Zoonotic Malaria Risk in Serra Do Mar, Atlantic Forest, Brazil.

Here, the main goal is to assess natural infections of Plasmodium spp. in anophelines in a forest reserve from the same region where we previously found a surprisingly high rate (5.2%) of plasmodia infections (n = 25) in Kerteszia mosquitoes (N = 480) on the slopes of Serra do Mar, Atlantic Forest, Brazil. The mosquito collection sampling was carried out at the Legado das Águas Forest Reserve using CDC light traps and Shannon traps at night (5-10 pm) in 3-day collections in November 2021 and March, April, May, and November 2022. The captured specimens were morphologically identified at the species level and had their genomic DNA extracted in pools of up to 10 mosquitoes/pool. Each pool was tested using 18S qPCR and cytb nested PCR plus sequencing. A total of 5301 mosquitoes, mostly belonging to the genus Kerteszia (99.7%), were sampled and sorted into 773 pools. Eight pools positive for Plasmodium spp. were identified: four for Plasmodium spp., one for P. vivax or P. simium, one for P. malariae or P. brasilianum, and two for the P. falciparum-like parasite. After Sanger sequencing, two results were further confirmed: P. vivax or P. simium and P. malariae or P. brasilianum. The minimum infection rate for Kerteszia mosquitoes was 0.15% (eight positive pools/5285 Kerteszia mosquitoes). The study reveals a lower-than-expected natural infection rate (expected = 5.2% vs. observed = 0.15%). This low rate relates to the absence of Alouatta monkeys as the main simian malaria reservoir in the studied region. Their absence was due to a significant population decline following the reemergence of yellow fever virus outbreaks in the Atlantic Forest from 2016 to 2019. However, this also indicates the existence of alternative reservoirs to infect Kerteszia mosquitoes. The found zoonotic species of Plasmodium, including the P. falciparum-like parasite, may represent a simian malaria risk and thus a challenge for malaria elimination in Brazil.

Investigação sobre a presença de Plasmodium spp. em Anophelinae (Diptera: Culicidae) do Vale do Ribeira, sudeste da Mata Atlântica, Estado de São Paulo, Brasil / Investigation of the presence of Plasmodium spp. in Anophelinae (Diptera: Culicidae) from Vale do Ribeira, southeastern Atlantic Forest, State of São Paulo, Brazil

Objetivos: Identificar espécies de anofelinos presentes na região do Vale do Ribeira, Sudeste de São Paulo, e a circulação de plasmódios por estes anofelinos. Método: Foram utilizadas armadilhas CDC com atração luminosa e armadilhas de Shannon no período que compreende das 17:00 às 22:00 horas e atração por humano protegido realizadas das 9:00 às 12:00 horas durante três dias consecutivos em seis campanhas, realizadas de setembro de 2021 a novembro de 2022. Os espécimes capturados foram identificados morfologicamente, separados por espécie e tiveram seu DNA genômico extraído em grupos de até 10 mosquitos, considerando os métodos de captura, locais, datas e horas. Os grupos de DNA genômico foram analisados utilizando tecnologia de PCR em tempo real para amplificar fragmento de 157 a 165 pares de bases da unidade 18S do DNA ribossômico, que permite identificar a presença de plasmódios. As amostras positivas foram submetidas a técnicas de PCR aninhado de fragmento do gene citocromo b do genoma mitocondrial. De oito amostras analisadas, duas foram positivas e tiveram seus amplicons sequenciados empregando tecnologia de Sanger. Resultados: Foram coletados 5.577 mosquitos classificados no gênero Kerteszia (99,62%) e gênero Anopheles (0,38%), agrupados em 811 grupos. Foram identificados oito grupos positivos para plasmódios, sendo um para Plasmodium vivax / Plasmodium simium, um para Plasmodium malariae / Plasmodium brasilianum, dois para Plasmodium falciparum e quatro grupos positivos apenas para Plasmodium spp. O sequenciamento Sanger confirmou a infecção por P. vivax / P. simium em uma amostra, e P. malariae / P. brasilianum em outra. A proporção total de anofelinos infectados por Plasmodium spp. foi 0,5% (4/811), 0,2% dos anofelinos coletados apresentavam infecção por P. falciparum (2/811), 0,1% apresentavam infecção por P. vivax (1/811) e a mesma proporção de infectados por P. malariae (1/811). A razão mínima de infecção (RMI) para Plasmodium spp. nos anofelinos coletados foi 0,125, para P. vivax foi 0,02, assim como para P. malariae e, para P. falciparum foi 0,035. As estimativas de razão entre mosquitos infectados e mosquitos coletados foram 0,007 para Plasmodium spp., 0,0002 para P. vivax, 0,0005 para P. falciparum e 0,0005 para P. malariae. A razão de infecção absoluta por P. vivax foi 0,02 em março de 2022 e 0,01 por P. falciparum em novembro de 2022 no mesmo ponto de coleta. Conclusão: Foi identificada a presença de plasmódios zoonóticos em área de reserva da Mata Atlântica do sudeste brasileiro, onde a presença de hospedeiros humanos é restrita aos moradores, turistas, trabalhadores da reserva e pesquisadores. A densidade de mosquitos infectados por P. falciparum foi maior do que a de P. vivax e P. malariae. O encontro de mosquitos, naturalmente, infectados por estas espécies de plasmódios indica a necessidade de programas de educação ambiental delineados aos frequentadores e moradores da região da reserva e do entorno. Acresce considerar a necessidade de pesquisas detalhadas sobre as taxas de Plasmodium ocorrendo na área estudada e na Mata Atlântica, com sequenciamentos dos genomas das espécies que são encontradas em localidades diversas do Brasil e América Latina.

Objectives: Identify species of anophelines present in the Vale do Ribeira region, Southeast of São Paulo, and the circulation of plasmodia by these anophelines. Method: CDC light traps and Shannon traps were used in the period from 5:00 p.m. to 10:00 p.m. and attraction by protected human carried out from 9:00 a.m. to 12:00 p.m. for three consecutive days in 6 campaigns, carried out from September 2021 to November 2022. The captured specimens were morphologically identified, separated by species, and had their genomic DNA extracted in pools of up to 10 mosquitoes, considering capture methods, locations, dates and time. By utilizing real-time PCR technology, a 157 to 165 base pairs segment of the 18S ribosomal DNA unit was amplified to identify plasmodia within the genomic DNA pools. The positive samples were submitted to nested PCR techniques of cytochrome b gene fragment of the mitochondrial genome. Of 8 samples analyzed, two were positive and had their amplicons sequenced using Sanger technology. Results: A total of 5,577 mosquitoes belonging to the genus Kerteszia (99.62%) and to the genus Anopheles (0.38%) were collected, grouped into 811 pools. Eight positive pools for plasmodia were identified, one for Plasmodium vivax / Plasmodium simium, one for Plasmodium malariae / Plasmodium. brasilianum, two for Plasmodium falciparum and four pools positive only for Plasmodium spp. Sanger sequencing confirmed P. vivax / P. simium infection in one sample, and P. malariae / P. brasilianum in the other. The total proportion of anophelines infected with Plasmodium spp. was 0.5% (4/811), 0.2% of the collected anophelines had P. falciparum infection (2/811), 0.1% had P. vivax infection (1/811) and the same proportion of infected with P. malariae (1/811). The minimum infection rate (MIR) for Plasmodium spp. in anophelines collected was 0.125, for P. vivax was 0.02, as well as for P. malariae, and for P. falciparum it was 0.035. The ratio estimates between infected and collected mosquitoes were 0.007 for Plasmodium spp., 0.0002 for P. vivax, 0.0005 for P. falciparum, and 0.0005 for P. malariae. The absolute infection ratio for P. vivax was 0.02 in March 2022 and 0.01 for P. falciparum infection in November 2022 at the same collection point. Conclusion: The presence of zoonotic plasmodia was identified in a reserve area of the Atlantic Forest in southeastern Brazil, where the presence of human hosts is restricted to residents, tourists, reserve workers and researchers. The density of mosquitoes infected with P. falciparum was higher than that of P. vivax and P. malariae. The finding of mosquitoes naturally infected by these species of plasmodia shows the need for environmental education programs designed for visitors and residents of the reserve region and surroundings. In addition, consider the need for detailed research on the rates of Plasmodium occurring in the studied area and in the Atlantic Forest, with sequencing of the genomes of the species that are found in different locations in Brazil and Latin America.

Dengue-2 and Guadeloupe Mosquito Virus RNA Detected in Aedes (Stegomyia) spp. Collected in a Vehicle Impound Yard in Santo André, SP, Brazil.

In 2018-2019, we conducted mosquito collections in a municipal vehicle impound yard, which is 10 km from the Serra do Mar Environmental Protection Area in Santo André, SP, Brazil. Our aim is to study arboviruses in the impound yard, to understand the transmission of arboviruses in an urban environment in Brazil. We captured the mosquitoes using human-landing catches and processed them for arbovirus detection by conventional and quantitative RT-PCR assays. We captured two mosquito species, Aedes aegypti (73 total specimens; 18 females and 55 males) and Ae. albopictus (34 specimens; 27 females and 7 males). The minimum infection rate for DENV-2 was 11.5 per 1000 (CI95%: 1-33.9). The detection of DENV-2 RNA in an Ae. albopictus female suggests that this virus might occur in high infection rates in the sampled mosquito population and is endemic in the urban areas of Santo André. In addition, Guadeloupe mosquito virus RNA was detected in an Ae. aegypti female. To our knowledge, this was the first detection of the Guadeloupe mosquito virus in Brazil.

Implication of galanin gene rs948854 polymorphism in depressive symptoms in adolescents.

Genetic, social, and environmental conditions contribute to the development of depression, but the pathophysiological mechanisms are still unclear. Data accumulated in recent years provide significant evidence for a direct role of galanin (GAL). This study aimed to investigate the relation between SNPs in the galaninergic system and depressive symptoms in adolescents. A total of112 adolescents aged 10-18years participated in this study. The Children Depression Inventory (CDI) was used to evaluate depressive symptoms. The effects of rs948854 and rs4432027 SNPs, both located within the promoter region of the GAL gene, rs11665337 in the GALR1 receptor, and rs8836 in the GALR2 receptor on depressive symptoms were examined. The results indicated that 30.4% of the participants had depression. We found that girls were significantly more likely to be depressive than boys. Furthermore, rs948854 minor (G) allele was associated with depressive symptoms. Adolescents carrying the GG and AG genotype for the A/G (rs948854) SNP showed higher CDI scores than those carrying homozygous AA. The binomial logistic regression analysis revealed that adolescents carrying the GG genotype at SNP rs948854 had a higher likelihood of being depressive than adolescents carrying the AA or AG genotypes (P=0.033). Moreover, individuals whose mothers had a positive history for depression and who were sedentary were more likely to display depressive symptoms (P=0.013 and P=0.032, respectively). In conclusion, the SNP rs948854 in the GAL gene seems to be involved in the modulation of depressive state, especially in individuals with GG genotype.

Modification of epigenetic patterns in low birth weight children: importance of hypomethylation of the ACE gene promoter.

There is a growing body of evidence that epigenetic alterations are involved in the pathological mechanisms of many chronic disorders linked to fetal programming. Angiotensin-converting enzyme (ACE) appears as one candidate gene that brings new insights into the epigenetic control and later development of diseases. In this view, we have postulated that epigenetic modifications in the ACE gene might show different interactions between birth weight (BW), blood pressure levels, plasma ACE activity and ACE I/D polymorphism. To explore this hypothesis, we performed a cross-sectional study to evaluate the DNA methylation of 3 CpG sites using pyrosequencing within the ACE gene promoter of peripheral blood leukocytes from 45 LBW children compared with 70 NBW children. Our results have revealed that LBW children have lower methylation levels (P<0.001) in parallel with a higher ACE activity (P = 0.001). Adjusting for prematurity, gender, age, body mass index, and family history of cardiovascular disease did not alter these findings. We have also performed analyses of individual CpG sites. The frequency of DNA methylation was significantly different at two CpG sites (site 1: nucleotide position +555; and site 3: nucleotide position +563). In addition, we have found a significant inverse correlation between degree of DNA methylation and both ACE activity (P<0.001) and systolic blood pressure levels (P<0.001). We also observed that the methylation level was significantly lower in LBW children who are carriers of the DD genotype compared to NBW children with DD genotype (P<0.024). In conclusion, we are able to demonstrate that the hypomethylation in the 3 CpG sites of ACE gene promoter is associated with LBW in 6 to 12 year-old children. The magnitude of these epigenetic changes appears to be clinically important, which is supported by the observation that discrete changes in DNA methylation can affect systolic blood pressure and ACE protein activity levels.

Influence of birth weight on the renal development and kidney diseases in adulthood: experimental and clinical evidence.

Several clinical and experimental studies support the hypothesis that foetal programming is an important determinant of nephropathy, hypertension, coronary heart disease, and type 2 diabetes during adulthood. In this paper, the renal repercussions of foetal programming are emphasised, and the physiopathological mechanisms are discussed. The programming of renal diseases is detailed based on the findings of kidney development and functional parameters.

Association of ACE gene insertion/deletion polymorphism with birth weight, blood pressure levels, and ACE activity in healthy children.

BACKGROUND: The human angiotensin-converting enzyme (ACE) gene contains a polymorphism consisting of either an insertion (I) or a deletion (D) of a 287 bp Alu repetitive sequence in intron 16. The potential role of ACE polymorphism in the risk of developing hypertension or other cardiovascular disorders has not been determined in relation to birth weight (BW). METHODS: The ACE genotype and plasma ACE activity were determined in 167 children. Among these children, 60 were identified with low BW (LBW), and 107 were of normal BW (NBW). RESULTS: ACE activity levels were significantly elevated in LBW children compared with the NBW group (P < 0.001). There was a significant association of the ACE activity with systolic blood pressure (SBP) levels in our population (P < 0.001). Among the ACE genotypes, no significant differences were found with respect to BW (P = 0.136). However, our results revealed that LBW children had a higher D allele frequency than NBW children (P = 0.036). When analyzed by quartiles of SBP or ACE activity, we found a greater frequency of both the LBW children and those carrying the DD genotype in the highest quartiles of these parameters, whereas the NBW children tended to be in the lowest quartile (P < 0.001). Similar results were observed with the heterozygote ID children after categorization by quartiles of both SBP (P < 0.001) and ACE activity (P = 0.004). CONCLUSIONS: The ACE I/D polymorphism, especially the DD genotype, can be interpreted as a major factor in association between LBW and high BP levels.

Leukemia, non-Hodgkin's lymphoma, and Wilms tumor in childhood: the role of birth weight.

There is emerging evidence that higher birth weight is associated with increased risk of cancer, in particular childhood leukemia. The purpose of this paper is to study whether this correlation is also significant with other childhood cancer. For this, we conducted a case-control study including 410 childhood cancer patients and 1,575 matched controls to investigate birth weight as a risk factor for leukemia, Wilms tumor, and non-Hodgkin's lymphoma. The estimated risk for all cancers has been found to be statistically and significantly higher in birth weight of more than 4,000 g (odds ratio, 2.50 and 95% confidence intervals (CI), 1.72-3.63). For leukemia, the estimated risk was 1.86 (95% CI, 1.04-3.30), for non-Hodgkin lymphoma, 1.99 (95% CI, 1.08-3.69), and being more remarkable for Wilms tumor, 4.76 (95% CI, 2.73-8.28). Moreover, moderate increased risk of both leukemia and non-Hodgkin lymphoma was also associated with birth weight between 3,000 and 3,999 g. High birth weight was associated with all cancers also when adjusted by gestational age, length at birth, and gender (odds ratio, 6.10 and 95% CI, 1.15-32.57). No associations were found for maternal alcohol consumption during pregnancy, maternal smoking, or smoking by other people at home or presence of obstetric variables (e.g., gestational diabetes, preeclampsia, and abruptio placentae). The present study supports the hypothesis that high birth weight is an independent risk factor for childhood Wilms tumor, leukemia, and non-Hodgkin lymphoma. Further studies should explore biological reasons to explain this relationship and, ultimately, to expand our knowledge about prenatal influences on the occurrence of this disease.