Silvia Maria Di Santi

Malaria in Brazil: what happens outside the Amazonian endemic region

Brazil, a country of continental proportions, presents three profiles of malaria transmission. The first and most important numerically, occurs inside the Amazon. The Amazon accounts for approximately 60% of the nation’s territory and approximately 13% of the Brazilian population. This region hosts 99.5% of the nation’s malaria cases, which are predominantly caused by Plasmodium vivax (i.e., 82% of cases in 2013). The second involves imported malaria, which corresponds to malaria cases acquired outside the region where the individuals live or the diagnosis was made. These cases are imported from endemic regions of Brazil (i.e., the Amazon) or from other countries in South and Central America, Africa and Asia. Imported malaria comprised 89% of the cases found outside the area of active transmission in Brazil in 2013. These cases highlight an important question with respect to both therapeutic and epidemiological issues because patients, especially those with falciparum malaria, arriving in a region where the health professionals may not have experience with the clinical manifestations of malaria and its diagnosis could suffer dramatic consequences associated with a potential delay in treatment. Additionally, because the Anopheles vectors exist in most of the country, even a single case of malaria, if not diagnosed and treated immediately, may result in introduced cases, causing outbreaks and even introducing or reintroducing the disease to a non-endemic, receptive region. Cases introduced outside the Amazon usually occur in areas in which malaria was formerly endemic and are transmitted by competent vectors belonging to the subgenus Nyssorhynchus (i.e., Anopheles darlingi, Anopheles aquasalis and species of the Albitarsis complex). The third type of transmission accounts for only 0.05% of all cases and is caused by autochthonous malaria in the Atlantic Forest, located primarily along the southeastern Atlantic Coast. They are caused by parasites that seem to be (or to be very close to) P. vivax and, in a less extent, by Plasmodium malariae and it is transmitted by the bromeliad mosquito Anopheles (Kerteszia) cruzii. This paper deals mainly with the two profiles of malaria found outside the Amazon: the imported and ensuing introduced cases and the autochthonous cases. We also provide an update regarding the situation in Brazil and the Brazilian endemic Amazon.

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 SUCENs 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.

Análise da freqüência de recaídas de malária por Plasmodium vivax em região não endêmica (São Paulo, Brasil)

Very few well-established information is available about the frequency and timeliness of relapses in cases of Plasmodium vivax malaria acquired in Brazil. So, we analysed a series of correctly treated patients observed out of endemic areas. The rate of relapses seen in Sao Paulo, which may represent that of the parasitosis in the whole country, was high, ranging from 7.5% to 24.5%, and early in most cases, i.e. appearing by three months, what anticipates a high endemicity.

In vivo and in vitro Plasmodium falciparum resistance to chloroquine, amodiaquine and quinine in the Brazilian Amazon.

In order to study the chemoresistance of Plasmodium falciparum to commonly used antimalarial drugs in Brazil the authors have studied ten patients with falciparum malaria, acquired in the Brazilian Amazon region. Patients were submitted to in vivo study of drug sensitivity, after chemotherapy with either 4-aminoquinolines (chloroquine or amodiaquine) or quinine. Adequate drug absorption was confirmed by standard urine excretion tests for antimalarials. Eight patients could be followed up to 28 days. Among these in vivo resistance (R I and R II responses) was seen in all patients who received 4-amino-quinolines. One patient treated with quinine exhibited a R III response. Peripheral blood samples of the same patients were submitted to in vitro microtests for sensitivity to antimalarials. Out of nine successful tests, resistance to chloroquine and amodiaquine was found in 100% and resistance to quinine in 11.11% of isolates. Probit analysis of log dose-response was used to determine effective concentrations EC50, EC90 and EC99 to the studied drugs. Good correlation between in vivo and in vitro results was seen in six patients. The results emphasize high levels of P. falciparum resistance to 4-aminoquinolines and suggest an increase in resistance to quinine in the Brazilian Amazon region, reinforcing the need for continuous monitoring of drug sensitivity to adequate chemotherapy according to the most efficacious drug regimens.

Malária autóctone notificada no Estado de São Paulo: aspectos clínicos e epidemiológicos de 1980 a 2007

INTRODUCTION: Autochthonous malaria in the State of Sao Paulo is characterized by sporadic outbreaks in the western region and by persistent transmission in the eastern region, where oligosymptomatic cases with low parasitemia due to Plasmodium vivax occur. The objectives of this study were to assess the completeness of autochthonous malaria notification forms; to estimate the incidence trends of autochthonous cases in the State of Sao Paulo from 1980 to 2007; and to analyze the clinical and epidemiological patterns of cases in two autochthonous regions over this period. METHODS: This was a descriptive study that analyzed 18 variables on the notification form for malaria in the State of Sao Paulo, comparing these two regions over two periods (1980-1993 and 1994-2007). The data sources were SUCEN/SES/SP, SINAN/CVE/SES/SP and DATASUS. RESULTS: The completeness was greater than 85% for 11 variables. The incidence trend was decreasing. There were 821 autochthonous cases: 91.6% occurred in the eastern region, predominantly due to Plasmodium vivax. Asymptomatic infection had a higher percentage in the second period (p < 0.001). CONCLUSIONS: The completeness of the information was satisfactory. The clinical differences observed deserve attention from epidemiological surveillance agencies, which need to deal with the challenge of asymptomatic infection by Plasmodium.

Malaria diagnosis from pooled blood samples: comparative analysis of real-time PCR, nested PCR and immunoassay as a platform for the molecular and serological diagnosis of malaria on a large-scale

Malaria diagnoses has traditionally been made using thick blood smears, but more sensitive and faster techniques are required to process large numbers of samples in clinical and epidemiological studies and in blood donor screening. Here, we evaluated molecular and serological tools to build a screening platform for pooled samples aimed at reducing both the time and the cost of these diagnoses. Positive and negative samples were analysed in individual and pooled experiments using real-time polymerase chain reaction (PCR), nested PCR and an immunochromatographic test. For the individual tests, 46/49 samples were positive by real-time PCR, 46/49 were positive by nested PCR and 32/46 were positive by immunochromatographic test. For the assays performed using pooled samples, 13/15 samples were positive by real-time PCR and nested PCR and 11/15 were positive by immunochromatographic test. These molecular methods demonstrated sensitivity and specificity for both the individual and pooled samples. Due to the advantages of the real-time PCR, such as the fast processing and the closed system, this method should be indicated as the first choice for use in large-scale diagnosis and the nested PCR should be used for species differentiation. However, additional field isolates should be tested to confirm the results achieved using cultured parasites and the serological test should only be adopted as a complementary method for malaria diagnosis.

Evaluation of a rapid dipstick test, Malar-CheckTM, for the diagnosis of Plasmodium falciparum malaria in Brazil

The present study was carried out to evaluate the Malar-Check trade mark Pf test, an immunochromatographic assay that detects Plasmodium falciparum Histidine Rich Protein II, does not require equipment, and is easy and rapid to perform. In dilution assays performed to test sensitivity against known parasite density, Malar-Check were compared with thick blood smear (TBS), the gold standard for diagnosis. Palo Alto isolate or P. falciparum blood from patients with different parasitemias was used. The average cut-off points for each technique in three independent experiments were 12 and 71 parasites/mm3 (TBS and Malar-Check, respectively). In the field assays, samples were collected from patients with fever who visited endemic regions. Compared to TBS, Malar-Check yielded true-positive results in 38 patients, false-positive results in 3, true-negative results in 23, and false-negative result in 1. Malar-Check performed with samples from falciparum-infected patients after treatment showed persistence of antigen up to 30 days. Malar-Check should aid the diagnosis of P. falciparum in remote areas and improve routine diagnosis even when microscopy is available. Previous P. falciparum infection, which can determine a false-positive test in cured individuals, should be considered. The prompt results obtained with the Malar-Check for early diagnosis could avoid disease evolution to severe cases.

In vitro chloroquine resistance modulation study on fresh isolates of Brazilian Plasmodium falciparum: Intrinsic antimalarial activity of phenothiazine drugs

Phenothiazine drugs - fluphenazine, chlorpromazine, methotrimeprazine and trifluoperazine - were evaluated as modulating agents against Brazilian chloroquine-resistant fresh isolates of Plasmodium falciparum. Aiming to simulate therapeutic schedules, chloroquine was employed at the concentration used for sensitive falciparum malaria treatment and anti-psychotic therapeutic concentrations of the phenothiazine drugs were adopted in two-fold serial dilutions. The in vitro microtechnique for drug susceptibility was employed. Unlike earlier reported data, the phenothiazine modulating effect was not observed. However, all the drugs demonstrated intrinsic antiplasmodial activity in concentrations lower than those described in the literature. In addition, IC50 estimates have been shown to be inferior to the usual anti-psychotic therapeutic concentrations. Statistical analysis also suggested an increase in the parasitaemia rate or, even, a predominant antiparasitic effect of phenothiazine over chloroquine when used in combination.

Unexpected detection of Plasmodium vivax and Plasmodium falciparum DNA in asymptomatic blood donors: fact or artifact?

A study searching for Plasmodium vivax and Plasmodium falciparum DNA among blood donors from the non-endemic area in Brazil reported a rate of 7.41%. This number is at least three times higher than what has been observed in blood donors from the Amazon, an endemic area concentrating >99% of all malaria cases in Brazil. Moreover, the majority of the donors were supposedly infected by P. falciparum, a rare finding both in men and anophelines from the Atlantic forest. These findings shall be taken with caution since they disagree with several publications in the literature and possibly overestimate the actual risk of malaria transmission by blood transfusion in São Paulo city.

Survey of Plasmodium spp. in free-ranging neotropical primates from the Brazilian Amazon region impacted by anthropogenic actions.

This study investigated Plasmodium spp. infection in free-ranging neotropical primates from Brazilian Amazon regions under the impact of major anthropogenic actions. Blood samples from 19 new world primates were collected and analyzed with microscopic and molecular procedures. The prevalence of Plasmodium infection was 21.0% (4/19) and PCR positive samples were identified as P. brasilianum. Considering the social-economic changes that the Amazon is facing, the prevalence of P. brasilianum infection highlights the necessity to closely monitor the movement of both human and non-human primate populations, in order to mitigate pathogen exposure and the introduction of new agents into previously naïve areas.