more than half. The material is also suitable for genetic analysis in either fresh or stored specimens prepared by this method.

Malaria vectors in the Brazilian Amazon: Anopheles of the subgenus Nyssorhynchus

Various species of Anopheles (Nyssorhynchus) were studied in the Amazon with the objective of determining their importance as malaria vectors. Of the 33 known Anopheles species occurring in the Amazon, only 9 were found to be infected with Plasmodium. The different species of this subgenus varied both in diversity and density in the collection areas. The populations showed a tendency towards lower density and diversity in virgin forest than in areas modified by human intervention. The principal vector, An. darlingi, is anthropophilic with a continuous activity cycle lasting the entire night but peaking at sunset and sunrise. These species (Nyssorhynchus) are peridomiciliary, entering houses to feed on blood and immediately leaving to settle on nearby vegetation. Anopheles nuneztovari proved to be zoophilic, crepuscular and peridomiciliary. These habits may change depending on a series of external factors, especially those related to human activity. There is a possibility that sibling species exist in the study area and they are being studied with reference to An. darlingi, An. albitarsis and An. nuneztovari. The present results do not suggest the existence of subpopulations of An. darlingi in the Brazilian Amazon.

An initial examination of the epidemiology of malaria in the State of Roraima, in the Brazilian Amazon Basin

This study firstly describes the epidemiology of malaria in Roraima, Amazon Basin in Brazil, in the years from 1991 to 1993: the predominance of plasmodium species, distribution of the blood slides examined, the malaria risk and seasonality; and secondly investigates whether population growth from 1962 to 1993 was associated with increasing risk of malaria. Frequency of malaria varied significantly by municipality. Marginally more malaria cases were reported during the dry season (from October to April), even after controlling for by year and municipality. Vivax was the predominant type in all municipalities but the ratio of plasmodium types varied between municipalities. No direct association between population growth and increasing risk of malaria from 1962 to 1993 was detected. Malaria in Roraima is of the "frontier" epidemiological type with high epidemic potential.

In vitro evaluation of quinidine sensitivity in brazilian Plasmodium falciparum isolates: comparative analysis to quinine and chloroquine

Falciparum malaria represents a serious and an increasing world public health problem due to the acquired parasite's resistance to the most available drugs. In some endemic areas, quinidine, a diastereoisomer of the antimalarial quinine, has been employed for replacing the latter. In order to evaluate the use of quinidine as an alternative to the increasing loss of quinine effectiveness in Brazilian P. falciparum strains, as has been observed in the Amazon area, we have assayed quinidine, quinine and chloroquine. The in vitro microtechnique was employed. All isolates showed to be highly resistant to chloroquine. Resistance to quinine was not noted although high MIC (minimal inhibitory concentration) values have been observed. These data corroborate the decreasing sensitivity to quinine in strains from Brazil. Quinidine showed IC50 from 0.053 to 4.577 mumol/L of blood while IC50 from 0.053 to 8.132 mumol/L of blood was estimated for quinine. Moreover, clearance of the parasitemia was observed in concentrations lower than that used for quinidine in antiarrhythmic therapy, confirming our previous data. The results were similar to African isolate.

Clinical and laboratory findings of Plasmodium vivax malaria in Colombia, 2001

A descriptive study was carried out in 104 patients with Plasmodium vivax malaria, from the region of Turbo (Antioquia, Colombia). Clinical features and levels of hemoglobin, glycemia, serum bilirubin, alanine-aminotransferase (ALT), aspartate-aminotransferase (AST), creatinine and complete blood cell profile were established. 65% of the studied individuals were men and their mean age was 23. Of all individuals 59% had lived in the region for > 1 year and 91% were resident in the rural area. 42% were farmers and 35% had a history of malaria. The mean parasitaemia was 5865 parasites/mm³. The evolution of the disease was short (average of 4.0 days). Fever, headache and chills were observed simultaneously in 91% of the cases while the most frequent signs were palmar pallor (46%), jaundice (15%), hepatomegaly (17%), and spleen enlargement (12%). Anemia was found in 39% of the women and in 51% of the men, 8% of individuals had thrombocytopaenia and 41% had hypoglycemia.

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.

Retrospective study of malaria prevalence and Anopheles genus in the area of influence of the binational Itaipu reservoir

The importance of hydroelectric dams beside the human interchange in the maintenance of malarious foci and the occurrence of the Anopheles genus on the Binational Itaipu Reservoir were the main points of this retrospective study. Data were collected from existing registrations at National, State and Municipal Health Departments and literature systematic overview, from January 1984 to December 2003. The occurrence of some outbreak of malaria, mainly by Plasmodium vivax, and the prevalence of species of the Anopheles genus different from Anopheles darlingi in the region are discussed. The malaria in the left bank of Paraná River is a focal problem, which must be approached locally through health, educational and social actions to prevent the continuity of outbreaks in the area. Concomitantly, it is necessary to plan and apply effective surveillance measures in the influence area of the Itaipu Reservoir.

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.

Salivary gland proteins of the human malaria vector, Anopheles dirus B (Diptera: Culicidae)

Salivary gland proteins of the human malaria vector, Anopheles dirus B were determined and analyzed. The amount of salivary gland proteins in mosquitoes aged between 3 - 10 days was approximately 1.08 ± 0.04 µg/female and 0.1 ± 0.05 µg/male. The salivary glands of both sexes displayed the same morphological organization as that of other anopheline mosquitoes. In females, apyrase accumulated in the distal regions, whereas alpha-glucosidase was found in the proximal region of the lateral lobes. This differential distribution of the analyzed enzymes reflects specialization of different regions for sugar and blood feeding. SDS-PAGE analysis revealed that at least seven major proteins were found in the female salivary glands, of which each morphological region contained different major proteins. Similar electrophoretic protein profiles were detected comparing unfed and blood-fed mosquitoes, suggesting that there is no specific protein induced by blood. Two-dimensional polyacrylamide gel analysis showed the most abundant salivary gland protein, with a molecular mass of approximately 35 kilodaltons and an isoelectric point of approximately 4.0. These results provide basic information that would lead to further study on the role of salivary proteins of An. dirus B in disease transmission and hematophagy.

High prevalence of malaria infection in Amazonas state, Venezuela

This study was carried out to determine the incidence of malaria in an endemic region of Amazonas State, Venezuela. For this, 200 random samples were collected from symptomatic and asymptomatic individuals from San Fernando de Atabapo and Santa Barbara. Epidemiological factors were related to malaria infection, which was diagnosed by microscopy observation and amplification of the 18S rDNA sequence by PCR. Malaria prevalence in these populations was 28.5%, whilst P. vivax and P. falciparum prevalences were 12 and 17%, respectively. No infection by P. malariae was found. A mixed infection was found on an asymptomatic individual. Prevalence patterns differed between age groups depending on the Plasmodium species. We found that 34.8% of the P. vivax and 15.2% of the P. falciparum infections were asymptomatic. The use of nets was helpful to prevent P. vivax infection, but did not protect against P. falciparum infection. The results suggest the presence of more than one mosquito vector in the area, displaying a differential pattern of infection for each Plasmodium species. There appear to be risk factors associated with malaria infections in some individuals. The population based approach and PCR diagnosis improved the accuracy of the statistical analysis in the study.

Leishmania spp. parasite isolation through inoculation of patient biopsy macerates in interferon gamma knockout mice

Isolation of Leishmania parasite and species identification are important for confirmation and to help define the epidemiology of the leishmaniasis. Mice are often used to isolate pathogens, but the most common mouse strains are resistant to infection with parasites from the Leishmania (Viannia) subgenus. In this study we tested the inoculation of interferon gamma knockout (IFNγ KO) mice with biopsy macerates from Leishmania-infected patients to increase the possibility of isolating parasites. Biopsies from twenty five patients with clinical signs of leishmaniasis were taken and tested for the presence of parasites. Immunohistochemical assay (IHC) and conventional histopathology detected the parasite in 88% and 83% of the patients, respectively. Leishmania sp. were isolated in biopsy macerates from 52% of the patients by culture in Grace's insect medium, but 13% of isolates were lost due to contamination. Inoculation of macerates in IFNγ KO mice provides isolation of parasites in 31.8% of the biopsies. Most isolates belong to L. (Viannia) subgenus, as confirmed by PCR, except one that belongs to L. (Leishmania) subgenus. Our preliminary results support the use of IFNγ KO mice to improve the possibility to isolate New World Leishmania species.