Gisely Cardoso de Melo

Concurrent Helminthic Infection Protects Schoolchildren with Plasmodium vivax from Anemia

Background Plasmodium vivax is responsible for a significant portion of malaria cases worldwide, especially in Asia and Latin America, where geo-helminthiasis have a high prevalence. Impact of the interaction between vivax malaria and intestinal helminthes has been poorly explored. The objective of this study was to evaluate the influence of intestinal helminthiasis on the concentration of hemoglobin in children with Plasmodium vivax malaria in rural areas in the municipality of Careiro, in the Western Brazilian Amazon. Methodology/Principal Findings A cohort study was conducted from April to November 2008, enrolling children from 5 to 14 years old in two rural areas endemic for malaria. A cross-sectional evaluation was performed in April to actively detect cases of malaria and document baseline hemoglobin and nutritional status. Children were followed-up for six months through passive case detection of malaria based on light microscopy. Throughout the follow-up interval, hemoglobin value and stool examination (three samples on alternate days) were performed on children who developed P. vivax malaria. For 54 schoolchildren with a single infection by P. vivax, hemoglobin during the malaria episode was similar to the baseline hemoglobin for children co-infected with Ascaris lumbricoides (n = 18), hookworm (n = 11) and Trichuris trichiura (n = 9). In children without intestinal helminthes, a significant decrease in the hemoglobin during the malarial attack was seen as compared to the baseline concentration. In the survival analysis, no difference was seen in the time (in days) from the baseline cross-sectional to the first malarial infection, between parasitized and non-parasitized children. Conclusion/Significance For the first time, a cohort study showed that intestinal helminthes protect against hemoglobin decrease during an acute malarial attack by P. vivax.

Thrombocytopenia in Plasmodium vivax Malaria Is Related to Platelets Phagocytosis

Background Although thrombocytopenia is a hematological disorder commonly reported in malarial patients, its mechanisms are still poorly understood, with only a few studies focusing on the role of platelets phagocytosis. Methods and Findings Thirty-five malaria vivax patients and eight healthy volunteers (HV) were enrolled in the study. Among vivax malaria patients, thrombocytopenia (<150,000 platelets/µL) was found in 62.9% (22/35). Mean platelet volume (MPV) was higher in thrombocytopenic patients as compared to non- thrombocytopenic patients (p = 0.017) and a negative correlation was found between platelet count and MPV (r = −0.483; p = 0.003). Platelets from HV or patients were labeled with 5-chloromethyl fluorescein diacetate (CMFDA), incubated with human monocytic cell line (THP-1) and platelet phagocytosis index was analyzed by flow cytometry. The phagocytosis index was higher in thrombocytopenic patients compared to non-thrombocytopenic patients (p = 0.042) and HV (p = 0.048). A negative correlation was observed between platelet count and phagocytosis index (r = −0.402; p = 0.016). Platelet activation was assessed measuring the expression of P-selectin (CD62-P) in platelets’ surface by flow cytometry. No significant difference was found in the expression of P-selectin between thrombocytopenic patients and HV (p = 0.092). After evaluating the cytokine profile (IL-2, IL-4, IL-6, IL-10, TNF-α, IFN-γ and IL-17) in the patients’ sera, levels of IL-6, IL-10 and IFN-γ were elevated in malaria patients compared to HV. Moreover, IL-6 and IL-10 values were higher in thrombocytopenic patients than non-thrombocytopenic ones (p = 0.044 and p = 0.017, respectively. In contrast, TNF-α levels were not different between the three groups, but a positive correlation was found between TNF-α and phagocytosis index (r = −0.305; p = 0.037). Conclusion/Significance Collectively, our findings indicate that platelet phagocytosis may contribute to thrombocytopenia found in vivax malaria. Finally, we believe that this study opens new avenues to explore the mechanisms involved in platelet dysfunction, commonly found in vivax malaria patients.

Clinical complications of G6PD deficiency in Latin American and Caribbean populations: systematic review and implications for malaria elimination programmes

BackgroundAlthough G6PDd individuals are generally asymptomatic throughout their life, the clinical burden of this genetic condition includes a range of haematological conditions, including acute haemolytic anaemia (AHA), neonatal jaundice (NNJ) and chronic non-sphaerocytic anaemia (CNSA). In Latin America (LA), the huge knowledge gap regarding G6PDd is related to the scarce understanding of the burden of clinical manifestation underlying G6PDd carriage. The aim of this work was to study the clinical significance of G6PDd in LA and the Caribbean region through a systematic review.MethodsA systematic search of the published literature was undertaken in August 2013. Bibliographies of manuscripts were also searched and additional references were identified. Only original research was included. All study designs were included, as long as any clinical information was present. Studies were eligible for inclusion if they reported clinical information from populations living in LA or Caribbean countries or about migrants from these countries living in countries outside this continent.ResultsThe Medline search generated 487 papers, and the LILACS search identified 140 papers. After applying the inclusion criteria, 100 original papers with any clinical information on G6PDd in LA were retrieved. Additionally, 16 articles were included after reading the references from these papers. These 116 articles reported data from 18 LA and Caribbean countries. The major clinical manifestations reported from LA countries were those related to AHA, namely drug-induced haemolysis. Most of the published works regarding drug-induced haemolysis in LA referred to haemolytic crises in P. vivax malaria patients during the course of the treatment with primaquine (PQ). Favism, infection-induced haemolysis, NNJ and CNSA appear to play only a minor public health role in this continent.ConclusionHaemolysis in patients using PQ seems to be the major clinical manifestation of G6PDd in LA and contributes to the morbidity of P. vivax infection in this continent, although the low number of reported cases, which could be linked to under-reporting of complications. These results support the need for better strategies to diagnose and manage G6PDd in malaria field conditions. Additionally, Malaria Control Programmes in LA should not overlook this condition in their national guidelines.

High levels of IgG3 anti ICB2-5 in Plasmodium vivax-infected individuals who did not develop symptoms

BackgroundPlasmodium vivax has the potential to infect 2.85 billion individuals worldwide. Nevertheless, the limited number of studies investigating the immune status of individuals living in malaria-endemic areas, as well as the lack of reports investigating serological markers associated with clinical protection, has hampered development of vaccines for P. vivax. It was previously demonstrated that naturally total IgG against the N-terminus of P. vivax merozoite surface protein 1 (Pv-MSP1) was associated with reduced risk of malarial infection.MethodsImmune response against Pv-MSP1 (N-terminus) of 313 residents of the Rio Pardo rural settlement (Amazonas State, Brazil) was evaluated in a cross-sectional and longitudinal follow up over two months (on site) wherein gold standard diagnosis by thick blood smear and rRNA gene-based nested real-time PCR were used to discriminate symptomless Plasmodium vivax-infected individuals who did not develop clinical symptoms during a 2-months from those uninfected ones or who have had acute malaria. The acquisition of antibodies against Pv-MSP1 was also evaluated as survival analysis by prospective study over a year collecting information of new malaria infections in surveillance database.ResultsThe majority of P. vivax-infected individuals (52-67%) showed immune recognition of the N-terminus of Pv-MSP1. Interesting data on infected individuals who have not developed symptoms, total IgG levels against the N-terminus Pv-MSP1 were age-dependent and the IgG3 levels were significantly higher than levels of subjects had acute malaria or those uninfected ones. The total IgG anti ICB2-5 was detected to be an important factor of protection against new malaria vivax attacks in survival analysis in a prospective survey (p = 0.029).ConclusionsThe study findings illustrate the importance of IgG3 associated to 2-months of symptomless in P. vivax infected individuals and open perspectives for the rationale of malaria vaccine designs capable to sustain high levels of IgG3 against polymorphic malaria antigens.

Integrated vector management targeting Anopheles darlingi populations decreases malaria incidence in an unstable transmission area, in the rural Brazilian Amazon.

BackgroundStudies on vector behaviour should be conducted in order to evaluate the effectiveness of vector control measures on malaria protection in endemic areas of Latin America, where P. vivax predominates. This work aims to investigate the fauna of anopheline mosquitoes and verify the impact of integrated vector management in two colonization projects in the Careiro Municipality, Western Brazilian Amazon.MethodsFour mosquitoes’ captures were carried out from August 2008 to March 2010, with an interval of six months between each collection. Since September 2009 a large programme to reduce the burden of malaria has started in the two communities by distribution of insecticide-treated bed nets (ITN) and intensification of indoor residual spraying (IRS). Human biting rates (HBRs), entomological inoculation rates (EIRs), malaria incidence rate (MIR) and Plasmodium carrier’s prevalence were used as outcomes to estimate the impact of the control measures.ResultsA total of 3,189 anophelines were collected, belonging to 13 species. Anopheles darlingi was the predominant species in the period (42.6%), followed by Anopheles albitarsis (38.4%). An. darlingi HBRs showed a notable decreasing trend from the start to the end of the study. Conversely, An. albitarsis increased its contribution to overall HBRs throughout the study. For An. darlingi there was a significant positive correlation between HBRs and MIR (p = 0.002). Anopheles albitarsis HBRs showed a significant negative correlation with the corresponding MIR (p = 0.045). EIR from total anophelines and from An. darlingi and An. albitarsis presented decreasing patterns in the successive collections. Four species of anophelines (An. darlingi, An. albitarsis, Anopheles braziliensis and Anopheles nuneztovari) were naturally infected with Plasmodium, albeit at very low infection rates. There were a decrease in the MIR for both vivax and falciparum malaria and in the prevalence of Plasmodium vivax and Plasmodium falciparum carriers during the period of study.ConclusionsThere is strong evidence of association between the density of An. darlingi and the incidence of malaria in the studies sites, further highlighting the importance of this vector in malaria transmission in this region. An. darlingi susceptibility to control using ITN and IRS is likely to be high in the rural settlements studied.

Qualidade de hortaliças comercializadas no noroeste do Paraná, Brasil

This work had the objective of performing a parasitological evaluation of fresh vegetables colletedfrom production places and free markets of Maringa, Parana. From July/2002 to July/2004, 66samples were collected in 13 smallholdings and 181 samples in 17 free markets. Heads of lettuce(varieties flat, curly and American), watercress and rocket salad, picked up randomly and in triplicate,were submitted to Oliveira & Germano’s and Kynioun staining techniques and to the research oftotal and thermotolerant coliforms. The total index of parasitism of the vegetables of free markets(63%) and smallholdings (71.1%) didn’t differ statistically. The fresh vegetables from smallholdingshave shown more protozoa than (10/47) that from the free markets (19/114). Rhabditoidea larvaeand Ancylostomatoidea eggs were equally distributed in the fresh vegetables of both places. Parasiticforms of other species were also found. All the curly lettuce samples collected in the smallholdingspresented total and thermotolerant bacteria index incompatible with the human consumption. Thedifficult in doing laboratorial diagnose at the genre and specie level of the human parasites in freshvegetables and the sanitary surveillance challenges in the obtaining of good quality products werediscussed.

G6PD deficiency in Latin America: systematic review on prevalence and variants

Plasmodium vivax radical cure requires the use of primaquine (PQ), a drug that induces haemolysis in glucose-6-phosphate dehydrogenase deficient (G6PDd) individuals, which further hampers malaria control efforts. The aim of this work was to study the G6PDd prevalence and variants in Latin America (LA) and the Caribbean region. A systematic search of the published literature was undertaken in August 2013. Bibliographies of manuscripts were also searched and additional references were identified. Low prevalence rates of G6PDd were documented in Argentina, Bolivia, Mexico, Peru and Uruguay, but studies from Curaçao, Ecuador, Jamaica, Saint Lucia, Suriname and Trinidad, as well as some surveys carried out in areas of Brazil, Colombia and Cuba, have shown a high prevalence (> 10%) of G6PDd. The G6PD A-202A mutation was the variant most broadly distributed across LA and was identified in 81.1% of the deficient individuals surveyed. G6PDd is a frequent phenomenon in LA, although certain Amerindian populations may not be affected, suggesting that PQ could be safely used in these specific populations. Population-wide use of PQ as part of malaria elimination strategies in LA cannot be supported unless a rapid, accurate and field-deployable G6PDd diagnostic test is made available.

Expression levels of pvcrt-o and pvmdr-1 are associated with chloroquine resistance and severe Plasmodium vivax malaria in patients of the Brazilian Amazon.

Molecular markers associated with the increase of chloroquine resistance and disease severity in Plasmodium vivax are needed. The objective of this study was to evaluate the expression levels of pvcrt-o and pvmdr-1 genes in a group of patients presenting CQRPv and patients who developed severe complications triggered exclusively by P. vivax infection. Two different sets of patients were included to this comprehensive study performed in the Brazilian Amazon: 1) patients with clinically characterized chloroquine-resistant P. vivax compared with patients with susceptible parasites from in vivo studies and 2) patients with severe vivax malaria compared with patients without severity. Quantitative real-time PCR was performed to compare the transcript levels of two main transporters genes, P. vivax chloroquine resistance transporter (pvcrt-o) and the P. vivax multidrug resistance transporter (pvmdr-1). Twelve chloroquine resistant cases and other 15 isolates from susceptible cases were included in the first set of patients. For the second set, seven patients with P. vivax-attributed severe and 10 mild manifestations were included. Parasites from patients with chloroquine resistance presented up to 6.1 (95% CI: 3.8–14.3) and 2.4 (95% CI: 0.53–9.1) fold increase in pvcrt-o and pvmdr-1 expression levels, respectively, compared to the susceptible group. Parasites from the severe vivax group had a 2.9 (95% CI: 1.1–8.3) and 4.9 (95% CI: 2.3–18.8) fold increase in pvcrt-o and pvmdr-1 expression levels as compared to the control group with mild disease. These findings suggest that chloroquine resistance and clinical severity in P. vivax infections are strongly associated with increased expression levels of the pvcrt-o and pvmdr-1 genes likely involved in chloroquine resistance.

In vitro chloroquine resistance for Plasmodium vivax isolates from the Western Brazilian Amazon

BackgroundChloroquine (CQ) and primaquine (PQ) are still the drugs of choice to treat Plasmodium vivax malaria in many endemic areas, Brazil included. There is in vivo evidence for the P. vivax resistance to CQ in the Brazilian Amazon, where the increase in the proportion of P. vivax malaria parallels the increase of unusual clinical complications related to this species. In this study, in vitro CQ and mefloquine (MQ)-susceptibility of P. vivax isolates from the Western Brazilian Amazon was tested using the double-site enzyme-linked lactate dehydrogenase immunodetection (DELI) assay.MethodsA total of 112 P. vivax isolates were tested in vitro for CQ-susceptibility and out of these 47 were also tested for MQ-susceptibility. The DELI assay was used to detect P. vivax growth at 48-hour short-term culture in isolates with ring stages ranging from 50 to %. Each isolate was tested in triplicate and geometric means of IC50’s was obtained. Nineteen isolates were genetically characterized for pvdhfr, pvmrp1, pvmdr1 and pvdhps candidate genes likely related to CQ resistance (10 with IC50<40 nM and 9 with IC50 >100 nM).ResultsTwelve out of 112 isolates were considered resistant to CQ, resulting in 10.7% (IC95% 5.0-16.4), while 3 out of 47 (6.4%; IC95% 0.0-12.8) were resistant to MQ. A discrete correlation was observed between IC50’s of CQ and MQ (Spearman=0.294; p=0.045). For pvdhps gene, a non-synonymous mutation was found at codon 382 (S→C) in 5/8 CQ-sensitive samples and 1/9 CQ-resistant samples (p=0.027). The other molecular markers were not associated to CQ-susceptibility.ConclusionsIn vitro CQ-resistance estimated in this study, estimated by the DELI test, was very similar to that observed in clinical trials, suggesting that in vitro procedures developed by capable local laboratories are useful in the surveillance of CQ-resistance in the Amazon; concurrent Amazon P. vivax strains with both CQ and MQ resistance may be common; and a non-synonymous mutation at pvdhps codon 382 (S→C) was associated to in vitro susceptibility to CQ, needing further studies to be confirmed.

P. vivax malaria and dengue fever co-infection: a cross-sectional study in the Brazilian Amazon.

Background Malaria and dengue are the most prevalent vector-borne diseases worldwide and represent major public health problems. Both are endemic in tropical regions, propitiating co-infection. Only few co-infection cases have been reported around the world, with insufficient data so far to enhance the understanding of the effects of co-infection in the clinical presentation and severity. Methodology/Principal Findings A cross-sectional study was conducted (2009 to 2011) in hospitalized patients with acute febrile syndrome in the Brazilian Amazon. All patients were submitted to thick blood smear and PCR for Plasmodium sp. detection, ELISA, PCR and NS1 tests for dengue, viral hepatitis, HIV and leptospirosis. In total, 1,578 patients were recruited. Among them, 176 (11.1%) presented P. vivax malaria mono-infection, 584 (37%) dengue fever mono-infection, and 44 (2.8%) were co-infected. Co-infected patients had a higher chance of presenting severe disease (vs. dengue mono-infected), deep bleeding (vs. P. vivax mono-infected), hepatomegaly, and jaundice (vs. dengue mono-infected). Conclusions/Significance In endemic areas for dengue and malaria, jaundice (in dengue patients) and spontaneous bleeding (in malaria patients) should raise the suspicion of co-infection. Besides, whenever co-infection is confirmed, we recommend careful monitoring for bleeding and hepatic complications, which may result in a higher chance of severity, despite of the fact that no increased fatality rate was seen in this group.