Introducing automation to the molecular diagnosis of Trypanosoma cruzi infection: A comparative study of sample treatments, DNA extraction methods and real-time PCR assays

Background Polymerase chain reaction (PCR) has become a useful tool for the diagnosis of Trypanosoma cruzi infection. The development of automated DNA extraction methodologies and PCR systems is an important step toward the standardization of protocols in routine diagnosis. To date, there are only two commercially available Real-Time PCR assays for the routine laboratory detection of T. cruzi DNA in clinical samples: TCRUZIDNA.CE (Diagnostic Bioprobes Srl) and RealCycler CHAG (Progenie Molecular). Our aim was to evaluate the RealCycler CHAG assay taking into account the whole process. Methodology/Principal findings We assessed the usefulness of an automated DNA extraction system based on magnetic particles (EZ1 Virus Mini Kit v2.0, Qiagen) combined with a commercially available Real-Time PCR assay targeting satellite DNA (SatDNA) of T. cruzi (RealCycler CHAG), a methodology used for routine diagnosis in our hospital. It was compared with a well-known strategy combining a commercial DNA isolation kit based on silica columns (High Pure PCR Template Preparation Kit, Roche Diagnostics) with an in-house Real-Time PCR targeting SatDNA. The results of the two methodologies were in almost perfect agreement, indicating they can be used interchangeably. However, when variations in protocol factors were applied (sample treatment, extraction method and Real-Time PCR), the results were less convincing. A comprehensive fine-tuning of the whole procedure is the key to successful results. Guanidine EDTA-blood (GEB) samples are not suitable for DNA extraction based on magnetic particles due to inhibition, at least when samples are not processed immediately. Conclusions/Significance This is the first study to evaluate the RealCycler CHAG assay taking into account the overall process, including three variables (sample treatment, extraction method and Real-Time PCR). Our findings may contribute to the harmonization of protocols between laboratories and to a wider application of Real-Time PCR in molecular diagnostic laboratories associated with health centers.Fil: Abras, Alba. Universidad de Barcelona; España. Universidad de Girona; España. Instituto de Salud Global de Barcelona; EspañaFil: Ballart, Cristina. Universidad de Barcelona; España. Instituto de Salud Global de Barcelona; EspañaFil: Llovet, Teresa. Universitat Autònoma de Barcelona; España. Hospital de la Santa Creu I Sant Pau; EspañaFil: Roig, Carme. Hospital de la Santa Creu I Sant Pau; EspañaFil: Gutiérrez, Cristina. Hospital de la Santa Creu I Sant Pau; EspañaFil: Tebar, Silvia. Universidad de Barcelona; España. Instituto de Salud Global de Barcelona; EspañaFil: Berenguer, Pere. Hospital de la Santa Creu I Sant Pau; EspañaFil: Pinazo, María-Jesús. Instituto de Salud Global de Barcelona; EspañaFil: Posada, Elizabeth. Instituto de Salud Global de Barcelona; EspañaFil: Gascón, Joaquim. Instituto de Salud Global de Barcelona; EspañaFil: Schijman, Alejandro Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Gállego, Montserrat. Instituto de Salud Global de Barcelona; España. Universidad de Barcelona; EspañaFil: Muñoz, Carmen. Hospital de la Santa Creu I Sant Pau; España. Universitat Autònoma de Barcelona; Españ

Male Deep-Sea Shrimps Aristeus antennatus at Fishing Grounds: Growth and First Evaluation of Recruitment by Multilocus Genotyping

The population biology of the deep-sea shrimp Aristeus antennatus, as with other exploited demersal species, is usually studied using data from fishery statistics. Such statistical analyses have shown female-biased sex ratios during the spawning season in this species. Because the abundance of males increases at greater depths that are not exploited by fisheries (virgin grounds), knowledge on their recruitment is limited. Here, the growth and recruitment of A. antennatus males at fishing grounds was evaluated. This was achieved by integrating information on previously identified breeding behaviours and by tracing the young-of-year cohort through genotyping at 10 microsatellite loci. Using a codend and a codend cover with distinct meshed windows, four groups of males were collected in winter and in a subsequent spawning summer season. Summer collections were mostly composed of pre-adult males, reaching sizes that are to be expected from the growth of winter juveniles; however, many specimens also originated from nearby grounds. This result indicates the horizontal dispersal of male juveniles via intermediate and deep oceanographic currents. Such dispersal complements passive larval dispersal in surface waters, and contributes to the weak genetic divergence among regional fishing grounds. These features could be shared by other deep-sea crustacean and fish species, and should be considered for the sustainable exploitation of demersal fisheriesThis work was supported by a grant from Spanish Ministerio de Economia y Competitividad (CTM2014-54648-C2-2-R) to M.I.R. L.P. and M.A. benefited from predoctoral fellowship from the Universitat de Girona (BR2014 and IFUdG2018, respectively)S

Seroprevalence of canine Leishmania infantum infection in the Mediterranean region and identification of risk factors : The example of North-Eastern and Pyrenean areas of Spain

This project received funding from the European Union's Horizon 2020 research and innovation programme under Marie Sklodowska-Curie grant agreement nº 642609 and by Ministerio de Ciencia e Innovación, Spain (CG12010-22368-CO2-01).Altres ajuts: RICET/RD12/0018/0010The Mediterranean basin is an endemic region for canine leishmaniosis (CanL), where it represents a major veterinary problem and raises human health concerns. However, the distribution of the disease is heterogeneous and not all countries and locations have been equally studied and characterized. This work describes the situation of CanL in Girona province (Catalonia, Spain), for which no data has been previously reported, and presents a relevant study to exemplify other areas with similar characteristics across the region. Four cross-sectional seroprevalence surveys were performed from 2012 to 2016 throughout the province, including 36 sampling stations in 26 localities and a total of 593 dogs. For each animal, individual and location variables were also collected. Additionally, each dog owner answered a questionnaire about their knowledge of CanL and preventive methods used. Blood samples were analysed by an in-house ELISA and a mixed logistic regression model was used to assess the relationship between pre-determined variables and dog seropositivity. A Spearman's correlation was used to assess the association between dog owners' perceived risk of CanL and Leishmania infantum seropositivity in dogs at a given location. The overall true seroprevalence estimated for Girona province was 19.5% (95%CI: 15.5-23.5), of which only 6.8% (10/146) were considered symptomatic. Age of the dog [OR = 1.21 (95%CI: 1.11-1.31); p < 0.001] and altitude [OR = 0.02 (95%CI: 0.001-0.19); p = 0.001] were identified as risk factors for the infection. The results obtained in this study are expected to aid in the implementation of directed control programmes in CanL endemic areas throughout Europe, as well as to provide suitable data for the design of better risk assessment maps of the disease

The Leishmania donovani species complex: A new insight into taxonomy.

Among the 20 or so Leishmania spp. described as pathogenic for humans, those of the Leishmania donovani complex are the exclusive causative agents of systemic and fatal visceral leishmaniasis. Although well studied, the complex is taxonomically controversial, which hampers clinical and epidemiological research. In this work, we analysed 56 Leishmania strains previously identified as L. donovani, Leishmania archibaldi or Leishmania infantum, isolated from humans, dogs and sandfly vectors throughout their distribution area. The strains were submitted to biochemical and genetic analyses and the resulting data were compared for congruence. Our results show: i) a partial concordance between biochemical and genetic-based data, ii) very limited genetic variability within the L. donovani complex, iii) footprints of frequent genetic exchange along an east-west gradient, marked by a widespread diffusion of alleles across the geographical range, and iv) a large-scale geographical spreading of a few genotypes. From a taxonomic point of view, considering the absence of relevant terminology in existing classes, the L. donovani complex could be treated as a single entit

Seroprevalence of canine Leishmania infantum infection in the Mediterranean region and identification of risk factors: The example of North-Eastern and Pyrenean areas of Spain

The Mediterranean basin is an endemic region for canine leishmaniosis (CanL), where it represents a major veterinary problem and raises human health concerns. However, the distribution of the disease is heterogeneous and not all countries and locations have been equally studied and characterized. This work describes the situation of CanL in Girona province (Catalonia, Spain), for which no data has been previously reported, and presents a relevant study to exemplify other areas with similar characteristics across the region. Four cross-sectional seroprevalence surveys were performed from 2012 to 2016 throughout the province, including 36 sampling stations in 26 localities and a total of 593 dogs. For each animal, individual and location variables were also collected. Additionally, each dog owner answered a questionnaire about their knowledge of CanL and preventive methods used. Blood samples were analysed by an in-house ELISA and a mixed logistic regressio nmodel was used to assess the relationship between pre-determined variables and dog seropositivity. A Spearman's correlation was used to assess the association between dog owners'perceived risk of CanL an dLeishmania infantums eropositivity in dogs at a given location. The overall true seroprevalence estimated forGirona province was 19.5% (95%CI: 15.5-23.5), of which only 6.8% (10/146) were considered symptomatic. Age of the dog [OR = 1.21 (95%CI: 1.11-1.31); p < 0.001] and altitude [OR = 0.02 (95%CI: 0.001-0.19);p = 0.001] were identified as risk factors for the infection. The results obtained in this study are expected to aid in the implementation of directed control programmes in CanL endemic areas throughout Europe, as well as to provide suitable data for the design of better risk assessment maps of the diseas

Usefulness of Matrix-Assisted Laser Desorption/Ionization-Time of Flight Mass Spectrometry in the Characterization of Leishmania Strains Causing Tegumentary Leishmaniasis in Bolivia versus hsp70 Gene Sequencing

Matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) is a proteomic technique with proven efficiency in the identification of microorganisms, such as bacteria, fungi, and parasites. The present study aimed to evaluate the usefulness of MALDI-TOF MS for the characterization of Leishmania species circulating in Bolivia using hsp70 gene sequencing as a reference technique. 55 Leishmania strains that were isolated from patients with tegumentary leishmaniasis were analyzed. MALDI-TOF MS identified two species of the L. braziliensis complex (L. braziliensis, n = 26; L. braziliensis outlier, n = 18), one species of the L. guyanensis complex (L. guyanensis, n = 1), one species of the L. lainsoni complex (L. lainsoni, n = 2), and two species of the complex (, n = 5; and L. garnhami, n = 3). All of the strains were correctly identified at the subgenus, genus, and complex level, but 10 of them (18%) were misidentified as other species within the same complex by the hsp70 gene sequencing, with 7 of these corresponding to possible hybrids. Thus, one L. braziliensis corresponded to L. peruviana, two L. braziliensis corresponded to L. braziliensis / L. peruviana possible hybrids, two corresponded to , and three L. garnhami and two corresponded to / possible hybrids. Accordingly, MALDI-TOF MS could be used as an alternative to molecular techniques for the identification of Leishmania spp., as it is low cost, simple to apply, and able to quickly produce results. In Bolivia, its application would allow for the improvement of the management of patient follow-ups, the updating of the epidemiological data of the Leishmania species, and a contribution to the control of tegumentary leishmaniasis. IMPORTANCE The objective of the study was to evaluate the usefulness of MALDI-TOF MS for the characterization of Leishmania species circulating in Bolivia, in comparison with the sequencing of the hsp70 gene. In our study, all of the isolates could be identified, and no misidentifications were observed at the complex level. Although the equipment implies a high initial investment in our context, MALDI-TOF MS can be used in different areas of microbiology and significantly reduces the cost of testing. Once the parasite culture is obtained, the technique quickly yields information by accessing a free database that is available online. This would allow for the improvement of the management of patients and follow-ups, the updating of the epidemiological data of the species, and a contribution to the control of tegumentary leishmaniasis in Bolivia. Likewise, it can be used to determine a specific treatment to be given, according to the causal species of Leishmania, when there are protocols in this regard in the area

Parasitemia Levels in Trypanosoma cruzi Infection in Spain, an Area Where the Disease Is Not Endemic: Trends by Different Molecular Approaches

Trypanosoma cruzi infection has expanded globally through human migration. In Spain, the mother-to-child route is the mode of transmission contributing to autochthonous Chagas disease (CD); however, most people acquired the infection in their country of origin and were diagnosed in the chronic phase (imported chronic CD). In this context, we assessed the quantitative potential of the Loopamp Trypanosoma cruzi detection kit (Sat-TcLAMP) based on satellite DNA (Sat-DNA) to determine parasitemia levels compared to those detected by real-time quantitative PCRs (qPCRs) targeting Sat-DNA (Sat-qPCR) and kinetoplast DNA minicircles (kDNA-qPCR). This study included 173 specimens from 39 autochthonous congenital and 116 imported chronic CD cases diagnosed in Spain. kDNA-qPCR showed higher sensitivity than Sat-qPCR and Sat-TcLAMP. According to all quantitative approaches, parasitemia levels were significantly higher in congenital infection than in chronic CD (1 × 10-1 to 5 × 105 versus >1 × 10-1 to 6 × 103 parasite equivalents/mL, respectively [P < 0.001]). Sat-TcLAMP, Sat-qPCR, and kDNA-qPCR results were equivalent at high levels of parasitemia (P = 0.381). Discrepancies were significant for low levels of parasitemia and older individuals. Differences between Sat-TcLAMP and Sat-qPCR were not qualitatively significant, but estimations of parasitemia using Sat-TcLAMP were closer to those by kDNA-qPCR. Parasitemia changes were assessed in 6 individual cases in follow-up, in which trends showed similar patterns by all quantitative approaches. At high levels of parasitemia, Sat-TcLAMP, Sat-qPCR, and kDNA-qPCR worked similarly, but significant differences were found for the low levels characteristic of late chronic CD. A suitable harmonization strategy needs to be developed for low-level parasitemia detection using Sat-DNA- and kDNA-based tests. IMPORTANCE: Currently, molecular equipment has been introduced into many health care centers, even in low-income countries. PCR, qPCR, and loop-mediated isothermal amplification (LAMP) are becoming more accessible for the diagnosis of neglected infectious diseases. Chagas disease (CD) is spreading worldwide, and in countries where the disease is not endemic, such as Spain, the parasite Trypanosoma cruzi is transmitted from mother to child (congenital CD). Here, we explore why LAMP, aimed at detecting T. cruzi parasite DNA, is a reliable option for the diagnosis of congenital CD and the early detection of reactivation in chronic infection. When the parasite load is high, LAMP is equivalent to any qPCR. In addition, the estimations of T. cruzi parasitemia in patients living in Spain, a country where the disease is not endemic, resemble natural evolution in areas of endemicity. If molecular tests are introduced into the diagnostic algorithm for congenital infection, early diagnosis and timely treatment would be accomplished, so the interruption of vertical transmission can be an achievable goal.This research was supported by the Foundation for Innovative New Diagnostics (FIND), Geneva, Switzerland (WO klob-0003), and the Surveillance Program of Chagas Disease of the National Centre for Microbiology (CNM), Instituto de Salud Carlos III (ISCIII). CNM-ISCIII research team is supported by Fundación Mundo Sano, Spain (MVP 237/19). The ISGlobal research team is supported by the Agència de Gestió d’Ajuts Universitaris i de Recerca AGAUR) (2017 SGR 00924). ISGlobal is a member of the Centres de Recerca de Catalunya (CERCA) Programme, Government of Catalonia (Spain).S

Altered Hypercoagulability Factors in Patients with Chronic Chagas Disease: Potential Biomarkers of Therapeutic. Response

Thromboembolic events were described in patients with Chagas disease without cardiomyopathy. We aim to confirm if there is a hypercoagulable state in these patients and to determine if there is an early normalization of hemostasis factors after antiparasitic treatment. Ninety-nine individuals from Chagas disease-endemic areas were classified in two groups: G1, with T.cruzi infection (n = 56); G2, healthy individuals (n = 43). Twenty-four hemostasis factors were measured at baseline. G1 patients treated with benznidazole were followed for 36 months, recording clinical parameters and performance of conventional serology, chemiluminescent enzyme-linked immunosorbent assay (trypomastigote-derived glycosylphosphatidylinositol-anchored mucins), quantitative polymerase chain reaction, and hemostasis tests every 6-month visits. Prothrombin fragment 1+2 (F1+2) and endogenous thrombin potential (ETP) were abnormally expressed in 77% and 50% of infected patients at baseline but returned to and remained at normal levels shortly after treatment in 76% and 96% of cases, respectively. Plasmin-antiplasmin complexes (PAP) were altered before treatment in 32% of G1 patients but normalized in 94% of cases several months after treatment. None of the patients with normal F1+2 values during follow-up had a positive qRT-PCR result, but 3/24 patients (13%) with normal ETP values did. In a percentage of chronic T. cruzi infected patients treated with benznidazole, altered coagulation markers returned into normal levels. F1+2, ETP and PAP could be useful markers for assessing sustained response to benznidazole

Serological diagnosis of chronic Chagas disease: Is it time for a change?

Chagas disease has spread to non-endemic areas with human migration. Since no single reference standard test is available, serological diagnosis of chronic Chagas disease requires at least two tests. New generation techniques have significantly improved the accuracy of Chagas disease diagnosis by the use of a large mixture of recombinant antigens with different detection systems, such as chemiluminescence. The aim of the present study was to assess the overall accuracy of a new generation kit, Architect Chagas (cut-off ≥ 1 S/CO, sample relative light units/cut-off value), as a single technique in the diagnosis of chronic Chagas disease. Architect Chagas showed a sensitivity of 100% (95% confidence interval, CI = 99.5-100) and a specificity of 97.6% (95% CI = 95.2-99.9). Five out of six false-positive sera were a consequence of cross-reactivity with Leishmania spp. and all of them achieved results < 5 S/CO. We propose Architect Chagas as a single technique for screening in blood banks and for routine diagnosis in clinical laboratories. Only grey zone and positive sera with a result ≤ 6 S/CO would need to be confirmed by a second serological assay, thus avoiding false-positive sera and the problem of cross-reactivity with Leishmania spp. The application of this proposal would result in important savings in the cost of Chagas disease diagnosis and therefore in the management and control of the disease