Molecular Diagnosis of Leishmaniasis in Spain: Development and Validation of Ready-To-Use Gel-Form Nested and Real-Time PCRs To Detect Leishmania spp.

Leishmaniasis is an endemic parasitic disease in at least 98 countries. In Spain, it is considered a zoonosis caused by Leishmania infantum, with an annual incidence of 0.62 cases/100,000 inhabitants. The predominant clinical manifestations are the cutaneous (CL) and visceral forms (VL), and the diagnosis is performed by parasitological, serological, and molecular tests. At the WHO Collaborating Center for Leishmaniasis (WHOCCLeish), routine diagnostic tests are based on a nested PCR (Ln-PCR), culture, and serological tests. To simplify our PCR protocol, we aimed to develop and validate a ready-to-use nested gel-form PCR (LeishGelPCR) and a duplex real-time PCR (qPCR) that allowed simultaneous detection of Leishmania and mammalian DNA as an internal control (Leish-qPCR). Clinical validation was performed in 200 samples from the WHOCCLeish collection; 92 and 85 out of 94 and 87 samples were positive by LeishGelPCR and Leish-qPCR, respectively, showing a sensitivity of 98% in both approaches. The specificity was 100% for LeishGelPCR and 98% for Leish-qPCR. The limits of detection of both protocols were similar (0.5 and 0.2 parasites/reaction). Parasite loads in VL and CL forms were similar, although high loads were observed when invasive samples were tested. In conclusion, LeishGelPCR and Leish-qPCR showed excellent performance in the diagnosis of leishmaniasis. These new forms of 18S rRNA gene PCR are equivalent to Ln-PCR and can be introduced in the algorithm for CL and VL diagnosis. IMPORTANCE Although the gold standard for diagnosis of leishmaniasis is the microscopic observation of amastigotes, molecular techniques are becoming a cost-efficient alternative. Currently, PCR is a routine resource that is used in many reference microbiology laboratories. In this article, we have described two ways to improve the reproducibility and usability of the molecular detection of Leishmania spp. These new approaches could be introduced even in middle- and low-resource laboratories; one is a ready-to-use gel-form system of a nested PCR and the other is a real-time PCR. We show why molecular diagnosis is the best methodology to confirm a clinical suspicion of leishmaniasis with higher sensitivity than traditional methods, thus facilitating early diagnosis and timely treatment of human leishmaniasis.

Zoonotic Enterocytozoon bieneusi genotypes in free-ranging and farmed wild ungulates in Spain.

Microsporidia comprises a diverse group of obligate, intracellular, and spore-forming parasites that infect a wide range of animals. Among them, Enterocytozoon bieneusi is the most frequently reported species in humans and other mammals and birds. Data on the epidemiology of E. bieneusi in wildlife are limited. Hence, E. bieneusi was investigated in eight wild ungulate species present in Spain (genera Ammotragus, Capra, Capreolus, Cervus, Dama, Ovis, Rupicapra, and Sus) by molecular methods. Faecal samples were collected from free-ranging (n = 1058) and farmed (n = 324) wild ungulates from five Spanish bioregions. The parasite was detected only in red deer (10.4%, 68/653) and wild boar (0.8%, 3/359). Enterocytozoon bieneusi infections were more common in farmed (19.4%, 63/324) than in wild (1.5%, 5/329) red deer. A total of 11 genotypes were identified in red deer, eight known (BEB6, BEB17, EbCar2, HLJD-V, MWC_d1, S5, Type IV, and Wildboar3) and three novel (DeerSpEb1, DeerSpEb2, and DeerSpEb3) genotypes. Mixed genotype infections were detected in 15.9% of farmed red deer. Two genotypes were identified in wild boar, a known (Wildboar3) and a novel (WildboarSpEb1) genotypes. All genotypes identified belonged to E. bieneusi zoonotic Groups 1 and 2. This study provides the most comprehensive epidemiological study of E. bieneusi in Spanish ungulates to date, representing the first evidence of the parasite in wild red deer populations worldwide. Spanish wild boars and red deer are reservoir of zoonotic genotypes of E. bieneusi and might play an underestimated role in the transmission of this microsporidian species to humans and other animals.

The fungal-related intracellular parasite Enterocytozoon bieneusi is a worldwide public health and veterinary problem. Here we demonstrated that it was present in wild boar, and wild and farmed red deer in Spain, with genotypes potentially capable of infecting humans, posing a public health risk.

Wild micromammal host spectrum of zoonotic eukaryotic parasites in Spain. Occurrence and genetic characterisation.

Micromammals have historically been recognized as highly contentious species in terms of the maintenance and transmission of zoonotic pathogens to humans. Limited information is currently available on the epidemiology and potential public health significance of intestinal eukaryotes in wild micromammals. We examined 490 faecal samples, grouped into 155 pools, obtained from 11 micromammal species captured in 11 Spanish provinces for the presence of DNA from Cryptosporidium spp., Giardia duodenalis, Enterocytozoon bieneusi and Blastocystis sp. The presence of Leishmania spp. was investigated in individual spleen samples. All micromammal species investigated harboured infections by at least one eukaryotic parasite, except Apodemus flavicollis, Myodes glareolus, Sorex coronatus and Sciurus vulgaris, but the sample size for these host species was very low. Cryptosporidium spp. was the most prevalent species found (3.7%, 95% confidence interval [CI]: 2.2-5.7), followed by G. duodenalis (2.8%, 95% CI: 1.6-4.6) and E. bieneusi (2.6%, 95% CI: 1.4-4.3). All pooled faecal samples tested negative for Blastocystis sp. Leishmania infantum was identified in 0.41% (95% CI: 0.05-1.46) of the 490 individual spleen samples analysed. Sequence analyses allowed the identification of Cryptosporidium andersoni (5.9%), C. ditrichi (11.7%), C. muris (5.9%), C. parvum (5.9%), C. tyzzeri (5.9%), rat genotypes CR97 (5.9%) and W19 (5.9%), vole genotypes V (11.7%) and VII (5.9%) and Cryptosproridium spp. (35.3%) within Cryptosporidium (n = 17). Known genotypes C (66.7%) and Peru11 (25.0%) and a novel genotype (named MouseSpEb1, 8.3%) were detected within E. bieneusi (n = 12). None of the G. duodenalis-positive samples could be genotyped at the assemblage level. Molecular data indicate that wild micromammals were primarily infected by rodent-adapted species/genotypes of eukaryotic pathogens and thereby have a limited role as a source of human infections. The presence of ruminant-adapted species C. andersoni along with finding C. parvum is indicative of an overlap between domestic/peri-domestic and sylvatic transmission cycles of these agents.

Parental leave in medical school: supporting students as parents.

CONTEXT: The overlap between medical school, residency, and childbearing potential increases the likelihood a woman will pursue parenthood within her, or her partner's, medical training. Parental leave benefits mothers, fathers, and infants. Adequate parental leave promotes physical recovery, mental health, infant bonding, improved breastfeeding, appropriate childhood immunization, and familial engagement. Despite the risks and benefits, the United States does not have national paid maternity, paternity, or parental leave requirements. Complicating matters for medical trainees, parental leave policies are not well-defined within the undergraduate (UME) and graduate medical education (GME) realms. Significant policy advancements are on the horizon for GME; however, medical schools are left without evidence to support policy formation. OBJECTIVES: This study aims to identify the presence and nature of maternal/paternal leave policies and procedures within UME. Given the authors' close association with osteopathic medical education, only osteopathic medical schools were considered to lay the framework for future study in UME. METHODS: Investigators searched university websites for student handbooks outlining rules and policies surrounding parental leave. The following terms were utilized to investigate these documents: "parental," "maternity," "paternity," "pregnant," "pregnancy," and "leave of absence" (LOA). Administrative personnel were contacted, and subjective data were documented. A parental leave policy was defined as explicitly dedicated to expectant parents or those parents planning on adoption. Medical leave or other short- and long-term LOA policies were not considered a parental leave policy. RESULTS: A total of 42 osteopathic medical schools were identified. Investigators established email communication with 17 schools (40.5%). Neither a student handbook nor email contact could be made with one institution. Two (4.9%) osteopathic medical schools overtly described parental leave in their policies. The majority of schools recommended students seeking parental leave follow short- or long-term LOA policies. CONCLUSIONS: Without protected leave time, students must decide whether to begin a family or delay medical education. As GME begins prioritizing policy change, the authors call on UME to follow suit. Parenthood and medicine must be intertwined.

Diarrhoea-causing enteric protist species in intensively and extensively raised pigs (Sus scrofa domesticus) in Southern Spain. Part II: Association with Hepatitis E virus susceptibility.

Enteropathogenic parasites can infect a wide range of mammals, including humans, supposing an important zoonotic risk. Hepatitis E virus (HEV) is an emerging foodborne pathogen of increasing public health relevance, affecting both humans and animal populations. Because both microorganisms share faecal-oral transmission route they may constitute an excellent model to evaluate the interplay between them. Thus, we aim to evaluate the viral-parasite interactions at the enteric interface in swine. We included pigs of two different breeds farming in South Spain under different production systems. We compared the HEV prevalence by the presence of Giardia duodenalis, Cryptosporidium spp., Balantioides coli, Blastocystis sp. and Enterocytozoon bieneusi in faecal samples. The HEV prevalence was 13.1 (62 out 475, 95% CI: 10.2-16.4). Those pigs infected with Cryptosporidium spp. showed a higher prevalence of HEV (30.8 vs. 12%; p = .012). In the same way, animals bearing E. bieneusi seem to have a higher rate of HEV infection (24.2 vs. 12.2%; p = .06). According to their location in the gut, animals bearing intracellular enteroparasites showed a higher HEV prevalence than those uninfected (29.6 vs. 12.7%; p = .038), meanwhile those carrying extracellular enteroparasites had a lower likelihood to be infected by HEV than those uninfected (12.1 vs. 23.1%; p = .071). Those animals bearing both types of enteroparasites showed a similar prevalence of HEV infection than those exhibiting negative for both (20.8 vs. 26.1%; p = .763). Our study provides evidence that intracellular and extracellular enteroparasites modulate the susceptibility to HEV infection in pigs. Meanwhile, the presence of extracellular enteroparasites shows a protective effect on the risk of HEV acquisition in swine, whereas intracellular enteroparasites seems to have the opposite effect, favouring the HEV infection.

Diarrhoea-causing enteric protist species in intensively and extensively raised pigs (Sus scrofa domesticus) in Southern Spain. Part I: Prevalence and genetic diversity.

Numerous protist species are shared between humans and pigs. Among those, Giardia duodenalis, Cryptosporidium spp. and Balantioides coli have a clear public and animal health significance. For others such as Enterocytozoon bieneusi and Blastocystis sp., their impact on animal health has not been fully established. Little information is currently available on the molecular diversity of these protists in swine populations. To fill this gap, we molecularly assessed G. duodenalis, Cryptosporidium spp., B. coli, Blastocystis sp. and E. bieneusi in faecal samples from Iberian and Large White pigs raised under different (intensive and/or extensive) management systems in southern Spain. A total of 151 extensively raised Iberian pigs, 140 intensively raised Iberian pigs, and 184 intensively raised Large White pigs were investigated. Blastocystis sp. was the agent most prevalently found (47.8%), followed by B. coli (45.5%), G. duodenalis (10.7%), E. bieneusi (6.9%), and Cryptosporidium spp. (5.5%). Blastocystis sp. was significantly less prevalent in intensively raised Iberian pigs (22.9%) than in their extensively raised counterparts (51.0%) or in intensively raised Large White pigs (64.1%). A significantly higher prevalence was found for G. duodenalis, Cryptosporidium spp., and E. bieneusi in Large White pigs than Iberian pigs. Balantioides coli was similarly distributed (40.0-51.1%) in all three investigated swine populations. Sequence analyses revealed the presence of G. duodenalis assemblage E, two Cryptosporidium species (Cryptosporidium scrofarum and Cryptosporidium suis), B. coli (genotypes A and B), Blastocystis sp. (ST1, ST3, and ST5), and E. bieneusi (EbpA, EbpC, EbpD, O, and a novel genotype named PigSpEb2). Novel genotype PigSpEb2 was found alone or in combination with EbpA. Data suggest a widespread exposure to protist enteroparasites in domestic pig populations irrespectively of breed and raising management system. Many of the species/genotypes identified have a zoonotic potential and might represent a public health concern.

Molecular Detection and Characterization of Blastocystis sp. and Enterocytozoon bieneusi in Cattle in Northern Spain.

Some enteric parasites causing zoonotic diseases in livestock have been poorly studied or even neglected. This is the case in stramenopile Blastocystis sp. and the microsporidia Enterocytozoon bieneusi in Spain. This transversal molecular epidemiological survey aims to estimate the prevalence and molecular diversity of Blastocystis sp. and E. bieneusi in cattle faecal samples (n = 336) in the province of Álava, Northern Spain. Initial detection of Blastocystis and E. bieneusi was carried out by polymerase chain reaction (PCR) and Sanger sequencing of the small subunit (ssu) rRNA gene and internal transcribed spacer (ITS) region, respectively. Intra-host Blastocystis subtype diversity was further investigated by next generation amplicon sequencing (NGS) of the ssu rRNA gene in those samples that tested positive by conventional PCR. Amplicons compatible with Blastocystis sp. and E. bieneusi were observed in 32.1% (108/336, 95% CI: 27.2-37.4%) and 0.6% (2/336, 95% CI: 0.0-1.4%) of the cattle faecal samples examined, respectively. Sanger sequencing produced ambiguous/unreadable sequence data for most of the Blastocystis isolates sequenced. NGS allowed the identification of 10 Blastocystis subtypes including ST1, ST3, ST5, ST10, ST14, ST21, ST23, ST24, ST25, and ST26. All Blastocystis-positive isolates involved mixed infections of 2-8 STs in a total of 31 different combinations. The two E. bieneusi sequences were confirmed as potentially zoonotic genotype BEB4. Our data demonstrate that Blastocystis mixed subtype infections are extremely frequent in cattle in the study area. NGS was particularly suited to discern underrepresented subtypes or mixed subtype infections that were undetectable or unreadable by Sanger sequencing. The presence of zoonotic Blastocystis ST1, ST3, and ST5, and E. bieneusi BEB4 suggest cross-species transmission and a potential risk of human infection/colonization.

Loop-Mediated Isothermal Amplification Allows Rapid, Simple and Accurate Molecular Diagnosis of Human Cutaneous and Visceral Leishmaniasis Caused by Leishmania infantum When Compared to PCR.

Loop-mediated isothermal amplification allows the rapid, sensitive and specific amplification of DNA without complex and expensive equipment. We compared the diagnostic performance of Loopamp™ Leishmania Detection Kit (Eiken Chemical Co., Ltd., Tokyo, Japan) with conventional and real-time polymerase chain reaction (PCR) for human cutaneous and visceral leishmaniasis caused by L. infantum. A total of 230 DNA samples from cutaneous (CL) and visceral (VL) leishmaniasis cases and controls from Spain, characterized by Leishmania nested PCR (LnPCR) were tested by: (i) the Loopamp™ Leishmania Detection Kit (Loopamp), run on Genie III real-time fluorimeter (OptiGene, UK); and (ii) real-time quantitative PCR (qPCR). The Loopamp test returned 98.8% (95% confidence interval-CI: 96.0-100.00) sensitivity and specificity of 97.7% (95% CI: 92.2-100) on VL samples, and 100% (95% CI: 99.1-100) sensitivity and 100.0% (95% CI: 98.8-100.0) specificity on CL samples. The Loopamp time-to-positivity (Tp) obtained by real-time fluorimetry showed excellent concordance (C = 97.91%) and strong correlation (r = 0.799) with qPCR's cycle threshold (Ct). The performance of Loopamp is comparable to that of LnPCR and qPCR in the diagnosis of cutaneous and visceral leishmaniasis due to L. infantum. The excellent correlation between the Tp and Ct should be further investigated to determine the accuracy of Loopamp to quantify parasite load in tissues.

Prevalence of asymptomatic Leishmania infection and associated risk factors, after an outbreak in the south-western Madrid region, Spain, 2015.

BackgroundA large outbreak of leishmaniasis with 758 cutaneous and visceral leishmaniasis cases occurred in 2009 in Fuenlabrada, in the south-west of the Madrid region of Spain.AimWe aimed to determine the prevalence of asymptomatic Leishmania infection after this outbreak, and its associated risk factors.MethodsA cross-sectional study of 804 healthy individuals living in Fuenlabrada who had no history of leishmaniasis, was conducted between January and July 2015. Asymptomatic infections were sought by either a combination of PCR, immunofluorescent antibody titre, and direct agglutination tests, or by whole blood stimulation assay (WBA) with interleukin-2 (IL-2) quantification.ResultsUsing the first approach, prevalence of asymptomatic individuals was 1.1% (9/804), while the second returned a value of 20.7% (143/804). Older age, being male, proximity to the park where the focus of infection was identified, and living in a detached house, were all strongly associated with the prevalence of asymptomatic infection.ConclusionsThe true number of infected individuals may be underestimated if only serological methods are used. The combination of WBA with IL-2 quantification may allow to better determine the prevalence of asymptomatic Leishmania infection, which would be useful in establishing control measures and in quantifying their impact. In our study, the use of WBA with IL-2 quantification also helped establish the risk factors that influence exposure to and infection by Leishmania.

Cellular Markers of Active Disease and Cure in Different Forms of Leishmania infantum-Induced Disease.

Increased numbers of peripheral blood mononucleocytes (PBMC) and increased IFN-γ secretion following in vitro challenge of blood samples with soluble Leishmania antigen (SLA), have been proposed as biomarkers of specific cell-mediated immunity, indicating that treatment of visceral leishmaniasis (VL) has been successful. However, Leishmania infantum infection may manifest as cutaneous leishmaniasis (CL), and less commonly as localized leishmanial lymphadenopathy (LLL) or mucosal leishmaniasis (ML). The present work examines the value of these biomarkers as indicators of cured leishmaniasis presenting in these different forms. Blood samples were collected before and after treatment from patients living in Fuenlabrada (Madrid, Spain), an L. infantum-endemic area recently the center of a leishmaniasis outbreak. All samples were subjected to Leishmania-specific PCR, serological tests (IFAT and rK39-ICT), and the SLA-cell proliferation assay (SLA-CPA), recording PBMC proliferation and the associated changes in IFN-γ production. Differences in the results recorded for the active and cured conditions were only significant for VL. PCR returned positive results in 67% of patients with active VL and in 3% of those with cured leishmaniasis. Similarly, rK39-ICT returned a positive result in 77% of active VL samples vs. 52% in cured VL samples, and IFAT in 90% vs. 56%; in the SLA-CPA, PBMC proliferation was seen in 16% vs. 90%, and an associated increase in IFN-γ production of 14 and 84%, respectively. The present findings reinforce the idea that PBMC proliferation and increased IFN-γ production in SLA-stimulated PBMC provide biomarkers of clinical cure in VL. Other tests are urgently needed to distinguish between the cured and active forms of the other types of clinical leishmaniasis caused by L. infantum.

Validation of rK39 immunochromatographic test and direct agglutination test for the diagnosis of Mediterranean visceral leishmaniasis in Spain.

BACKGROUND: Visceral leishmaniasis (VL), the most severe form of leishmaniasis, is endemic in Europe with Mediterranean countries reporting endemic status alongside a worrying northward spread. Serological diagnosis, including immunochromatographic test based on the recombinant antigen rK39 (rK39-ICT) and a direct agglutination test (DAT) based on the whole parasite antigen, have been validated in regions with high VL burden, such as eastern Africa and the Indian subcontinent. To date, no studies using a large set of patients have performed an assessment of both methods within Europe. METHODOLOGY/PRINCIPAL FINDINGS: We selected a range of clinical serum samples from patients with confirmed VL (including HIV co-infection), Chagas disease, malaria, other parasitic infections and negative samples (n = 743; years 2009-2015) to test the performance of rK39-ICT rapid test (Kalazar Detect Rapid Test; InBios International, Inc., USA) and DAT (ITM-DAT/VLG; Institute of Tropical Medicine Antwerp, Belgium). An in-house immunofluorescence antibody test (IFAT), was included for comparison. Estimated sensitivities for rK39-ICT and DAT in HIV-negative VL patients were 83.1% [75.1-91.2] and 84.2% [76.3-92.1], respectively. Sensitivity was reduced to 67.3% [52.7-82.0] for rK39 and increased to 91.3% [82.1-100.0] for DAT in HIV/VL co-infected patients. The in-house IFAT was more sensitive in HIV-negative VL patients, 84.2% [76.3-92.1] than in HIV/VL patients, 79.4% [73.3-96.2]. DAT gave 32 false positives in sera from HIV-negative VL suspects, compared to 0 and 2 for rK39 and IFAT, respectively, but correctly detected more HIV/VL patients (42/46) than rK39 (31/46) and IFAT (39/46). CONCLUSIONS/SIGNIFICANCE: Though rK39-ICT and DAT exhibited acceptable sensitivity and specificity a combination with other tests is required for highly sensitive diagnosis of VL cases in Spain. Important variation in the performance of the tests were seen in patients co-infected with HIV or with other parasitic infections. This study can help inform the choice of serological test to be used when screening or diagnosing VL in a European Mediterranean setting.

Application of real-time PCR for the detection of Strongyloides spp. in clinical samples in a reference center in Spain.

Strongyloidiasis is one of the major intestinal helminthic infections in humans with a worldwide distribution, affecting especially tropical and subtropical regions. This disease can occur without any symptoms or as a potentially fatal hyperinfection or disseminated infection. Definitive diagnosis of Strongyloides stercoralis infection relies mainly on demonstration of larvae in stool, but at present there is no gold standard for this diagnosis. Our main objective was to evaluate a real-time PCR targeting the 18S rRNA gene of Strongyloides spp. and to compare it with routine parasitological methods. DNA from Strongyloides venezuelensis was used to optimize PCR protocols obtaining an analytical sensitivity of 0.1 pg of parasite DNA per sample. Sensitivity and specificity of real-time PCR on fecal samples from 231 patients screened for suspected strongyloidiasis attending two hospitals in Madrid were 93.8% and 86.5%, respectively. No significant differences were found when comparing Ct-values of positive PCR between parasitological positive and negative samples. This study showed that real-time PCR is an effective tool for diagnosing strongyloidiasis and could be applied in association with parasitological methods in epidemiological studies in endemic areas. It would be also important to assess its performance in immunocompromised populations who are at risk of fatal disease.