Licia Natal Fernandes

Natural infection in anopheline species and its implications for autochthonous malaria in the Atlantic forest in Brazil

BackgroundA descriptive study was carried out in an area of the Atlantic Forest with autochthonous malaria in the Parelheiros subdistrict on the periphery of the municipality of São Paulo to identify anopheline fauna and anophelines naturally infected with Plasmodium as well as to discuss their role in this peculiar epidemiological context.MethodsEntomological captures were made from May 2009 to April 2011 using Shannon traps and automatic CDC traps in four areas chosen for their different patterns of human presence and incidences of malaria (anthropic zone 1, anthropic zone 2, transition zone and sylvatic zone). Natural Plasmodium infection was detected by nested PCR based on amplification of the 18S rRNA gene.ResultsIn total, 6,073 anophelines were collected from May 2009 to April 2011, and six species were identified in the four zones. Anopheles cruzii was the predominant species in the three environments but was more abundant in the sylvatic zone.Anopheles (Kerteszia) cruzii specimens from the anthropic and sylvatic zones were positive for P. vivax and P. malariae. An. (Ker.) bellator, An. (Nys.) triannulatus, An. (Nys.) strodei, An. (Nys.) lu tzi and An. (Ano) maculipes were found in small numbers. Of these, An. (Nys.) triannulatus and An. (Nys.) lutzi, which were collected in the anthropic zone, were naturally infected with P. vivax while An. (Nys.) triannulatus from the anthropic zones and An. (Nys.) strodei from the transition zone were positive for P. malariae.ConclusionThese results confirm that Anopheles (Kerteszia) cruzii plays an important role as a major Plasmodium vector. However, the finding of other naturally infected species may indicate that secondary vectors are also involved in the transmission of malaria in the study areas. These findings can be expected to help in the implementation of new measures to control autochthonous malaria in areas of the Atlantic Forest.

Risk of Giardia infection for drinking water and bathing in a peri-urban area in Sao Paulo, Brazil.

A high incidence of waterborne diseases is observed worldwide and in order to address contamination problems prior to an outbreak, quantitative microbial risk assessment is a useful tool for estimating the risk of infection. The objective of this paper was to assess the probability of Giardia infection from consuming water from shallow wells in a peri-urban area. Giardia has been described as an important waterborne pathogen and reported in several water sources, including ground waters. Sixteen water samples were collected and examined according to the US EPA (1623, 2005). A Monte Carlo method was used to address the potential risk as described by the exponential dose response model. Giardia cysts occurred in 62.5% of the samples (<0.1–36.1 cysts/l). A median risk of 10−1 for the population was estimated and the adult ingestion was the highest risk driver. This study illustrates the vulnerability of shallow well water supply systems in peri-urban areas.

Genotypic Characterization of Cryptosporidium hominis from Water Samples in São Paulo, Brazil

The protozoan parasite Cryptosporidium has emerged as one of the most important water contaminants, causing waterborne outbreaks of diarrheal diseases worldwide. The small size of oocysts under the microscope and the possibility of changes in characteristics of oocysts, mainly in environmental samples, make the taxonomy of the genus difficult if morphologic characteristics are considered. This limitation encouraged the application of molecular methods to identify this microorganism. The aim of this study was to detect and identify by nested-polymerase chain reaction oocysts of Cryptosporidium present in water samples in the state of São Paulo, Brazil. Water samples were concentrated through a membrane filter, DNA was extracted by using a standard technique, and both amplification reactions used forward and reverse oligonucleotides that were complementary to Cryptosporidium 18S ribosomal RNA gene sequences. Thirty water samples from different sites of collection in the state of São Paulo were evaluated. Cryptosporidium oocysts were detected in 30% of the samples. By genoptyping, C. hominis and Cryptosporidium sp. were identified in recreational water and C. meleagridis was identified in surface water samples. This is the first report of C. hominis in environmental samples in Brazil. Although identification of Cryptosporidium is still a difficult task, molecular methods are essential for specific identification and are a helpful tool to aid to understand the epidemiology of this parasite in Brazil.

Detection of Culex flavivirus and Aedes flavivirus nucleotide sequences in mosquitoes from parks in the city of Sao Paulo, Brazil

The dengue viruses are widespread in Brazil and are a major public health concern. Other flaviviruses also cause diseases in humans, although on a smaller scale. The city of São Paulo is in a highly urbanized area with few green spaces apart from its parks, which are used for recreation and where potential vertebrate hosts and mosquito vectors of pathogenic Flavivirus species can be found. Although this scenario can contribute to the transmission of Flavivirus to humans, little is known about the circulation of members of this genus in these areas. In light of this, the present study sought to identify Flavivirus infection in mosquitoes (Diptera: Culicidae) collected in parks in the city of São Paulo. Seven parks in different sectors of the city were selected. Monthly mosquito collections were carried out in each park from March 2011 to February 2012 using aspiration and traps (Shannon and CD C-CO2). Nucleic acids were extracted from the mosquitoes collected and used for reverse-transcriptase and real-time polymerase chain reactions with genus-specific primers targeting a 200-nucleotide region in the Flavivirus NS5 gene. Positive samples were sequenced, and phylogenetic analyses were performed. Culex and Aedes were the most frequent genera of Culicidae collected. Culex flavivirus (CxFV)-related and Aedes flavivirus (AEFV)- related nucleotide sequences were detected in 17 pools of Culex and two pools of Aedes mosquitoes, respectively, among the 818 pools of non-engorged females analyzed. To the best of our knowledge, this is the first report of CxFV and AEFV in the city of São Paulo and Latin America, respectively. Both viruses are insect- specific flaviviruses, a group known to replicate only in mosquito cells and induce a cytopathic effect in some situations. Hence, our data suggests that CxFV and AEFV are present in Culex and Aedes mosquitoes, respectively, in parks in the city of São Paulo. Even though Flavivirus species of medical importance were not detected, surveillance is recommended in the study areas because of the presence of vertebrates and mosquitoes that could act as amplifying hosts and vectors of flaviviruses, providing the required conditions for circulation of these viruses.

Clustering symptoms of non-severe malaria in semi-immune Amazonian patients

Malaria is a disease that generates a broad spectrum of clinical features. The purpose of this study was to evaluate the clinical spectrum of malaria in semi-immune populations. Patients were recruited in Mâncio Lima, a city situated in the Brazilian Amazon region. The study included 171 malaria cases, which were diagnosed via the use of a thick blood smear and confirmed by molecular methods. A questionnaire addressing 19 common symptoms was administered to all patients. Multiple correspondence analysis and hierarchical cluster analysis were performed to identify clusters of symptoms, and logistic regression was used to identify factors associated with the occurrence of symptoms. The cluster analysis revealed five groups of symptoms: the first cluster, which included algic- and fever-related symptoms, occurred in up to 95.3% of the cases. The second cluster, which comprised gastric symptoms (nausea, abdominal pain, inappetence, and bitter mouth), occurred in frequencies that ranged between 35.1% and 42.7%, and at least one of these symptoms was observed in 71.9% of the subjects. All respiratory symptoms were clustered and occurred in 42.7% of the malaria cases, and diarrhea occurred in 9.9% of the cases. Symptoms constituting the fifth cluster were vomiting and pallor, with a 14.6% and 11.7% of prevalence, respectively. A higher parasitemia count (more than 300 parasites/mm3) was associated with the presence of fever, vomiting, dizziness, and weakness (P < 0.05). Arthralgia and myalgia were associated with patients over the age of 14 years (P < 0.001). Having experienced at least eight malaria episodes prior to the study was associated with a decreased risk of chills and fever and an increased risk of sore throat (P < 0.05). None of the symptoms showed an association with gender or with species of Plasmodium. The clinical spectrum of malaria in semi-immune individuals can have a broad range of symptoms, the frequency and intensity of which are associated with age, past exposure to malaria, and parasitemia. Understanding the full spectrum of nonsevere malaria is important in endemic areas to guide both passive and active case detection, for the diagnosis of malaria in travelers returning to non-endemic areas, and for the development of vaccines aimed to decrease symptom severity.

Reassessment of asymptomatic carriers of Plasmodium spp. in an endemic area with a very low incidence of malaria in extra-Amazonian Brazil

BackgroundRegions with residual transmission are potential obstacles to the elimination of malaria. It is, therefore, essential to understand the factors associated with the maintenance of endemic malaria in these areas. The objective was to investigate whether the status of asymptomatic carriers of Plasmodium spp. DNA is maintained in the long term in an extra-Amazonian region of Brazil with low incidence, residual malaria transmission.MethodsAsymptomatic carriers of Plasmodium DNA detected in a survey carried out between 2001 and 2004 were reassessed between 2010 and 2011 using questionnaires, PCR and thick and thin blood smear tests three times at 3-month intervals.ResultsOf the 48 carriers detected between 2001 and 2004, 37 were located. Of these, only two had positive PCR results and, as in the first survey, Plasmodium malariae DNA was detected.ConclusionThe findings suggest that untreated dwellers from this extra-Amazonian region, who initially harbour malaria parasites, may become negative without ever developing apparent symptoms of the disease. Although the possibility of re-infection cannot be ruled out, the finding of two individuals harbouring P. malariae, both in the first and in the second survey, may be compatible with a long-term carrier state for this parasite. Since most clinical cases of malaria in the region are a consequence of infection by Plasmodium vivax, the epidemiological impact of such long-term carriage would be limited.

A survey of flaviviruses in birds in green areas of São Paulo, Brazil

INTRODUCTION This study aimed to investigate Flavivirus infection in birds captured in green areas of São Paulo. METHODS Oropharyngeal swabs, cloacal swabs, and blood samples from 170 birds captured in two green areas in São Paulo, Brazil were subjected to real time-polymerase chain reaction (RT-qPCR) analysis of Flavivirus specific NS5 gene fragment. RESULTS All samples were negative for the presence of viral ribonucleic acid (RNA). CONCLUSIONS Despite the negative results, Flavivirus surveillance must be performed regularly due to favorable ecological conditions for virus circulation and transmission among birds in these areas and their close proximity to humans.