Molecular detection of Mayaro virus during a dengue outbreak in the state of Mato Grosso, Central-West Brazil

Mayaro virus (MAYV) is frequently reported in Pan-Amazonia. The aim of this study was to investigate the circulation of alphaviruses during a dengue outbreak in the state of Mato Grosso, Brazil. Serum samples from dengue-suspected patients were subjected to multiplex semi-nested reverse transcriptase polymerase chain reaction for 11 flaviviruses and five alphaviruses, to nucleotide sequencing and to viral isolation. MAYV was detected in 15 (2.5%) of 604 patients. Twelve were co-infected with dengue virus 4, which was isolated from 10 patients. The molecular detection of MAYV in dengue-suspected patients suggests that other arboviruses may be silently circulating during dengue outbreaks in Brazil.

Detection of Wuchereria bancrofti DNA in paired serum and urine samples using polymerase chain reaction-based systems

The Global Program for the Elimination of Lymphatic Filariasis (GPELF) aims to eliminate this disease by the year 2020. However, the development of more specific and sensitive tests is important for the success of the GPELF. The present study aimed to standardise polymerase chain reaction (PCR)-based systems for the diagnosis of filariasis in serum and urine. Twenty paired biological urine and serum samples from individuals already known to be positive for Wuchereria bancrofti were collected during the day. Conventional PCR and semi-nested PCR assays were optimised. The detection limit of the technique for purified W. bancrofti DNA extracted from adult worms was 10 fg for the internal systems (WbF/Wb2) and 0.1 fg by using semi-nested PCR. The specificity of the primers was confirmed experimentally by amplification of 1 ng of purified genomic DNA from other species of parasites. Evaluation of the paired urine and serum samples by the semi-nested PCR technique indicated only two of the 20 tested individuals were positive, whereas the simple internal PCR system (WbF/Wb2), which has highly promising performance, revealed that all the patients were positive using both samples. This study successfully demonstrated the possibility of using the PCR technique on urine for the diagnosis of W. bancrofti infection.

Optimisation of a quantitative polymerase chain reaction-based strategy for the detection and quantification of human herpesvirus 6 DNA in patients undergoing allogeneic haematopoietic stem cell transplantation

Human herpesvirus 6 (HHV-6) may cause severe complications after haematopoietic stem cell transplantation (HSCT). Monitoring this virus and providing precise, rapid and early diagnosis of related clinical diseases, constitute essential measures to improve outcomes. A prospective survey on the incidence and clinical features of HHV-6 infections after HSCT has not yet been conducted in Brazilian patients and the impact of this infection on HSCT outcome remains unclear. A rapid test based on real-time quantitative polymerase chain reaction (qPCR) has been optimised to screen and quantify clinical samples for HHV-6. The detection step was based on reaction with TaqMan® hydrolysis probes. A set of previously described primers and probes have been tested to evaluate efficiency, sensitivity and reproducibility. The target efficiency range was 91.4% with linearity ranging from 10-106 copies/reaction and a limit of detection of five copies/reaction or 250 copies/mL of plasma. The qPCR assay developed in the present study was simple, rapid and sensitive, allowing the detection of a wide range of HHV-6 loads. In conclusion, this test may be useful as a practical tool to help elucidate the clinical relevance of HHV-6 infection and reactivation in different scenarios and to determine the need for surveillance.

A sensitive, specific and reproducible real-time polymerase chain reaction method for detection of Plasmodium vivaxandPlasmodium falciparum infection in field-collected anophelines

We describe a simple method for detection of Plasmodium vivaxand Plasmodium falciparum infection in anophelines using a triplex TaqMan real-time polymerase chain reaction (PCR) assay (18S rRNA). We tested the assay on Anopheles darlingi and Anopheles stephensi colony mosquitoes fed withPlasmodium-infected blood meals and in duplicate on field collected An. darlingi. We compared the real-time PCR results of colony-infected and field collected An. darlingi, separately, to a conventional PCR method. We determined that a cytochromeb-PCR method was only 3.33% as sensitive and 93.38% as specific as our real-time PCR assay with field-collected samples. We demonstrate that this assay is sensitive, specific and reproducible.

Using immunoglobulin Y as an alternative antibody for the detection of hepatitis A virus in frozen liver sections

An increasing amount of research has been conducted on immunoglobulin Y (IgY) because the use of IgY offers several advantages with respect to diagnostic testing, including its easy accessibility, low cost and translatability to large-scale production, in addition to the fact that it can be ethically produced. In a previous work, immunoglobulin was produced and purified from egg yolks (IgY) reactive to hepatitis A virus (HAV) antigens. In the present work, this anti-HAV-specific IgY was used in an indirect immunofluorescence assay to detect viral antigens in liver biopsies that were obtained from experimentally infected cynomolgus monkeys. Fields that were positive for HAV antigen were detected in liver sections using confocal microscopy. In conclusion, egg yolks from immunised hens may be a reliable source for antibody production, which can be employed for immunological studies.

Detection of Oropouche virus segment S in patients and inCulex quinquefasciatus in the state of Mato Grosso, Brazil

This study aimed to investigate the circulation of Orthobunyavirus species in the state of Mato Grosso (MT) Brazil. During a dengue outbreak in 2011/2012, 529 serum samples were collected from patients with acute febrile illness with symptoms for up to five days and 387 pools of female Culex quinquefasciatuscaptured in 2013 were subjected to nested-reverse transcription-polymerase chain reaction for segment S of the Simbu serogroup followed by nucleotide sequencing and virus isolation in Vero cells. Patients (5/529; 0.9%) from Cuiabá (n = 3), Várzea Grande (n = 1) and Nova Mutum (n = 1) municipalities were positive for the S segment of Oropouche virus (OROV). Additionally, eight/387 Cx. quinquefasciatuspools were positive for the segment, with a minimum infection rate of 2.3. Phylogenetic analysis indicated that all the samples belong to the subgenotype Ia, presenting high homology with OROV strains obtained from humans and animals in the Brazilian Amazon. The present paper reports the first detection of an Orthobunyavirus, possibly OROV, in patients and in Cx. quinquefasciatus mosquitoes in MT. This finding reinforces the notion that arboviruses frequently reported in the Amazon Region circulate sporadically in MT during dengue outbreaks.

Polymerase chain reaction detection of LeishmaniaDNA in skin biopsy samples in Sri Lanka where the causative agent of cutaneous leishmaniasis is Leishmania donovani

Leishmania donovani is the known causative agent of both cutaneous (CL) and visceral leishmaniasis in Sri Lanka. CL is considered to be under-reported partly due to relatively poor sensitivity and specificity of microscopic diagnosis. We compared robustness of three previously described polymerase chain reaction (PCR) based methods to detectLeishmania DNA in 38 punch biopsy samples from patients presented with suspected lesions in 2010. Both, Leishmaniagenus-specific JW11/JW12 KDNA and LITSR/L5.8S internal transcribed spacer (ITS)1 PCR assays detected 92% (35/38) of the samples whereas a KDNA assay specific forL. donovani (LdF/LdR) detected only 71% (27/38) of samples. All positive samples showed a L. donovani banding pattern upon HaeIII ITS1 PCR-restriction fragment length polymorphism analysis. PCR assay specificity was evaluated in samples containing Mycobacterium tuberculosis, Mycobacterium leprae, and human DNA, and there was no cross-amplification in JW11/JW12 and LITSR/L5.8S PCR assays. The LdF/LdR PCR assay did not amplify M. leprae or human DNA although 500 bp and 700 bp bands were observed in M. tuberculosis samples. In conclusion, it was successfully shown in this study that it is possible to diagnose Sri Lankan CL with high accuracy, to genus and species identification, using Leishmania DNA PCR assays.

Detection of all four dengue serotypes in Aedes aegypti female mosquitoes collected in a rural area in Colombia

The Aedes aegypti vector for dengue virus (DENV) has been reported in urban and periurban areas. The information about DENV circulation in mosquitoes in Colombian rural areas is limited, so we aimed to evaluate the presence of DENV in Ae. aegypti females caught in rural locations of two Colombian municipalities, Anapoima and La Mesa. Mosquitoes from 497 rural households in 44 different rural settlements were collected. Pools of about 20 Ae. aegypti females were processed for DENV serotype detection. DENV in mosquitoes was detected in 74% of the analysed settlements with a pool positivity rate of 62%. The estimated individual mosquito infection rate was 4.12% and the minimum infection rate was 33.3/1,000 mosquitoes. All four serotypes were detected; the most frequent being DENV-2 (50%) and DENV-1 (35%). Two-three serotypes were detected simultaneously in separate pools. This is the first report on the co-occurrence of natural DENV infection of mosquitoes in Colombian rural areas. The findings are important for understanding dengue transmission and planning control strategies. A potential latent virus reservoir in rural areas could spill over to urban areas during population movements. Detecting DENV in wild-caught adult mosquitoes should be included in the development of dengue epidemic forecasting models.