The rapid spread of Zika virus (ZIKV) represents a global public health problem, especially in areas that harbor several mosquito species responsible for virus transmission, such as Brazil. In these areas, improvement in mosquito control needs to be a top priority, but mosquito viral surveillance occurs inefficiently in ZIKV-endemic countries. Quantitative reverse transcription PCR (qRT-PCR) is the gold standard for molecular diagnostic of ZIKV in both human and mosquito samples. However, the technique presents high cost and limitations for Point-of-care (POC) diagnostics, which hampers its application for a large number of samples in entomological surveillance programs. Here, we developed and validated a one-step reverse transcription LAMP (RT-LAMP) platform for detection of ZIKV in mosquito samples. The RT-LAMP assay was highly specific for ZIKV and up to 10,000 times more sensitive than qRT-PCR. Assay validation was performed using 60 samples from Aedes aegypti and Culex quinquefasciatus mosquitoes collected in Pernambuco State, Brazil, which is at the epicenter of the Zika epidemic. The RT-LAMP had a sensitivity of 100%, specificity of 91.18%, and overall accuracy of 95.24%. Thus, our POC diagnostics is a powerful and inexpensive tool to monitor ZIKV in mosquito populations and will allow developing countries to establish better control strategies for this devastating pathogen.

Multiple epicenters of the SARS-CoV-2 pandemic have emerged since the first pneumonia cases in Wuhan, China, such as Italy, USA, and Brazil. Brazil is the third-most affected country worldwide, but genomic sequences of SARS-CoV-2 strains are mostly restricted to states from the Southeast region. Pernambuco state, located in the Northeast region, is the sixth most affected Brazilian state, but very few genomic sequences from the strains circulating in this region are available. We sequenced 101 strains of SARS-CoV-2 from patients presenting Covid-19 symptoms that reside in Pernambuco. Phylogenetic reconstructions revealed that all genomes belong to the B lineage and most of the samples (88%) were classified as lineage B.1.1. We detected multiple viral introductions from abroad (likely from Europe) as well as six local B.1.1 clades composed by Pernambuco only strains. Local clades comprise sequences from the capital city (Recife) and other country-side cities, corroborating the community spread between different municipalities of the state. These findings demonstrate that different from Southeastern Brazilian states where the epidemics were majorly driven by one dominant lineage (B.1.1.28 or B.1.1.33), the early epidemic phase at the Pernambuco state was driven by multiple B.1.1 lineages seeded through both national and international traveling.

Introduction: Polymerase chain reaction (PCR) may offer an alternative diagnostic option when clinical signs and symptoms suggest visceral leishmaniasis (VL) but microscopic scanning and serological tests provide negative results. PCR using urine is sensitive enough to diagnose human visceral leishmaniasis (VL). However, DNA quality is a crucial factor for successful amplifi cation. Methods: A comparative performance evaluation of DNA extraction methods from the urine of patients with VL using two commercially available extraction kits and two phenol-chloroform protocols was conducted to determine which method produces the highest quality DNA suitable for PCR amplifi cation, as well as the most sensitive, fast and inexpensive method. All commercially available kits were able to shorten the duration of DNA extraction. Results: With regard to detection limits, both phenol: chloroform extraction and the QIAamp DNA Mini Kit provided good results (0.1 pg of DNA) for the extraction of DNA from a parasite smaller than Leishmania (Leishmania) infantum (< 100fg of DNA). However, among 11 urine samples from subjects with VL, better performance was achieved with the phenol:chloroform method (8/11) relative to the QIAamp DNA Mini Kit (4/11), with a greater number of positive samples detected at a lower cost using PCR. Conclusion: Our results demonstrate that phenol:chloroform with an ethanol precipitation prior to extraction is the most effi cient method in terms of yield and cost, using urine as a non-invasive source of DNA and providing an alternative diagnostic method at a low cost.

Conventional nested PCR is a very sensitive and specific method for the diagnosis of visceral leishmaniasis. However, this type of PCR is notorious for contamination problems related to the processing of the product between the first and the second PCR steps. In order to have a PCR method that is just as efficient but without the risk of contamination, we attempted the optimization of a single-tube nested PCR (STNPCR) method. During the first and the second PCR steps, we used the small subunit of ribosomal RNA (ssu rRNA) and the ribosomal internal transcribed spacer (ITS) as targets, respectively. The performances of STNPCR and nested PCR in detecting the DNA of Leishmania chagasi were compared. In the case of STNPCR, the inner primers were immobilized on the interior of the tube cap by means of adsorption microtubes and then were solubilized before the second reaction. This procedure eliminated the need to open the microtube, which could have led to false-positive results through cross-contamination. The detection limit for the purified L. chagasi DNA was 1 fg by using nested PCR and 10 fg by using STNPCR. We also tested the specificity of the system against other parasites, and observed that Trypanosoma cruzi DNA was amplified with a detection limit of up to 1 pg. This study not only presents a promising tool for the diagnosis of visceral leishmaniasis, but also provides a new tool for the diagnosis of Chagas disease, either in mono-infection by T. cruzi or in co-infection with Leishmania spp.

Abstract. After the emergence of the human immunodeficiency virus (HIV)/acquired immunodeficiency syndrome (AIDS), the number of visceral leishmaniasis (VL)-HIV/AIDS coinfections has increased worldwide. Herein, we assessed the usefulness of an rK39-based immunochromatographic test (rK39 ICT) (DiaMed-IT LEISH ® ; DiaMed AG, Cressier-sur-Morat, Switzerland) and a latex agglutination test (KAtex; Kalon Biological, Guildford, United Kingdom) for urinary antigen detection to diagnose VL in 15 HIV/AIDS patients from northeastern Brazil. VL diagnosis was based on clinical findings, cytology, serology, parasite DNA, and/or urinary antigen detection. VL was confirmed in seven out of 15 HIV/AIDS patients. Only three patients were positive in bone marrow cytology, three patients were conventional polymerase chain reaction (PCR) positive, while six were real-time PCR positive. All patients were direct agglutination test (DAT) (Royal Tropical Institute, Amsterdam, The Netherlands) positive; of these, four were positive by rK39 ICT and five by KAtex. Large-scale studies are needed to validate the use of the KAtex in the national public health laboratory network in Brazil, aiming at improving the diagnosis of VL in HIV/AIDS patients in this country.

The emergence of SARS-CoV-2 in the human population has caused a huge pandemic that is still unfolding in many countries around the world. Multiple epicenters of the pandemic have emerged since the first pneumonia cases in Wuhan, first in Italy followed by the USA and Brazil. Up to now, Brazil is the second most affected country, however, genomic sequences of SARS-CoV-2 strains circulating in the country are restricted to some highly impacted states. Although the Pernambuco state, located in the Northeast Region, is the sixth most affected brazilian state and the second considering lethality rate, there is a lack of high quality genomic sequences from the strains circulating in this region. Here, we sequenced 38 strains of SARS-CoV-2 from patients presenting Covid-19 symptoms. Phylogenetic reconstructions revealed that three lineages were circulating in the state and 36 samples belong to B1.1 lineage. We detected two introductions from European countries and five clades, corroborating the community spread of the virus between different municipalities of the state. Finally, we detected that all except one strain showed the D614G spike protein amino acid change that may impact virus infectivity in human cells. Our study brought new light to the spread of SARS-CoV-2 strains in one of the most heavily impacted states of Brazil.

Common in four continents, visceral leishmaniasis (VL) is an important but neglected disease. Human immunodeficiency virus (HIV) infection increases the risk of developing VL in people from leishmaniasis-endemic areas, with worse prognosis when there is coinfection. We conducted a cross-sectional study to determine the prevalence of HIV/VL coinfection in patients admitted in three referral hospitals for HIV/acquired immunodeficiency syndrome (AIDS) in Pernambuco, Brazil, and to compare epidemiological, clinical, and laboratory characteristics among HIV/VL coinfected and HIV mono-infected individuals. The sample consisted of HIV patients aged 18 years or more, in a period of data collection of 6 months. We performed four Leishmania tests-polymerase chain reaction (PCR), direct agglutination test, rK39, and latex agglutination test-and individuals with at least one positive test were considered coinfected. The HIV/VL coinfection prevalence we found was 16.9%. We observed large variation in prevalence according to the Leishmania test used, with low coincidence of positive tests. The most frequent symptoms found were weight loss (75.6%), fever (67.6%), and cough (55.3%). When we compared HIV/VL coinfected and HIV mono-infected groups we did not observe statistically significant differences. Low educational level (P = 0.004) and pallor (P = 0.009) were more frequent in the coinfected group. Serum albumin level was higher in coinfected individuals (P = 0.

BackgroundNeglected tropical diseases (NTDs) prevail in conditions of poverty and contribute to the maintenance of social inequality. Out of the NTDs prioritized by the Brazilian Ministry of Health, four parasitic infections require mandatory notification: acute Chagas disease, leishmaniasis, malaria, and schistosomiasis. Data on the behaviour of these NTDs in the young population are currently limited. This study seeks to analyse the epidemiological aspects of these parasitic infections in children and adolescents in Brazil.MethodsA retrospective exploratory ecological study was conducted. A spatial analysis of the cases reported between 2009 and 2013 in individuals aged between 0 and 19 years that were notified through the Health Notification Aggravation Information System (SINAN) was performed.ResultsIn total, 64,567 cases of cutaneous and visceral leishmaniasis, malaria, schistosomiasis, and acute Chagas disease were recorded in the SINAN database, representing a rate of 20.15 cases per 100,000 inhabitants. The average age of the cases was 12.2 years and 62.32% were male. Four hundred and three deaths related to these obligatorily reported parasites were recorded, indicating a case fatality rate of 0.62%. Visceral leishmaniasis and acute Chagas disease had the highest rates of lethality. A heterogeneous spatial distribution of the studied parasites was observed.ConclusionsThe number of cases and the lethality rate described in this study show that these diseases still represent a serious problem for public health in Brazil. This points to the need to encourage new research and the reformulation of social, economic, and public health policies aimed at ensuring better health and living conditions for all individuals, especially those among the populations considered vulnerable, as is the case of the young.Electronic supplementary materialThe online version of this article (10.1186/s40249-017-0369-0) contains supplementary material, which is available to authorized users.