Detecting Leishmania in dogs: A hierarchical-modeling approach to investigate the performance of parasitological and qPCR-based diagnostic procedures.

BACKGROUND: Domestic dogs are primary reservoir hosts of Leishmania infantum, the agent of visceral leishmaniasis. Detecting dog infections is central to epidemiological inference, disease prevention, and veterinary practice. Error-free diagnostic procedures, however, are lacking, and the performance of those available is difficult to measure in the absence of fail-safe "reference standards". Here, we illustrate how a hierarchical-modeling approach can be used to formally account for false-negative and false-positive results when investigating the process of Leishmania detection in dogs. METHODS/FINDINGS: We studied 294 field-sampled dogs of unknown infection status from a Leishmania-endemic region. We ran 350 parasitological tests (bone-marrow microscopy and culture) and 1,016 qPCR assays (blood, bone-marrow, and eye-swab samples with amplifiable DNA). Using replicate test results and site-occupancy models, we estimated (a) clinical sensitivity for each diagnostic procedure and (b) clinical specificity for qPCRs; parasitological tests were assumed 100% specific. Initial modeling revealed qPCR specificity < 94%; we tracked the source of this unexpected result to some qPCR plates having subtle signs of possible contamination. Using multi-model inference, we formally accounted for suspected plate contamination and estimated qPCR sensitivity at 49-53% across sample types and dog clinical conditions; qPCR specificity was high (95-96%), but fell to 81-82% for assays run in plates with suspected contamination. The sensitivity of parasitological procedures was low (~12-13%), but increased to ~33% (with substantial uncertainty) for bone-marrow culture in seriously-diseased dogs. Leishmania-infection frequency estimates (~49-50% across clinical conditions) were lower than observed (~60%). CONCLUSIONS: We provide statistical estimates of key performance parameters for five diagnostic procedures used to detect Leishmania in dogs. Low clinical sensitivies likely reflect the absence of Leishmania parasites/DNA in perhaps ~50-70% of samples drawn from infected dogs. Although qPCR performance was similar across sample types, non-invasive eye-swabs were overall less likely to contain amplifiable DNA. Finally, modeling was instrumental to discovering (and formally accounting for) possible qPCR-plate contamination; even with stringent negative/blank-control scoring, ~4-5% of positive qPCRs were most likely false-positives. This work shows, in sum, how hierarchical site-occupancy models can sharpen our understanding of the problem of diagnosing host infections with hard-to-detect pathogens including Leishmania.

Isolation, Identification, and Screening of Lactic Acid Bacteria with Probiotic Potential in Silage of Different Species of Forage Plants, Cocoa Beans, and Artisanal Salami.

The objective of this study was to isolate and characterize lactic acid bacteria with probiotic potential in silages of different species of forage plants, cocoa beans, and artisanal salami. The obtained isolates were submitted to the following evaluations: (i) screening for tolerance to pH 2 and bile salts, (ii) genotypic identification of isolates, (iii) survival in simulated gastric and pancreatic conditions, (iv) antimicrobial activity, (v) antibiotic susceptibility and safety, and (vi) properties associated with adhesion capacity. A total of 82 isolates were obtained and were screened for pH 2.0 tolerance and capacity to growth in the presence of bile salts (1.0 and 2.0%). Only 19 strains simultaneously presented tolerance to pH 2.0 and bile salts. These 19 strains were evaluated for genetic profile by Box-PCR. Subsequently, the selected strains were subjected to partial sequencing of the 16S rRNA gene. The species Lactobacillus plantarum was prevalent. The identified strains were evaluated for survival under simulated gastric and pancreatic conditions. Some strains have shown tolerance in both conditions. Different strains showed variations in antimicrobial activity, susceptibility to antibiotics, and properties associated with adhesion (hydrophobicity, autoaggregation, coaggregation, and adhesion to CaCo2 cells). All strains were negative for hemolysis, DNase, gelatinase, and biogenic amine synthesis activity. The L. plantarum SBR64.7 strain can be considered the most promising for it presented the lowest viability reduction when exposed to gastric and pancreatic juices.

The use of qPCR in human Chagas disease: a systematic review.

Introduction: The diagnosis in Chagas disease is a challenge because most infections with Trypanosoma cruzi are asymptomatic and currently serological tests have limitations, such as cross-reactivity with other trypanosomatids. Real-time PCR (qPCR) is a useful procedure that allows T. cruzi detection even when the parasitic load is very low and seems interesting for monitoring the response to trypanocidal treatment and elucidating cases with doubtful serological results. Areas covered: This systematic review aimed to investigate the applications and relevance of qPCR in human Chagas disease, and focus on the methodological aspects. Expert opinion: The results showed that blood samples with the TaqMan procedure direct to nuclear DNA (nDNA) sequences are used the most. However, a high variability among laboratories concerning the qPCR methods make it difficult to compare between studies and the use in routine surveillance laboratories, even if some works had performed an analytical validation of T. cruzi qPCR to try to counteract this. Nevertheless, the detection of T. cruzi by qPCR has multiple advantages including fast results, reduction of carryover contamination compared to conventional PCR, and high sensitivity and specificity. This study has given an overview of assays using qPCR in human Chagas disease and has shown the relevance of this technique in diagnosis.

The first Acanthamoeba keratitis case in the Midwest region of Brazil: diagnosis, genotyping of the parasite and disease outcome.

We report an Acanthamoeba keratitis case associated with the use of contact lens in a 28-year-old female from Brasília, Brazil. Samples from corneal scraping and contact lens case were used for culture establishment, PCR amplification, and partial sequencing (fragments of ~400kb) of small subunit rDNA; both culture and PCR were positive. The sequence analyses of the cornea and of isolates from the contact lens case showed similarity with the T4 genotype. To the best of our knowledge, this is the first report of T4 Acanthamoeba keratitis case from the Midwest region of Brazil.

The first Acanthamoeba keratitis case in the Midwest region of Brazil: diagnosis, genotyping of the parasite and disease outcome

Abstract We report an Acanthamoeba keratitis case associated with the use of contact lens in a 28-year-old female from Brasília, Brazil. Samples from corneal scraping and contact lens case were used for culture establishment, PCR amplification, and partial sequencing (fragments of ~400kb) of small subunit rDNA; both culture and PCR were positive. The sequence analyses of the cornea and of isolates from the contact lens case showed similarity with the T4 genotype. To the best of our knowledge, this is the first report of T4 Acanthamoeba keratitis case from the Midwest region of Brazil.

Author Correction: Surveillance of vector-borne pathogens under imperfect detection: lessons from Chagas disease risk (mis)measurement.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.

Surveillance of vector-borne pathogens under imperfect detection: lessons from Chagas disease risk (mis)measurement.

Vector-borne pathogens threaten human health worldwide. Despite their critical role in disease prevention, routine surveillance systems often rely on low-complexity pathogen detection tests of uncertain accuracy. In Chagas disease surveillance, optical microscopy (OM) is routinely used for detecting Trypanosoma cruzi in its vectors. Here, we use replicate T. cruzi detection data and hierarchical site-occupancy models to assess the reliability of OM-based T. cruzi surveillance while explicitly accounting for false-negative and false-positive results. We investigated 841 triatomines with OM slides (1194 fresh, 1192 Giemsa-stained) plus conventional (cPCR, 841 assays) and quantitative PCR (qPCR, 1682 assays). Detections were considered unambiguous only when parasitologists unmistakably identified T. cruzi in Giemsa-stained slides. qPCR was >99% sensitive and specific, whereas cPCR was ~100% specific but only ~55% sensitive. In routine surveillance, examination of a single OM slide per vector missed ~50-75% of infections and wrongly scored as infected ~7% of the bugs. qPCR-based and model-based infection frequency estimates were nearly three times higher, on average, than OM-based indices. We conclude that the risk of vector-borne Chagas disease may be substantially higher than routine surveillance data suggest. The hierarchical modelling approach we illustrate can help enhance vector-borne disease surveillance systems when pathogen detection is imperfect.

Synanthropic triatomines as potential vectors of Trypanosoma cruzi in Central Brazil

Abstract INTRODUCTION: Chagas disease surveillance requires current knowledge on synanthropic triatomines. We analyzed the occurrence and Trypanosoma cruzi infection rates of triatomine bugs in central Brazil, during 2012-2014. METHODS: Triatomines were collected inside or around houses, and T. cruzi infection was determined by optical microscopy and conventional/quantitative polymerase chain reaction. RESULTS: Of the 2706 triatomines collected, Triatoma sordida was the most frequent species in Goiás State, whereas Panstrongylus megistus predominated in the Federal District. Parasites identified were T. cruzi, T. rangeli, and Blastocrithidia sp. CONCLUSIONS: P. megistus and T. sordida sustained the risk of T. cruzi transmission to humans in central Brazil.

Synanthropic triatomines as potential vectors of Trypanosoma cruzi in Central Brazil.

INTRODUCTION: Chagas disease surveillance requires current knowledge on synanthropic triatomines. We analyzed the occurrence and Trypanosoma cruzi infection rates of triatomine bugs in central Brazil, during 2012-2014. METHODS: Triatomines were collected inside or around houses, and T. cruzi infection was determined by optical microscopy and conventional/quantitative polymerase chain reaction. RESULTS: Of the 2706 triatomines collected, Triatoma sordida was the most frequent species in Goiás State, whereas Panstrongylus megistus predominated in the Federal District. Parasites identified were T. cruzi, T. rangeli, and Blastocrithidia sp. CONCLUSIONS: P. megistus and T. sordida sustained the risk of T. cruzi transmission to humans in central Brazil.

Vector-borne transmission of Trypanosoma cruzi among captive Neotropical primates in a Brazilian zoo.

BACKGROUND: Neotropical primates are important sylvatic hosts of Trypanosoma cruzi, the etiological agent of Chagas disease. Infection is often subclinical, but severe disease has been described in both free-ranging and captive primates. Panstrongylus megistus, a major T. cruzi vector, was found infesting a small-primate unit at Brasília zoo (ZooB), Brazil. ZooB lies close to a gallery-forest patch where T. cruzi circulates naturally. Here, we combine parasitological and molecular methods to investigate a focus of T. cruzi infection involving triatomine bugs and Neotropical primates at a zoo located in the Brazilian Savannah. METHODS: We assessed T. cruzi infection in vectors using optical microscopy (n = 34) and nested PCR (n = 50). We used quantitative PCR (qPCR) to examine blood samples from 26 primates and necropsy samples from two primates that died during the study. We determined parasite lineages in five vectors and two primates by comparing glucose-6-phosphate isomerase (G6pi) gene sequences. RESULTS: Trypanosoma cruzi was found in 44 vectors and 17 primates (six genera and eight species); one Mico chrysoleucus and one Saguinus niger had high parasitaemias. Trypanosoma cruzi DNA was detected in three primates born to qPCR-negative mothers at ZooB and in the two dead specimens. One Callithrix geoffroyi became qPCR-positive over a two-year follow-up. All G6pi sequences matched T. cruzi lineage TcI. CONCLUSIONS: Our findings strongly suggest vector-borne T. cruzi transmission within a small-primate unit at ZooB - with vectors, and perhaps also parasites, presumably coming from nearby gallery forest. Periodic checks for vectors and parasites would help eliminate T. cruzi transmission foci in captive-animal facilities. This should be of special importance for captive-breeding programs involving endangered mammals, and would reduce the risk of accidental T. cruzi transmission to keepers and veterinarians.

Expanding the knowledge about Leishmania species in wild mammals and dogs in the Brazilian savannah.

BACKGROUND: Wild, synanthropic and domestic mammals act as hosts and/or reservoirs of several Leishmania spp. Studies on possible reservoirs of Leishmania in different areas are fundamental to understand host-parasite interactions and develop strategies for the surveillance and control of leishmaniasis. In the present study, we evaluated the Leishmania spp. occurrence in mammals in two conservation units and their surroundings in Brasília, Federal District (FD), Brazil. METHODS: Small mammals were captured in Brasília National Park (BNP) and Contagem Biological Reserve (CBR) and dogs were sampled in residential areas in their vicinity. Skin and blood samples were evaluated by PCR using different molecular markers (D7 24Sα rRNA and rDNA ITS1). Leishmania species were identified by sequencing of PCR products. Dog blood samples were subjected to the rapid immunochromatographic test (DPP) for detection of anti-Leishmania infantum antibodies. RESULTS: 179 wild mammals were studied and 20.1% had Leishmania DNA successfully detected in at least one sample. Six mammal species were considered infected: Clyomys laticeps, Necromys lasiurus, Nectomys rattus, Rhipidomys macrurus, Didelphis albiventris and Gracilinanus agilis. No significant difference, comparing the proportion of individuals with Leishmania spp., was observed between the sampled areas and wild mammal species. Most of the positive samples were collected from the rodent N. lasiurus, infected by L. amazonensis or L. braziliensis. Moreover, infections by Trypanosoma spp. were detected in N. lasiurus and G. agilis. All 19 dog samples were positive by DPP; however, only three (15.8%) were confirmed by PCR assays. DNA sequences of ITS1 dog amplicons showed 100% identity with L. infantum sequence. CONCLUSIONS: The results suggest the participation of six species of wild mammals in the enzootic transmission of Leishmania spp. in FD. This is the first report of L. amazonensis in N. lasiurus.

Occurrence and characterization of Acanthamoeba similar to genotypes T4, T5, and T2/T6 isolated from environmental sources in Brasília, Federal District, Brazil.

Species of Acanthamoeba can cause keratitis and brain infections. The characterization of environmental isolates is necessary to analyze the risk of human infection. We aimed at identifying and genotyping Acanthamoeba isolates from soil, swimming pools, and water features in Brasília, Federal District, Brazil, as well as determining their physiological characteristics and pathogenic potential. Among the 18 isolates studied, eight were similar to genotype T5, five to T4, and one to T2/T6, classified by the sequence analysis of 18S rDNA. Genotypes of four isolates were not determined. Ten isolates (55%) grew at 37 °C and seven (39%) grew in media with 1.5M mannitol, which are the physiological parameters associated with pathogenic Acanthamoeba; also, four isolates from swimming pools presented high pathogenic potential. Our results indicate a widespread distribution of potentially pathogenic Acanthamoeba T4, T5, and T2/T6 in different environmental sources in Brasília, revealing the potential risk of human infection and the need of preventive measures.