RESUMO
This study aims to assess microbiological contamination using a molecular tool for detection of multiple enteropathogens in a coastal ecosystem area in Rio de Janeiro, Brazil. Ten litres of superficial water samples were obtained during the spring ebb tide from sampling sites along the Jacarepaguá watershed. Samples were concentrated using skimmed milk flocculation method for TaqMan array card (TAC), designed to identify 35 enteric pathogens simultaneously, and single TaqMan qPCR analysis for detecting human adenovirus (HAdV) and JC human polyomavirus (JCPyV) as faecal indicator viruses (FIV). TAC results identified 17 enteric pathogens including 4/5 viral species investigated, 8/15 bacteria, 4/6 protozoa and 1/7 helminths. Escherichia coli concentration was also measured as faecal indicator bacteria (FIB) using Colilert Quanti-Tray System with positivity in all samples studied. HAdV and JCPyV qPCR were detected in 8 and 4 samples, respectively, with concentration ranging from 8 × 102 to 2 × 106 genome copies/L. Partial nucleotide sequencing demonstrated the occurrence of species HAdV A, C, D, and F, present in faeces of individuals with enteric and non-enteric infections, and JCPyV type 3 (Af2), prevalent in a high genetically mixed population like the Brazilian. The diversity of enteropathogens detected by TAC emphasizes the utility of this methodology for quick assessment of microbiological contamination of the aquatic ecosystems, speeding up mitigation actions where the risk of the exposed population is detected, as well as pointing out the infrastructure gaps in areas where accelerated urban growth is observed.
Assuntos
Ecossistema , Microbiologia da Água , Brasil , Monitoramento Ambiental , Floculação , HumanosRESUMO
BACKGROUND: Molecular assays for diagnosis of Flu A, Flu B, and RSV with short turn-around-time (TAT) are of considerable clinical importance. In addition, rapid and accurate diagnosis of a large panel of viral and atypical pathogens can be crucial in immunocompromised patients. OBJECTIVES: First, to evaluate the performance of the Simplexa™ Direct assay system in comparison with direct fluorescent antibody (DFA) and customized Taqman® Array Card (TAC) testing for RSV, Flu A, and Flu B in immunocompromised patients. Second, to evaluate different algorithms for the detection of respiratory pathogens in terms of cost, turn-around-time (TAT) and diagnostic yield. STUDY DESIGN: We collected 125 nasopharyngeal swabs (NTS) and 25 BAL samples from symptomatic immunocompromised patients. Samples for which Simplexa™ and TAC results were discordant underwent verification testing. The TAC assay is based on singleplex RT-PCR, targeting 24 viruses, 8 bacteria and 2 fungi simultaneously. RESULTS: The overall sensitivity was significantly lower for DFA testing than for the two molecular methods (p<0.05). Performance characteristics of Simplexa™ testing were not significantly different compared to TAC testing (p>0.1). For BAL samples only, the sensitivity and specificity of the Simplexa™ assay was 100%. In total, 6.7, 16 and 18% of samples were positive for Flu A, Flu B or RSV by DFA, Simplexa™ and TAC testing, respectively. When considering not only these pathogens but also all results for TAC, the method identified 93 samples with one or more respiratory pathogens (62%). A co-infection rate of 15.3% was found by TAC. The estimated costs and TAT were 8.2 and 2h for DFA, 31.8 and 1.5h for Simplexa™ and 55 and 3h for TAC testing. CONCLUSIONS: Performing the Simplexa™ test 24h a day/7 days a week instead of DFA would considerably improve the overall sensitivity and time-to-result, albeit at a higher cost generated in the laboratory. Performing the TAC would increase the diagnostic yield and detection of co-infections significantly.