Molecular detection and parasite load of Trypanosoma cruzi in digestive tract tissue of Chagas disease patients affected by megacolon.

Chagas disease, caused by the Trypanosoma cruzi parasite in the Americas affects ∼ 7 million people, 30% with cardiac tissue damage and 10-15% with digestive disorders. In this study, we have developed a protocol to detect the presence of the parasite and estimate its load in resected dysfunctional tissue segments of chronically infected patients with digestive megacolon. We have included samples from 43 individuals, 38/5 with positive/negative serology for Chagas disease and digestive syndromes. Samples of 1.5 to 2.0 cm2 were taken from different points of the dysfunctional digestive tract in specialized centres in Cochabamba, Bolivia. T. cruzi cultures were performed by inoculation with NNN-LIT culture medium, and genomic material was obtained from the samples for multiplex qPCR with TaqMan probes targeting satellite nuclear DNA. Cultures failed to isolate T. cruzi but qPCR reached a sensitivity of 42.1% (16/38) with all three spots and in triplicate. A new quantification methodology using synthetic satellite DNA as quantitation standard revealed parasite loads ranging from 2.2 × 102 to 1.0 × 106 satellite DNA copies/µl. Positive samples from the distal end showed a higher parasite load. The results of the present study strengthen and add further evidence to previous findings in an experimental mouse model of chronic T. cruzi infection, providing a valuable tool to improve scientific knowledge on the relevance of the digestive tract in parasite persistence, and underline the need of a better understanding of host-pathogen interaction in digestive tissues, considering pathophysiology, disease immunology and response to treatment.

Pan-stage real-time PCR for quantitation of Trypanosoma cruzi parasitic loads in blood samples.

Chagas disease is a complex zoonosis caused by Trypanosoma cruzi. The diagnosis of this infection is complex and molecular tools are suggested to detect the parasite in blood samples. A long-standing question arises in Chagas disease molecular diagnostics and is related to the feasibility of using epimastigotes in standard curves to quantify parasitic loads. Herein, we conducted experiments running standard curves with all the known life stages of T. cruzi. Our results indicate that regardless of the life stage employed, there are no statistically significant differences when calculating parasitic loads in blood samples. Our results have practical implications from a laboratory perspective in terms of the usability of epimastigotes to build standard curves for T. cruzi pan-stage assessment. Future studies are needed to further improve T. cruzi molecular diagnostic methods and enhance their impact in clinical practice.

Larvas de helmintos no solo de praças públicas de Caxias, Maranhão, Brasil / Helminth larvae in the soil of public squares in Caxias, Maranhão, Brazil / Larvas de helmintos en el suelo de plazas públicas en Caxias, Maranhão, Brasil

Solos de praças públicas são comumente contaminados por helmintos devido ao fácil acesso de cães e gatos infectados. Esses animais ao defecarem podem liberar ovos desses parasitos e, em condições ambientais favoráveis, tornam-se ovos embrionados ou larvas infectantes. O objetivo deste trabalho foi investigar a existência de larvas de helmintos no solo de duas praças públicas do município de Caxias, Maranhão, Brasil, durante a estação chuvosa e seca na região. A pesquisa foi realizada em março de 2018, considerado período chuvoso, e em outubro do mesmo ano, período seco, sendo que foram coletadas trinta amostras de areia, quinze de cada praça, nos dois períodos do ano. O material foi coletado e levado para o Laboratório de Parasitologia do Departamento de Parasitologia e Microbiologia da Universidade Federal do Piauí para análise. Na estação chuvosa, das quinze amostras analisadas na praça A, cinco foram positivas para larvas de ancilostomídeos e das quinze na praça B, três estavam contaminadas com os mesmos helmintos. No período seco, na praça A havia apenas uma amostra com essas larvas e na praça B não foram encontrados parasitos. Os resultados revelaram a presença de larvas de helmintos de caráter zoonótico no solo de praças públicas de Caxias, Maranhão, principalmente no período chuvoso, servindo de alerta à população local.(AU)

Soil in public squares is commonly contaminated by helminths due to the easy access of infected dogs and cats. These animals, when defecating, can release helminth eggs and, under favorable environmental conditions, those eggs can become embryonated or infective larvae. The purpose of this work was to investigate the existence of helminth larvae in the soil of two public squares in the city of Caxias, in the state of Maranhão, Brazil, during the rainy and dry seasons in the region. The study was carried out in March 2018, which is considered the rainy season, and in October of the same year, the dry season. A total of thirty sand samples were collected, fifteen from each square, in both periods of the year. The material was collected and taken to the Parasitology Laboratory of the Department of Parasitology and Microbiology of the Federal University of Piauí for analysis. In the rainy season, from the fifteen samples analyzed in square A, five were positive for hookworm larvae; and from the fifteen samples collected from square B, three were contaminated with the same helminths. During the dry period, only one sample from square A presented these larvae while no parasites were found in square B. The results revealed the presence of zoonotic helminth larvae in the soil of public squares in Caxias, Maranhão, mainly in the rainy season, which can be used as a warning sign to the local population.(AU)

Los suelos de las plazas públicas son comúnmente contaminados por helmintos debido al fácil acceso de perros y gatos infectados. Esos animales, al defecar, pueden liberar huevos de esos parásitos y, en condiciones ambientales favorables, convertirse en huevos embrionados o larvas infectantes. El objetivo de este trabajo fue investigar la existencia de larvas de helmintos en el suelo de dos plazas públicas de la ciudad de Caxias, Maranhão, Brasil, durante la estación lluviosa y seca de la región. La investigación se realizó en marzo de 2018, considerada época de lluvias, y en octubre del mismo año, época seca, y se recolectaron treinta muestras de arena, quince de cada plaza, en ambos períodos del año. El material fue recolectado y llevado al Laboratorio de Parasitología del Departamento de Parasitología y Microbiología de la Universidad Federal de Piauí para su análisis. En época de lluvias, de las quince muestras analizadas en la plaza A, cinco resultaron positivas a larvas de anquilostomiasis y de las quince de la plaza B, tres estaban contaminadas con los mismos helmintos. En el período poco lluvioso, en la plaza A solo hubo una muestra con esas larvas y en la plaza B no se encontraron parásitos. Los resultados revelaron la presencia de larvas de helmintos zoonóticos en el suelo de las plazas públicas de Caxias, Maranhão, principalmente en la época de lluvias, sirviendo de alerta a la población local.(AU)

Evaluation of Trypanosoma cruzi parasitic load by real-time PCR and blood culture in long-term kidney transplant recipients.

INTRODUCTION: Acute Chagas disease involving reactivation can occur after organ transplant, and follow-up by direct parasitological or molecular methods is essential for monitoring the parasitic load in such patients. In contrast, there is a little data on the parasitic load in long-term organ recipients. In this study, we examined the parasitic load in long-term kidney transplant patients and assessed the possibility of late Chagas disease reactivation. METHODOLOGY: Blood cultures and real-time PCR were used to assess the parasitic load in four immunosuppressed patients who underwent kidney transplants (between 1996 and 2014) and were also treated for parasites. RESULTS: There were no positive blood culture or real-time PCR results in Chagas disease patients who received kidney transplants. The real-time PCR presented detection limit of 0.1 parasite equivalent/mL. The time interval between the transplant and sample collection varied from one to 19 years. CONCLUSIONS: No parasites were detected in the evaluated patients. The use of benznidazole and immunosuppressive therapy may have contributed to control the T. cruzi infection. In transplanted patients with Chagas disease, the use of methods such real-time PCR and blood culture can monitor the parasitic load and prevent disease reactivation.

Correlates of Variation in Guinea Worm Burden among Infected Domestic Dogs.

The Guinea Worm Eradication Program has been extraordinarily successful-in 2019, there were 53 human cases reported, down from the estimated 3.5 million in 1986. Yet the occurrence of Guinea worm in dogs is a challenge to eradication efforts, and underlying questions about transmission dynamics remain. We used routine surveillance data to run negative binomial regressions predicting worm burden among infected dogs in Chad. Of 3,371 infected dogs reported during 2015-2018, 38.5% had multiple worms. A multivariable model showed that the number of dogs in the household was negatively associated with worm burden (adjusted incidence rate ratio [AIRR] = 0.95, 95% CI: 0.93-0.97, P < 0.0001) after adjusting for dog age (AIRR = 0.99, 95% CI: 0.96-1.01, P > 0.1). This could relate to the amount of infective inocula (e.g., contaminated food or water) shared by multiple dogs in a household. Other significant univariable associations with worm burden included dog history of Guinea worm infection (IRR = 1.30, 95% CI: 1.18-1.45) and dog owners who were hunters (IRR = 0.78, 95% CI: 0.62-0.99, P < 0.05) or farmers (IRR = 0.83, 95% CI: 0.77-0.90, P < 0.0001). Further analysis showed that the number of dogs in the household was significantly and positively correlated with nearly all other independent variables (e.g., owner occupation: farmer, fisherman, or hunter; dog age, sex, and history of Guinea worm). The associations we identified between worm burden and dogs per household, and dogs per household and owner characteristics should be further investigated with more targeted studies.

A sensitive and reliable quantitative immunohistochemistry technique to evaluate the percentage of Trypanosoma cruzi-infected tissue area.

Quantification of parasites in the context of Chagas disease is required to monitor the treatment with benznidazole, disease-associated cardiomyopathies and graft rejection after heart transplantation. As parasitological exams lack sensitivity, Real Time Polymerase Chain Reaction (rt-PCR) has emerged to evaluate the parasite load in blood samples and cardiac biopsies. However, despite its higher sensitivity, rt-PCR does not provide information on the location and distribution of amastigote nests within infected tissues, the characterization of inflammatory infiltrates or changes to tissue architecture. On the contrary, a sensitive immunohistochemistry technique (IHC) could fill these gaps. In the present study, a quantitative IHC exam was standardized and validated by testing adipose and cardiac tissues of experimentally infected mice containing variable parasite load levels of T. cruzi assessed by a sensitive Sybr Green rt-PCR with kDNA primers. Tissues were divided into four groups according to the parasite load: group A- 100 parasites/50 ng of DNA; group B -10 parasites; group C - around 1 parasite and group D - less than 1 parasite/50 ng/DNA. IHC was able to detect T. cruzi in the four groups, even in group D tissues containing fractions of a single parasite/50 ng of DNA sample according to rt-PCR. In conclusion, a highly sensitivity and reliable quantitative immunohistochemistry technique was developed and is proposed to estimate the percentage of T. cruzi-infected tissue area in chagasic patients presenting with cardiomyopathies, as a complementary test to rt-PCR.

Standardization of an LNA-based TaqMan assay qPCR analysis for Aspiculuris tetraptera DNA in mouse faeces.

BACKGROUND: Aspiculuris tetraptera, as a parasitic pinworm, is most frequently detected in laboratory mice, and transmission is mediated by the eggs contained in the faeces of infected mice. A highly sensitive and quantitative faeces-based diagnostic tool would be useful for the early detection of A. tetraptera to inhibit the expansion of infection. In this study, we developed a quantitative assay that exhibits high sensitivity in detecting A. tetraptera in faeces using PCR techniques. RESULTS: Endpoint PCR demonstrated the detection of A. tetraptera DNA in 0.5 ng genomic DNA extracted from the faeces of infected mice. To quantitatively detect the small amount of A. tetraptera DNA, locked nucleic acid (LNA)-based primers and LNA-based TaqMan probes were used for the quantitative PCR assay (qPCR). The combination of LNA-based DNA increased detection sensitivity by more than 100-fold compared to using normal oligo DNAs. The copy number of the A. tetraptera DNA detected was positively related to the infected faeces-derived genomic DNA with a simple linearity regression in the range of 20 pg to 15 ng of the genomic DNA. To more conveniently detect infection using faeces, the LNA-based TaqMan assay was applied to the crude fraction of the faeces without DNA purification. An assay using ethanol precipitation of the faeces yielded results consistent with those of direct microscopic observation. CONCLUSION: The LNA-TaqMan assay developed in this study quantitatively detects A. tetraptera infection in mouse faeces.

Mixed infections by different Trypanosoma cruzi discrete typing units among Chagas disease patients in an endemic community in Panama.

BACKGROUND: Trypanosoma cruzi, the hemoparasite that causes Chagas disease, is divided into six Discrete Typing Units or DTUs: TcI-TcVI plus Tcbat. This genetic diversity is based on ecobiological and clinical characteristics associated with particular populations of the parasite. The main objective of this study was the identification of DTUs in patients with chronic chagasic infections from a mountainous rural community in the eastern region of Panama. METHODS: A total of 106 patients were tested for Chagas disease with three serological tests (ELISA, rapid test, and Western blot). Molecular diagnosis and DTU typing were carried out by conventional PCRs and qPCR targeting different genomic markers, respectively. As a control sample for the typing, 28 patients suspected to be chagasic from the metropolitan area of Panama City were included. RESULTS: Results showed a positivity in the evaluated patients of 42.3% (33/78); high compared to other endemic regions in the country. In the control group, 20/28 (71.43%) patients presented positive serology. The typing of samples from rural patients showed that 78.78% (26/33) corresponded to TcI, while 9.09% (3/33) were mixed infections (TcI plus TcII/V/VI). Seventy-five percent (15/20) of the patients in the control group presented TcI, and in five samples it was not possible to typify the T. cruzi genotype involved. CONCLUSIONS: These results confirm that TcI is the main DTU of T. cruzi present in chronic chagasic patients from Panama. However, the circulation of other genotypes (TcII/V/VI) in this country is described for the first time. The eco-epidemiological characteristics that condition the circulation of TcII/V/VI, as well as the immune and clinical impact of mixed infections in this remote mountainous region should be investigated, which will help local action programs in the surveillance, prevention, and management of Chagas disease.

Comparison and clinical validation of qPCR assays targeting Leishmania 18S rDNA and HSP70 genes in patients with American Tegumentary Leishmaniasis.

Leishmaniasis is a worldwide neglected disease, encompassing asymptomatic infections and different clinical forms, such as American Tegumentary Leishmaniasis (ATL) which is part of the complex of diseases caused by protozoan parasites from Leishmania genus, transmitted by sand fly vectors. As a neglected disease, much effort is still needed in treatment and diagnosis. Currently, ATL diagnosis is mainly made by parasite detection by microscopy. The sensitivity of the method varies, and factors such as collection procedures interfere. Molecular approaches, specially based on Real Time PCR (qPCR) technique, has been widely used to detect Leishmania infection and to quantify parasite load, once it is a simple, rapid and sensitive methodology, capable to detect low parasite concentrations and less prone to variability. Although many studies have been already published addressing the use of this technique, an improvement on these methodologies, including an analytical validation, standardization and data association is demanded. Moreover, a proper validation by the assay by the use of clinical samples is still required. In this sense, the purpose of the present work is to compare the performance of qPCR using two commonly used targets (18S rDNA and HSP70) with an internal control (RNAse P) in multiplex reactions. Additionally, we validated reactions by assaying 88 samples from patients presenting different clinical forms of leishmaniasis (cutaneous, mucosal, recent and old lesions), representing the diversity found in Brazil's Amazon Region. Following the methodology proposed herein, the results indicate the use of both qPCR assays, 18S rDNA and HSP70, to achieve a very good net sensitivity (98.5%) and specificity (100%), performing simultaneous or sequential testing, respectively. With this approach, our main goal is to conclude the first step of a further multicenter study to propose the standardization of detection and quantification of Leishmania.

Comparación de tres métodos de concentración de enteroparásitos en muestras fecales humanas / Comparison between three enteroparasite concentration methods in human stool samples

Objetivo. Comparar tres métodos de concentración de enteroparásitos en muestras fecales humanas. Métodos. Se diseñó un estudio transversal en el que se evaluaron 154 muestras fecales categorizadas en dos grupos: parasitados (n= 127) y no parasitados (n= 27). Las muestras fueron sometidas a tres métodos: parasitológico directo, sedimentación simple y Ritchie modificado, y a la observación microscópica en lugol y suero fisiológico a un aumento de 40X. Resultados. Se observó mayor frecuencia en la presencia de estructuras parasitarias por el método de Ritchie modificado (37 por ciento), seguido de la sedimentación simple (14,8 por ciento) en el grupo de no parasitados; mientras que en el grupo de parasitados, se observó mayor carga parasitaria obtenida por el método de Ritchie (3+ (15,8 por ciento) y 2+ (23,6 por ciento), que en la sedimentación simple (3+ (10,2 por ciento) y 2+ (22,8 por ciento). Las especies parasitarias con mayor frecuencia fueron Entamoeba coli (20,3 por ciento), Giardia lamblia (18,8 por ciento), Blastocystis hominis (15,9 por ciento) y Endomlimax nana (15,2 por ciento); además, se presentó 48,7 por ciento casos con poliparasitismo. El área bajo la curva (AUC) para los métodos de Ritchie modificado, sedimentación simple y parasitológico directo fue de 0,870; 0.648 y 0,796, respectivamente. El AUC del método de Ritchie modificado fue mayor en los varones (0,933) que en las mujeres (0.,92); así como en aquellos menores de 12 años (0,867), comparados con personas entre 12-37 años (0,833) y 18-39 años (0,800). Conclusiones. El método de Ritchie modificado presenta alto rendimiento diagnóstico y permite concentrar mayor cantidad de parásitos intestinales que el método de sedimentación simple. Además, presenta la ventaja de utilizar insumos de fácil acceso y baja toxicidad, lo que genera mayor posibilidad de implementación en los laboratorios de parasitología(AU)

Objective: Compare three enteroparasite concentration methods in human stool samples. Methods: A cross-sectional study was conducted of 154 stool samples divided into two groups: with parasites (n= 127) and without parasites (n= 27). The samples were subjected to three methods: direct parasitological examination, simple sedimentation and modified Ritchie's, and to microscopic observation in lugol and saline solution to a 40x increase. Results: In the non-parasite group the highest frequency in the presence of parasite structures was observed with the modified Ritchie's method (37 percent), followed by simple sedimentation (14.8 percent). In the parasite group a greater parasite load was obtained by Ritchie's method (3+ (15.8 percent) and 2+ (23.6 percent) than by simple sedimentation (3+ (10.2 percent) and 2+ (22.8 percent). The parasite species showing the highest frequency were Entamoeba coli (20.3 percent), Giardia lamblia (18.8 percent), Blastocystis hominis (15.9 percent) and Endomlimax nana (15.2 percent), whereas polyparasitism was found in 48.7 percent of the cases. The area under the curve (AUC) for the modified Ritchie's method, simple sedimentation technique and direct parasitological examination was 0.870, 0.648 and 0.796, respectively. In the modified Ritchie's method the AUC was greater in male (0.933) than in female subjects (0.92), as well as in subjects aged under 12 years (0.867) in comparison with people aged 12-37 years (0.833) and 18-39 years (0.800). Conclusions: The modified Ritchie's method has a high diagnostic yield and makes it possible to concentrate a larger number of intestinal parasites than the simple sedimentation method. Additionally, it has the advantage of using inputs of easy access and low toxicity, broadening the possibility of its implementation in parasitology laboratories(AU)

Sensitivity and specificity of malaria rapid diagnostic test (mRDT CareStatTM) compared with microscopy amongst under five children attending a primary care clinic in southern Nigeria.

BACKGROUND: Malaria diagnosis using microscopy is currently the gold standard. However, malaria rapid diagnostic tests (mRDTs) were developed to simplify the diagnosis in regions without access to functional microscopy. AIM: The objective of this study was to compare the diagnostic accuracy of mRDT CareStatTM with microscopy. SETTING: This study was conducted in the paediatric primary care clinic of the Federal Medical Centre, Asaba, Nigeria. METHODS: A cross-sectional study for diagnostic accuracy was conducted from May 2016 to October 2016. Ninety-eight participants were involved to obtain a precision of 5%, sensitivity of mRDT CareStatTM of 95% from published work and 95% level of confidence after adjusting for 20% non-response rate or missing data. Consecutive participants were tested using both microscopy and mRDT. The results were analysed using EPI Info Version 7. RESULTS: A total of 98 children aged 3-59 months were enrolled. Malaria prevalence was found to be 53% (95% confidence interval [CI] = 46% - 60%), whilst sensitivity and specificity were 29% (95% CI = 20% - 38%) and 89% (95% CI = 83% - 95%), respectively. The positive and negative predictive values were 75% (95% CI = 66.4% - 83.6%) and 53% (95% CI = 46% - 60%), respectively. CONCLUSION: Agreement between malaria parasitaemia using microscopy and mRDT positivity increased with increase in the parasite density. The mRDT might be negative when malaria parasite density using microscopy is low.

Quantification of Toxocara canis DNA by qPCR in mice inoculated with different infective doses.

The nematode Toxocara canis is of public health importance and is the main causative agent of toxocariasis in humans. This disease is difficult to diagnose due to several factors, including the possibility of cross-reactions with other nematodes in the ELISA. To overcome this problem, molecular tests have been recommended as an alternative to identify the parasite. The quantitative real-time polymerase chain reaction (qPCR) technique was used in this study to identify and quantify the parasite load of T. canis in the mouse brain. To this end, 24 mice were divided into six groups, five of which were challenged with different infective doses of T. canis larvae (L3) (1000, 500, 250, 100 and 50 larvae), while the sixth group, uninfected, acted as negative control. Forty-five days after infection, the animals were euthanized to collect the brain, from which two portions of 20 mg of tissue were taken for DNA extraction, while the rest of the brain tissue was digested to quantify the number of larvae by microscopy. The number of DNA copies was calculated from the standard DNA quantification curve, showing values of E = 93.4%, R2 = 0.9655 and Y = -3.415. A strong positive correlation (R = 0, 81; p < .001) was found between the number of copies and the recovery of larvae from brain. However, the parasite's DNA was also identified even in animals from whose brain no larvae were recovered after tissue digestion. The results of this study therefore confirm that the qPCR technique can be a valuable tool for the detection and quantification of T. canis DNA in murine hosts, even in animals whose with tissues contain very few parasites.

Determining the viability of Schistosoma mansoni cercariae using fluorescence assays: An application for water treatment.

BACKGROUND: Schistosome cercariae are the human-infectious stage of the Schistosoma parasite. They are shed by snail intermediate hosts living in freshwater, and penetrate the skin of the human host to develop into schistosomes, resulting in schistosomiasis infection. Water treatment (e.g. filtration or chlorination) is one way of cutting disease transmission; it kills or removes cercariae to provide safe water for people to use for activities such as bathing or laundry as an alternative to infested lakes or rivers. At present, there is no standard method for assessing the effectiveness of water treatment processes on cercariae. Examining cercarial movement under a microscope is the most common method, yet it is subjective and time-consuming. Hence, there is a need to develop and verify accurate, high-throughput assays for quantifying cercarial viability. METHOD: We tested two fluorescence assays for their ability to accurately determine cercarial viability in water samples, using S. mansoni cercariae released from infected snails in the Schistosomiasis Collection at the Natural History Museum, London. These assays consist of dual stains, namely a vital and non-vital dye; fluorescein diacetate (FDA) and Hoechst, and FDA and Propidium Iodide. We also compared the results of the fluorescence assays to the viability determined by microscopy. CONCLUSION: Both fluorescence assays can detect the viability of cercariae to an accuracy of at least 92.2% ± 6.3%. Comparing the assays to microscopy, no statistically significant difference was found between the method's viability results. However, the fluorescence assays are less subjective and less time-consuming than microscopy, and therefore present a promising method for quantifying the viability of schistosome cercariae in water samples.

Tick burden on European roe deer (Capreolus capreolus) from Saxony, Germany, and detection of tick-borne encephalitis virus in attached ticks.

Southern Germany is known as tick-borne encephalitis (TBE) risk area; however, the north of the country is almost free of human TBE cases. Due to its location in the transition zone between TBE risk areas and areas with only sporadic cases, Saxony is of importance in the surveillance of TBE. Roe deer (Capreolus capreolus), showing high seroprevalence of TBE virus (TBEV) antibodies, are considered to be sentinels for TBE risk assessment. Thus, roe deer could be used as indicators helping to better understand the focality of the TBEV in nature and as a possible source to isolate TBEV. Therefore, the aims of this study were to examine roe deer coats for the presence of ticks to establish the tick burden and to detect the TBEV in attached ticks. One hundred thirty-four roe deer coats were provided by hunters from the Hunting Association in Saxony (August 2017-January 2019). The coats were frozen at - 80 °C and after de-freezing examined on both sides-inside and outside. Attached and nonattached ticks were collected, morphologically identified and tested using real-time RT-PCR for the presence of TBEV. In total, 1279 ticks were found on 48 coats. The predominant species was Ixodes ricinus (99.76%; n = 1276). Three remaining specimens were Ixodes spp. (0.16%, 1 female and 1 nymph) and Dermacentor reticulatus (0.08%, 1 male). The average infestation rate was 26.7 (SD = 69.5), with maximum of 439 ticks per animal. Females were the dominant life stage of ticks (n = 536; 42%), followed by nymphs (n = 397; n = 31.1%), males (n = 175; 13.7%), and larvae (n = 168; 13.2%). Only half of collected ticks were attached (n = 662; 51.8%). TBEV was detected only in one tick out of 1279 tested ticks. It was a female infesting a roe deer from Saxon Switzerland-East Ore Mountain. The results show that the method used in this study is not sufficient as a sentinel marker to predict TBEV spreading in nature. Although previous studies demonstrated the usefulness of serological testing of roe deer in order to trace TBE-endemic regions, using ticks attached to them to get virus isolates is not productive.

Parasitic load determination by differential expressions of 5-lipoxygenase and PGE2 synthases in visceral leishmaniasis.

Infection with L. donovani affects mainly visceral organs. Importantly, the parasitic load differs in different visceral organs; therefore there is a need to understand the organ specific immune regulation, particularly in the spleen and liver. Comparative studies between these organs in Leishmania infected hamster (Mesocricetus auratus) are lacking. Our study highlights the importance of eicosanoids in the organ specific pathology of visceral leishmaniasis. Among other immune cells, macrophages (mφ) which harbor Leishmania parasite are major producers of eicosanoids. In this study, we intend to explore linkage between organ specific immune response and eicosanoids. We suggest that eicosanoids (early immune modulators) and their organ specific expressions, possibly tune the outcome of mφ differently at different sites. We have observed that liver showed better containment of parasitic load than spleen, where we have found higher expression of 5-lipoxygenase (5-LO) enzyme along with IL-12 and iNOS. However, in spleen, enzymes of the PGE2 pathway i.e. PGE2 synthases (cytosolic and microsomal) along with IL-10 were predominantly higher. To further corroborate our findings, in vitro assays were carried out using purified eicosanoids (LTB4 and PGE2) and the inhibitors of these pathways. Findings establish that the 5-lipoxygenase pathway (i.e. LTB4) is anti-parasitic and its inhibition increases the parasitic load (qPCR based kDNA detection). On the contrary, PGES pathway (i.e. PGE2) supports establishment of infection in mφ. Taken together, 5-LO pathway plays a protective role in liver during L. donovani infection. However, the PGES pathway favors the parasite growth, particularly in the spleen at a later stage.

New promising method to assess microfilarial Loa loa load on the peripheral blood.

Loiasis is a vector-borne parasitic disease caused by the filarial Loa loa (L. loa). Definitive diagnosis can be done by identifying and counting microfilariae in the peripheral blood by microscopy and with L.loa-specific PCR. An additional diagnostic method is the detection of L.loa-specific antibodies. Accurate methods are needed to automate quantification of microfilaria (mf) in peripheral blood. Indeed, the treatment procedure depends on the microfilarial L. loa load in blood. We report the first documented use of flow cytometry as a new method to count microfilaraemia in peripheral blood from a patient with L. loa infection. The diagnosis of loiasis was strongly suspected based on clinical presentation and rapidly confirmed by identifying typical features of L. loa in the peripheral blood. This diagnosis was achieved by flow cytometry using a specific fluorescence pattern for microfilaraemia count. The current report highlights the potential of flow cytometry to assess microfilarial L. loa load from a patient with loiasis infection.

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.

Comparison on simultaneous caillary and venous parasite density and genotyping results from children and adults with uncomplicated malaria: a prospective observational study in Uganda.

BACKGROUND: Blood smear microscopy remains the gold-standard method to diagnose and quantify malaria parasite density. In addition, parasite genotyping of select loci is the most utilized method for distinguishing recrudescent and new infections and to determine the number of strains per sample. In research settings, blood may be obtained from capillary or venous compartments, and results from these matrices have been used interchangeably. Our aim was to compare quantitative results for parasite density and strain complexity from both compartments. METHODS: In a prospective observational study, children and adults presenting with uncomplicated Plasmodium falciparum malaria, simultaneous capillary and venous blood smears and dried blood spots were collected over 42-days following treatment with artemether-lumefantrine. Blood smears were read by two microscopists, any discrepancies resolved by a third reader. Parasite DNA fingerprinting was conducted using six microsatellites. Bland Altman analysis and paired t-test/McNemar's test were used to assess the difference in density readings and measurements. RESULTS: Two hundred twenty-three participants were included in the analysis (177 children (35 HIV-infected/142 HIV-uninfected), 21 HIV-uninfected pregnant women, and 25 HIV-uninfected non-pregnant adults). Parasite density measurements did not statistically differ between capillary and venous blood smears at the time of presentation, nor over the course of 42-day follow-up. Characterization of merozoite surface protein-2 (MSP-2) genetic polymorphism demonstrated a higher level of strain diversity at the time of presentation in venous samples, as compared with capillary specimens (p = 0.02). There was a high degree of variability in genotype-corrected outcomes when pairs of samples from each compartment were compared using MSP-2 alone, although the variability was reduced with the use of multiple markers. CONCLUSIONS: Parasite density measurements do not statistically differ between capillary and venous compartments in all studied demographic groups at the time of presentation with malaria, or over the course of follow-up. More strains were detected by MSP-2 genotyping in venous samples than in capillary samples at the time of malaria diagnosis. The use of multiple polymorphic markers reduces the impact of variability in strain detection on genotype-corrected outcomes. This study confirms that both capillary and venous compartments can be used for sampling with confidence in the clinical research setting. TRIAL REGISTRATION: The trial was registered at ClinicalTrials.gov under registration no. NCT01717885 .

Quantification of Parasite Loads by Automated Microscopic Image Analysis.

High content analysis enables automated, robust, and unbiased evaluation of in vitro Leishmania infection. Here, we describe a protocol based on the infection of THP-1 macrophages with Leishmania promastigotes and the quantification of parasite load by high content analysis. The technique is capable of detecting and quantifying intracellular amastigotes, providing a multiparametric readout of the total number of cells, ratio of infected cells, total number of parasites, and number of parasites per infected cells. The technique can be used to quantitate infection of any Leishmania species in virtually all types of permissive host cells and can be applied to quantification of drug activity and studies of the Leishmania intracellular life cycle stage.

Quantification of Leishmania Parasites in Murine Models of Visceral Infection.

Visceral leishmaniasis (VL) is mainly caused by Leishmania donovani (India and East Africa), and Leishmania infantum (Mediterranean Basin and South America) infections. Although murine models of visceral infection lack the clinicopathological aspects of VL in humans, they have been proven useful at advancing our knowledge in the Leishmania field. Indeed, these models have been used not only to better understand the pathophysiology of the infection but also in drug and vaccine development. This chapter focuses on the protocols used to experimentally infect mice and to quantify parasite burdens in mice infected with L. infantum using limiting dilution methodology of target organs and whole-mouse in vivo imaging.