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The analytical validation of sensitive, accurate and standardized Real-Time PCR methods for Trypanosoma cruzi quantification is crucial to provide a reliable laboratory tool for diagnosis of recent infections as well as for monitoring treatment efficacy. We have standardized and validated a multiplex Real-Time quantitative PCR assay (qPCR) based on TaqMan technology, aiming to quantify T. cruzi satellite DNA as well as an internal amplification control (IAC) in a single-tube reaction. IAC amplification allows rule out false negative PCR results due to inhibitory substances or loss of DNA during sample processing. The assay has a limit of detection (LOD) of 0.70 parasite equivalents/mL and a limit of quantification (LOQ) of 1.53 parasite equivalents/mL starting from non-boiled Guanidine EDTA blood spiked with T. cruzi CL-Brener stock. The method was evaluated with blood samples collected from Chagas disease patients experiencing different clinical stages and epidemiological scenarios: 1- Sixteen Venezuelan patients from an outbreak of oral transmission, 2- Sixty three Bolivian patients suffering chronic Chagas disease, 3- Thirty four Argentinean cases with chronic Chagas disease, 4- Twenty seven newborns to seropositive mothers, 5- A seronegative receptor who got infected after transplantation with a cadaveric kidney explanted from an infected subject. The performing parameters of this assay encourage its application to early assessment of T. cruzi infection in cases in which serological methods are not informative, such as recent infections by oral contamination or congenital transmission or after transplantation with organs from seropositive donors, as well as for monitoring Chagas disease patients under etiological treatment.

Background A significant reduction in parasite clearance rates following artesunate treatment of falciparum malaria, and increased failure rates following artemisinin combination treatments (ACT), signaled emergent artemisinin resistance in Western Cambodia. Accurate measurement of parasite clearance is therefore essential to assess the spread of artemisinin resistance in Plasmodium falciparum . The slope of the log-parasitaemia versus time relationship is considered to be the most robust measure of anti-malarial effect. However, an initial lag phase of numerical instability often precedes a steady exponential decline in the parasite count after the start of anti-malarial treatment. This lag complicates the clearance estimation, introduces observer subjectivity, and may influence the accuracy and consistency of reported results. Methods To address this problem, a new approach to modelling clearance of malaria parasites from parasitaemia-time profiles has been explored and validated. The methodology detects when a lag phase is present, selects the most appropriate model (linear, quadratic or cubic) to fit log-transformed parasite data, and calculates estimates of parasite clearance adjusted for this lag phase. Departing from previous approaches, parasite counts below the level of detection are accounted for and not excluded from the calculation. Results Data from large clinical studies with frequent parasite counts were examined. The effect of a lag phase on parasite clearance rate estimates is discussed, using individual patient data examples. As part of the World Wide Antimalarial Resistance Network's (WWARN) efforts to make innovative approaches available to the malaria community, an automated informatics tool: the parasite clearance estimator has been developed. Conclusions The parasite clearance estimator provides a consistent, reliable and accurate method to estimate the lag phase and malaria parasite clearance rate. It could be used to detect early signs of emerging resistance to artemisinin derivatives and other compounds which affect ring-stage clearance.

Due to the efforts to control schistosomiasis transmission in tropical countries, a large proportion of individuals from endemic areas present low parasite loads, which hinders diagnosis of intestinal schistosomiasis by the Kato-Katz (KK) method. Therefore, the development of more sensitive diagnostic methods is essential for efficient control measures. The aim was to evaluate the accuracy of a real-time polymerase chain reaction (RT-PCR) to detect Schistosoma mansoni DNA in fecal samples of individuals with low parasite loads. A cross-sectional population-based study was conducted in a rural community (n = 257) in Brazil. POC-CCA® was performed in urine and feces were used for RT-PCR. In addition, fecal exams were completed by 18 KK slides, saline gradient and Helmintex techniques. The combined results of the three parasitological tests detected schistosome eggs in 118 participants (45.9%) and composed the consolidated reference standard (CRS). By RT-PCR, 117 out of 215 tested samples were positive, showing 91.4% sensitivity, 80.2% specificity and good concordance with the CRS (kappa = 0.71). RT-PCR identified 86.9% of the individuals eliminating less than 12 eggs/g of feces, demonstrating much better performance than POC-CCA® (50.8%). Our results showed that RT-PCR is a valuable alternative for the diagnosis of intestinal schistosomiasis in individuals with very low parasite loads.

Chagas disease is still a major public health issue in many Latin American countries. One of the current major challenges is to find an association between Trypanosoma cruzi discrete typing units (DTUs) and clinical manifestations of the disease. In this study, we used a multilocus conventional PCR and quantitative real time PCR (qPCR) approaches to perform the molecular typing and parasite load quantification directly from blood specimens of 65 chronic Chagas disease patients. All patients were recruited at the same health center, but their place of birth were widely distributed in different geographic regions of Brazil. Of the 65 patients, 35 (53.8%) presented positive amplification by real time qPCR, being 20 (30.7%) with the clinical indeterminate form and 15 (23.1%) with the cardiac form of the disease. The parasite load median for all positive patients was 2.54 [1.43-11.14] parasite equivalents/mL (par. Eq./mL), with the load ranging from 0.12 to 153.66 par. Eq./mL. Noteworthy, the parasite load was significantly higher in patients over 70 years old (median 20.05 [18.29-86.86] par. Eq./mL). Using guanidine-EDTA blood samples spiked with reference T. cruzi strains, belonging to the six DTUs, it was possible to genotype the parasite up to 0.5 par. Eq./mL, with high specificity. Of the patients with positive qPCR, it was possible to identify the T. cruzi DTU in 28 patients (80%). For the remaining patients (20%), at least a partial result was obtained. Analysis of specimens showed prevalences of TcVI, TcII and mixed infection TcVI+TcII equal to 40%, 17.1% and 14.3%, respectively. In addition, two patients were infected by TcV, and one patient was coinfected by TcIII+TcVI, These last three patients were in stage A of chronic chagasic cardiomyopathy (CCC), and they were born at the Bahia State (northeast region of Brazil). When T. cruzi genotypes were compared with the parasite load, more elevated parasite loads were observed in patients infected by TcII in general (parasite load median of 7.56 par. Eq./mL) in comparison to patients infected by TcVI (median of 2.35 par. Eq./mL). However, while the frequency of CCC was 50% in patients infected by TcVI and TcV, only 16.7% of patients infected by TcII evolved to CCC. Taking together, our results contribute to update the epidemiological knowledge of T. cruzi DTUs in Brazil, and highlight the age of patient and infection by TcII as important features that lead to the observation of higher parasitemia levels.

Background The distribution of parasite load across hosts may modify the transmission dynamics of infectious diseases. Chagas disease is caused by a multi-host protozoan, Trypanosoma cruzi , but the association between host parasitemia and infectiousness to the vector has not been studied in sylvatic mammalian hosts. We quantified T. cruzi parasite load in sylvatic mammals, modeled the association of the parasite load with infectiousness to the vector and compared these results with previous ones for local domestic hosts. Methods The bloodstream parasite load in each of 28 naturally infected sylvatic mammals from six species captured in northern Argentina was assessed by quantitative PCR, and its association with infectiousness to the triatomine Triatoma infestans was evaluated, as determined by natural or artificial xenodiagnosis. These results were compared with our previous results for 88 humans, 70 dogs and 13 cats, and the degree of parasite over-dispersion was quantified and non-linear models fitted to data on host infectiousness and bloodstream parasite load. Results The parasite loads of Didelphis albiventris (white-eared opossum) and Dasypus novemcinctus (nine-banded armadillo) were directly and significantly associated with infectiousness of the host and were up to 190-fold higher than those in domestic hosts. Parasite load was aggregated across host species, as measured by the negative binomial parameter, k , and found to be substantially higher in white-eared opossums, cats, dogs and nine-banded armadillos (range: k  = 0.3–0.5) than in humans ( k  = 5.1). The distribution of bloodstream parasite load closely followed the “80–20 rule” in every host species examined. However, the 20% of human hosts, domestic mammals or sylvatic mammals exhibiting the highest parasite load accounted for 49, 25 and 33% of the infected triatomines, respectively. Conclusions Our results support the use of bloodstream parasite load as a proxy of reservoir host competence and individual transmissibility. The over-dispersed distribution of T. cruzi bloodstream load implies the existence of a fraction of highly infectious hosts that could be targeted to improve vector-borne transmission control efforts toward interruption transmission. Combined strategies that decrease the parasitemia and/or host–vector contact with these hosts would disproportionally contribute to T. cruzi transmission control. Graphical Abstract

Background Normal-looking skin of dogs with leishmaniosis frequently shows microscopic lesions along with the presence of Leishmania amastigotes. However, histological lesions with or without detection of amastigotes might not occur in less severe clinical cases. In addition, comparative studies between paired clinically-lesioned and normal-looking skin samples from dogs with different disease severity are lacking. The objective of this study was to compare histological and parasitological findings by Leishmania immunohistochemistry (IHC) and quantitative PCR (qPCR) on paired clinically-lesioned and normal-looking skin biopsies from 25 dogs with different clinical stages of leishmaniosis, 11 with stage I-mild disease (papular dermatitis) and 14 with stage II-III (ulcerative or exfoliative dermatitis). Results The study demonstrated microscopic lesions in 14 out of 25 (56%) samples from normal-looking skin biopsies. In those samples, perivascular to interstitial dermatitis composed by macrophages with lymphocytes and plasma cells was observed mainly in the superficial and mid-dermis. The intensity of the dermatitis was mild to moderate and always less prominent than in the clinically-lesioned skin. In normal-looking skin samples, the presence of parasites was detected by histology, IHC and qPCR in 5/25 (20%), 8/25 (32%) and 18/25 (72%), respectively. Leishmania was encountered in 11/25 (44%), 23/25 (92%) and 25/25 (100%) of clinically-lesioned skin samples by histology, IHC and qPCR, respectively. Normal-looking skin from dogs with stage I-mild disease was less frequently inflamed ( P  = 0.0172). Furthermore, Leishmania was more easily demonstrated by histology ( P  = 0.0464), IHC ( P  = 0.0421) or qPCR ( P  = 0.0068) in normal-looking skin of dogs with stage II-III-moderate to severe disease. In addition, in the latter group, there was a significantly higher parasite load studied by means of qPCR than in dogs with less severe disease ( P  = 0.043). Clinically-lesioned skin from dogs with stage I disease was more frequently characterised by the nodular to diffuse pattern and granuloma formation ( P  = 0.0166) and by a lower parasite load studied by means of qPCR ( P  = 0.043) compared with more diseased dogs. Conclusions Normal-looking skin from dogs with stage I is less likely to present histological lesions as well as harbour the parasite when compared with dogs with moderate to severe leishmaniosis.

Very little is known about the influence of massive and long distance migration on parasite epidemiology. Migration can simultaneously minimize exposure to common parasites in their habitats and increase exposure to novel pathogens from new environments and habitats encountered during migration, while physiological stress during long distance movement can lead to immune suppression, which makes migrants vulnerable to parasites. In this paper, we investigated the diversity, prevalence, parasite load, co-infection patterns and predilection sites of adult gastrointestinal helminths in 130 migrating wildebeests and tested for their relation with animal age, sex and migration time (which also could indicate different migration routes), and compared them with the non-migratory wildebeest. Surprisingly, only four parasite species were found, Oesophagostomum columbianum , Haemonchus placei , Calicophoron raja and Moniezia expansa , which were lower than in non-migratory wildebeest reported in the literature. These parasites were generalists, infecting livestock, and suggests that wildebeest and livestock, because of their interaction during migration, have a cross-infection risk. There was a negative relation between parasites diversity, prevalence and intensity of infection, and host age, which suggests that wildebeests acquire protective immunity against these parasites as they get older. Prevalence and intensity of infection were higher among wildebeest crossing the Mara Bridge (early migrants) compared to those crossing the Serena (late migrants), which suggests that early migrants (or migrants originating from different areas) have varying infection intensities. The prevalence and intensity of infection were higher in males compared to females and may be due to ecological, behavioural, or physiological differences between males and females. Our findings compared to those of previous studies suggest that migration may provide a mechanism to minimize exposure of hosts to common parasites through migratory escape, but this result awaits examination of helminths epidemiology of non-migratory wildebeests from areas of migrant origins. The potential parasitic cross-infection between wildebeests and livestock is a real risk to be taken into account in the management of wildebeest migration corridors.

1. Predators affect prey populations not only through direct predation, but also by acting as definitive hosts for their parasites and completing parasite life cycles. Understanding the affects of parasitism on prey population dynamics requires knowing how their predators' parasite community is affected by diet and prey availability. Ungulates, such as moose (Alces americanus) and white-tailed deer (Odocoileus virginianus), are often important prey for wolves (Cards lupus), but wolves also consume a variety of alternative prey, including beaver (Castor canadensis) and snowshoe hare (Lepus americanus). 2. The use of alternative prey, which may host different or fewer parasites than ungulates, could potentially reduce overall abundance of ungulate parasites within the ecosystem, benefiting both wolves and ungulate hosts. 3. We examined parasites in wolf carcasses from eastern Manitoba and estimated wolf diet using stable isotope analysis. Taeniidae cestodes were present in most wolves (75%), reflecting a diet primarily comprised of ungulates, but nematodes were unexpectedly rare. 4. Cestode abundance was negatively related to the wolf's δ¹³C value, indicating diet affects parasite abundance. Wolves that consumed a higher proportion of beaver and caribou (Rangifer tarandus), estimated using Bayesian mixing models, had lower cestode abundance, suggesting the use of these alternative prey can reduce parasite loads. 5. Long-term consumption of beavers may lower the abundance of adult parasites in wolves, eventually lowering parasite density in the region and ultimately benefiting ungulates that serve as intermediate hosts. Thus, alternative prey can affect both predator-prey and hostparasite interactions and potentially affect food web dynamics.

Here, we assessed outcome of experimental infection by Neospora caninum in goats intravenously inoculated with 10 6 tachyzoites of the Nc-Spain7 isolate at 40 (G1), 90 (G2) and 120 (G3) days of gestation. Infected goats had fever between 5 and 9 days post inoculation (dpi); all were seropositive at the time of abortion/birth. Foetal death occurred in G1 from 10 to 21 dpi ( n  = 7) and in G2 from 27 to 35 dpi ( n  = 4). Goats in G2 also had seropositive stillbirth ( n  = 1) and healthy kids ( n  = 2). G3 goats ( n  = 7) had 3 seropositive and 3 seronegative weak kids, and 2 seronegative healthy kids. Parasite DNA detection in placentomes was 100% in G2, 85.7% in G3 and in G1 was detected only in placentomes from the goats with foetal losses from 17 dpi (100%). Parasites were detected in foetal/kid brain (>85.7%) and liver (≥50%) of G2 and G3, and in G1 after 17 dpi (100%). The highest parasite loads were detected in the placentomes of G1 from 17 dpi and G2, and in foetal tissues of G1 from 17 dpi and G3. Multifocal necrotic lesions were observed in the placentas of the three groups, but they were larger and more frequent in G1 and G2. Similar lesions were observed in foetal tissues, but they were more frequent in G3. These findings suggest that, as observed in cattle and sheep, the clinical consequences of N. caninum in pregnant goats are dependent in part on the time of gestation when animals were infected.

Captive-bred animals, widely used in reintroduction programmes, are often immunologically naïve and more susceptible to pathogens. We analysed infection of invasive blood-sucking nematode Ashworthius sidemi in captive-bred European bison (Bison bonasus) released to the wild in the Białowieża Forest (Poland). Mean A. sidemi infection intensity of released bison (29,137 nematodes) was over threefold higher than in wild bison (8756). It indicates a rapid acquisition and increase in the infection intensity in previously dewormed bison released from captivity. Thus, reintroduction programmes should consider the impact of pathogens and involve controlled exposure of captive animals to specific parasites prior to release.