Publications by authors named "Israel Cruz"

57 Publications

ENE-COVID nationwide serosurvey served to characterize asymptomatic infections and to develop a symptom-based risk score to predict COVID-19.

J Clin Epidemiol 2021 Jun 11. Epub 2021 Jun 11.

National Centre for Epidemiology, Instituto de Salud Carlos III (ISCIII), Monforte de Lemos 5, 28029 Madrid, Spain; Consortium for Biomedical Research in Epidemiology and Public Health (CIBERESP), Instituto de Salud Carlos III (ISCIII), Monforte de Lemos 5, 28029 Madrid, Spain. Electronic address:

Objectives: To characterize asymptomatic SARS-CoV-2 infections and develop a symptom-based risk score useful in primary healthcare.

Study Design And Setting: Sixty-one thousand ninty-two community-dwelling participants in a nationwide population-based serosurvey completed a questionnaire on COVID-19 symptoms and received an immunoassay for SARS-CoV-2 IgG antibodies between April 27 and June 22, 2020. Standardized prevalence ratios for asymptomatic infection were estimated across participant characteristics. We constructed a symptom-based risk score and evaluated its ability to predict SARS-CoV-2 infection.

Results: Of all, 28.7% of infections were asymptomatic (95% CI 26.1-31.4%). Standardized asymptomatic prevalence ratios were 1.19 (1.02-1.40) for men vs. women, 1.82 (1.33-2.50) and 1.45 (0.96-2.18) for individuals <20 and ≥80 years vs. those aged 40-59, 1.27 (1.03-1.55) for smokers vs. nonsmokers, and 1.91 (1.59-2.29) for individuals without vs. with case contact. In symptomatic population, a symptom-based score (weights: severe tiredness = 1; absence of sore throat = 1; fever = 2; anosmia/ageusia = 5) reached standardized seroprevalence ratio of 8.71 (7.37-10.3), discrimination index of 0.79 (0.77-0.81), and sensitivity and specificity of 71.4% (68.1-74.4%) and 74.2% (73.1-75.2%) for a score ≥3.

Conclusion: The presence of anosmia/ageusia, fever with severe tiredness, or fever without sore throat should serve to suspect COVID-19 in areas with active viral circulation. The proportion of asymptomatics in children and adolescents challenges infection control.
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http://dx.doi.org/10.1016/j.jclinepi.2021.06.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8192836PMC
June 2021

Prevalence of Leishmania infection in three communities of Oti Region, Ghana.

PLoS Negl Trop Dis 2021 May 27;15(5):e0009413. Epub 2021 May 27.

School of Public Health, University of Ghana, Accra, Ghana.

Background: Leishmaniasis is a neglected tropical disease caused by parasites of the genus Leishmania and is transmitted by various species of female phlebotomine sand flies. The first report of cutaneous leishmaniasis (CL) in Ghana refer to a cluster of cases in 1999-2003 in the Ho municipality of the Volta Region. We conducted an epidemiological assessment in the Oti Region, encouraged by recent reports of potential cases of CL.

Methodology/principal Findings: Using a cross-sectional study design, the exposure to Leishmania was investigated in three communities of the Oti Region based on the leishmanin skin test (LST). LST results for 3,071 participants comprising 1091, 848, and 1132 persons from the communities of Ashiabre, Keri, and Sibi Hilltop, indicated an overall prevalence of exposure to Leishmania infection of 41.8% and individual community prevalence of 39.4%, 55.1%, and 34.2% respectively. Being male [AOR = 1.27; CI: 1.09, 1.49], and living in Keri [AOR = 1.83; CI: 1.43, 2.34] were associated with an increase in the odds of exposure to Leishmania. Being 5-10 years old [AOR = 1.48; CI: 1.06, 2.05], 11-17 years old [AOR = 2.03; CI: 1.45, 2.85], 18-40 years old [AORR = 2.83; CI: 1.81, 4.43] and 41-65 years old [AOR = 5.08; CI: 2.98, 8.68] were also significantly associated with increased odds of being exposed to Leishmania.

Conclusions/significance: This study demonstrated exposure to Leishmania in the study communities and also identified associated factors. Future efforts aimed at reducing exposure to Leishmania infection in the study area should take the associated factors into consideration.
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http://dx.doi.org/10.1371/journal.pntd.0009413DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8158879PMC
May 2021

Evaluation of Loopamp™ Detection Kit and Antigen ELISA for Post-Elimination Detection and Management of Visceral Leishmaniasis in Bangladesh.

Front Cell Infect Microbiol 2021 26;11:670759. Epub 2021 Apr 26.

Laboratory Sciences and Services Division, International Centre for Diarrhoeal Disease Research, Bangladesh, Dhaka, Bangladesh.

With reduced prevalence of visceral leishmaniasis (VL) in the Indian subcontinent (ISC), direct and field deployable diagnostic tests are needed to implement an effective diagnostic and surveillance algorithm for post-elimination VL control. In this regard, here we investigated the diagnostic efficacies of a loop-mediated isothermal amplification (LAMP) assay (Loopamp™ Detection Kit, Eiken Chemical CO., Ltd, Japan), a real-time quantitative PCR assay (qPCR) and the antigen ELISA (CLIN-TECH, UK) with different sampling techniques and evaluated their prospect to incorporate into post-elimination VL control strategies. Eighty clinically and rK39 rapid diagnostic test confirmed VL cases and 80 endemic healthy controls were enrolled in the study. Peripheral blood and dried blood spots (DBS) were collected from all the participants at the time of diagnosis. DNA was extracted from whole blood (WB) and DBS silica columns (QIAGEN) and boil & spin (B&S) methods and tested with qPCR and Loopamp. Urine was collected from all participants at the time of diagnosis and was directly subjected to the antigen ELISA. 41 patients were followed up and urine samples were collected at day 30 and day 180 after treatment and ELISA was performed. The sensitivities of the Loopamp-WB(B&S) and Loopamp-WB(QIA) were 96.2% (95% CI 89·43-99·22) and 95% (95% CI 87·69-98·62) respectively. The sensitivity of Loopamp-DBS(QIA) was 85% (95% CI 75·26- 92·00). The sensitivities of the qPCR-WB(QIA) and qPCR-DBS(QIA) were 93.8% (95% CI 86·01-97·94) and 72.5% (95% CI 61·38-81·90) respectively. The specificity of all molecular assays was 100%. The sensitivity and specificity of the antigen ELISA were 97.5% (95% CI 91·47-99·70) and 91.95% (95% CI 84·12-96·70) respectively. The antigen ELISA depicted clinical cure at day 180 in all the followed-up cases. Efficacy and sustainability identify the Loopamp-WB(B&S) and the antigen ELISA as promising and minimally invasive VL diagnostic tools to support VL diagnostic and surveillance activities respectively in the post-elimination era.
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http://dx.doi.org/10.3389/fcimb.2021.670759DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8108992PMC
July 2021

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

Microorganisms 2021 Mar 16;9(3). Epub 2021 Mar 16.

WHO Collaborating Centre for Leishmaniasis, National Center for Microbiology, Instituto de Salud Carlos III, 28220 Majadahonda, Spain.

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™ 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 . A total of 230 DNA samples from cutaneous (CL) and visceral (VL) leishmaniasis cases and controls from Spain, characterized by nested PCR (LnPCR) were tested by: (i) the Loopamp™ 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 () obtained by real-time fluorimetry showed excellent concordance (C = 97.91%) and strong correlation (r = 0.799) with qPCR's cycle threshold (). The performance of Loopamp is comparable to that of LnPCR and qPCR in the diagnosis of cutaneous and visceral leishmaniasis due to . The excellent correlation between the and should be further investigated to determine the accuracy of Loopamp to quantify parasite load in tissues.
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http://dx.doi.org/10.3390/microorganisms9030610DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7999953PMC
March 2021

A Novel Sampling Model to Study the Epidemiology of Canine Leishmaniasis in an Urban Environment.

Front Vet Sci 2021 8;8:642287. Epub 2021 Mar 8.

Área de Parasitología, Departamento de Agroquímica y Medio Ambiente, Universidad Miguel Hernández de Elche, Alicante, Spain.

Visceral leishmaniasis (VL) is one of the most important parasitic diseases in the world. The domestic dog is the main reservoir of zoonotic VL and a high prevalence of canine leishmaniasis (CanL) is associated with transmission of infection to humans. Here we describe the methodology used to obtain a rapid and representative sample of domestic dogs in the city of Posadas, Misiones, and compare the prevalence of infection with a sample of shelter dogs. We used the city land registry to make a random selection of homes and systematically recruited 349 domestic dogs from the selected properties. We also included all dogs from the main canine shelter within the city. Dogs were examined by two experienced veterinarians who recorded the presence of clinical signs common in CanL using a standardized protocol. We extracted a blood sample from each dog and performed four different serological tests to reveal the presence of anti- antibodies. After clinical examination, 145 domestic dogs (41.5%) and 63 (90%) shelter dogs had clinical signs compatible with CanL ( < 0.001). The seroprevalence among domestic dogs was 20.1% (95% CI 16.1-24.6) which was significantly lower than among the abandoned dogs (38.6%, 95% CI 27.7-50.6, < 0.001). The spatial distribution of infected dogs was fairly homogenous throughout the city. Among domestic dogs, we observed a positive association between where the dog slept and presence of anti- antibodies ( = 0.034). Of the seropositive domestic dogs 38 (54.4%) were asymptomatic. Our findings demonstrate how seroprevalence results can be highly influenced by sampling methodology. We demonstrate how the land registry can be used to estimate the prevalence of CanL in representative sample of domestic dogs in an urban setting, allowing decision makers to deepen their understanding the epidemiology of CanL in a timely and efficient manner for the development of plans to address both human and canine disease.
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http://dx.doi.org/10.3389/fvets.2021.642287DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7982517PMC
March 2021

Evaluation of the Performance of the Loopamp Trypanosoma cruzi Detection Kit for the Diagnosis of Chagas Disease in an Area Where It Is Not Endemic, Spain.

J Clin Microbiol 2021 04 20;59(5). Epub 2021 Apr 20.

Foundation for Innovative New Diagnostics FIND, Geneva, Switzerland.

In Spain, PCR is the tool of choice for the diagnosis of congenital Chagas disease (CD) and serology for diagnosing chronic CD. A loop-mediated isothermal amplification test for DNA detection showed good analytical performance and ease of use. We aimed to evaluate the performance of the Loopamp detection kit (Eiken Chemical Co. Ltd., Japan) (-LAMP) for congenital and chronic CD diagnosis using well-characterized samples. We included samples from 39 congenital and 174 chronic CD cases and from 48 uninfected children born to infected mothers and 34 nonchagasic individuals. The sensitivity, specificity, and accuracy of -LAMP were estimated using standard case definitions for congenital CD (positive result by parasitological or PCR tests or serology after 9 months of age) and chronic CD (positive serology by at least two tests). The -LAMP results were read by visual examination and a real-time fluorimeter. For congenital CD, -LAMP sensitivity was 97% for both types of reading; specificity was 92% by visual examination and 94% by fluorimeter. For chronic CD, sensitivity was 47% and specificity 100%. The accuracy in congenital CD was >94% versus 56% in chronic CD. The agreement of -LAMP with PCR tests was better in congenital CD (kappa, 0.86 to 0.91) than in chronic CD (kappa, 0.67 to 0.83). The Loopamp detection kit showed good performance for the diagnosis of congenital CD. -LAMP, like PCR, can be useful for the screening and early diagnosis of congenital infection.
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http://dx.doi.org/10.1128/JCM.01860-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8091841PMC
April 2021

Infection fatality risk for SARS-CoV-2 in community dwelling population of Spain: nationwide seroepidemiological study.

BMJ 2020 11 27;371:m4509. Epub 2020 Nov 27.

National Centre for Epidemiology, Institute of Health Carlos III, Monforte de Lemos 5, 28029 Madrid, Spain

Objective: To estimate the infection fatality risk for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), based on deaths with confirmed coronavirus disease 2019 (covid-19) and excess deaths from all causes.

Design: Nationwide seroepidemiological study.

Setting: First wave of covid-19 pandemic in Spain.

Participants: Community dwelling individuals of all ages.

Main Outcome Measures: The main outcome measure was overall, and age and sex specific, infection fatality risk for SARS-CoV-2 (the number of covid-19 deaths and excess deaths divided by the estimated number of SARS-CoV-2 infections) in the community dwelling Spanish population. Deaths with laboratory confirmed covid-19 were obtained from the National Epidemiological Surveillance Network (RENAVE) and excess all cause deaths from the Monitoring Mortality System (MoMo), up to 15 July 2020. SARS-CoV-2 infections in Spain were derived from the estimated seroprevalence by a chemiluminescent microparticle immunoassay for IgG antibodies in 61 098 participants in the ENE-COVID nationwide seroepidemiological survey between 27 April and 22 June 2020.

Results: The overall infection fatality risk was 0.8% (19 228 of 2.3 million infected individuals, 95% confidence interval 0.8% to 0.9%) for confirmed covid-19 deaths and 1.1% (24 778 of 2.3 million infected individuals, 1.0% to 1.2%) for excess deaths. The infection fatality risk was 1.1% (95% confidence interval 1.0% to 1.2%) to 1.4% (1.3% to 1.5%) in men and 0.6% (0.5% to 0.6%) to 0.8% (0.7% to 0.8%) in women. The infection fatality risk increased sharply after age 50, ranging from 11.6% (8.1% to 16.5%) to 16.4% (11.4% to 23.2%) in men aged 80 or more and from 4.6% (3.4% to 6.3%) to 6.5% (4.7% to 8.8%) in women aged 80 or more.

Conclusion: The increase in SARS-CoV-2 infection fatality risk after age 50 appeared to be more noticeable in men than in women. Based on the results of this study, fatality from covid-19 was greater than that reported for other common respiratory diseases, such as seasonal influenza.
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http://dx.doi.org/10.1136/bmj.m4509DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7690290PMC
November 2020

Diagnostics and the neglected tropical diseases roadmap: setting the agenda for 2030.

Trans R Soc Trop Med Hyg 2021 01;115(2):129-135

Neglected Tropical Diseases Support Center, Task Force for Global Health, Atlanta, GA 30030, USA.

Accurate and reliable diagnostic tools are an essential requirement for neglected tropical diseases (NTDs) programmes. However, the NTD community has historically underinvested in the development and improvement of diagnostic tools, potentially undermining the successes achieved over the last 2 decades. Recognizing this, the WHO, in its newly released draft roadmap for NTD 2021-2030, has identified diagnostics as one of four priority areas requiring concerted action to reach the 2030 targets. As a result, WHO established a Diagnostics Technical Advisory Group (DTAG) to serve as the collaborative mechanism to drive progress in this area. Here, the purpose and role of the DTAG are described in the context of the challenges facing NTD programmes.
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http://dx.doi.org/10.1093/trstmh/traa118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7842105PMC
January 2021

Trypanosoma cruzi loop-mediated isothermal amplification (Trypanosoma cruzi Loopamp) kit for detection of congenital, acute and Chagas disease reactivation.

PLoS Negl Trop Dis 2020 08 14;14(8):e0008402. Epub 2020 Aug 14.

Laboratorio de Biología Molecular de la Enfermedad de Chagas, Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr Héctor Torres", (INGEBI-CONICET), Buenos Aires, Argentina.

A Trypanosoma cruzi Loopamp kit was recently developed as a ready-to-use diagnostic method requiring minimal laboratory facilities. We evaluated its diagnostic accuracy for detection of acute Chagas disease (CD) in different epidemiological and clinical scenarios. In this retrospective study, a convenience series of clinical samples (venous blood treated with EDTA or different stabilizer agents, heel-prick blood in filter paper or cerebrospinal fluid samples (CSF)) from 30 infants born to seropositive mothers (13 with congenital CD and 17 noninfected), four recipients of organs from CD donors, six orally-infected cases after consumption of contaminated guava juice and six CD patients coinfected with HIV at risk of CD reactivation (N = 46 patients, 46 blood samples and 1 CSF sample) were tested by T. cruzi Loopamp kit (Tc LAMP) and standardized quantitative real-time PCR (qPCR). T. cruzi Loopamp accuracy was estimated using the case definition in the different groups as a reference. Cohen's kappa coefficient (κ) was applied to measure the agreement between Tc LAMP (index test) and qPCR (reference test). Sensitivity and specificity of T. cruzi Loopamp kit in blood samples from the pooled clinical groups was 93% (95% CI: 77-99) and 100% (95% CI: 80-100) respectively. The agreement between Tc LAMP and qPCR was almost perfect (κ = 0.92, 95% CI: 0.62-1.00). The T. cruzi Loopamp kit was sensitive and specific for detection of T. cruzi infection. It was carried out from DNA extracted from peripheral blood samples (via frozen EDTA blood, guanidine hydrochloride-EDTA blood, DNAgard blood and dried blood spots), as well as in CSF specimens infected with TcI or TcII/V/VI parasite populations. The T. cruzi Loopamp kit appears potentially useful for rapid detection of T. cruzi infection in congenital, acute and CD reactivation due to HIV infection.
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http://dx.doi.org/10.1371/journal.pntd.0008402DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7458301PMC
August 2020

Prevalence of SARS-CoV-2 in Spain (ENE-COVID): a nationwide, population-based seroepidemiological study.

Lancet 2020 08 6;396(10250):535-544. Epub 2020 Jul 6.

Directorate, Institute of Health Carlos III, Madrid, Spain.

Background: Spain is one of the European countries most affected by the COVID-19 pandemic. Serological surveys are a valuable tool to assess the extent of the epidemic, given the existence of asymptomatic cases and little access to diagnostic tests. This nationwide population-based study aims to estimate the seroprevalence of SARS-CoV-2 infection in Spain at national and regional level.

Methods: 35 883 households were selected from municipal rolls using two-stage random sampling stratified by province and municipality size, with all residents invited to participate. From April 27 to May 11, 2020, 61 075 participants (75·1% of all contacted individuals within selected households) answered a questionnaire on history of symptoms compatible with COVID-19 and risk factors, received a point-of-care antibody test, and, if agreed, donated a blood sample for additional testing with a chemiluminescent microparticle immunoassay. Prevalences of IgG antibodies were adjusted using sampling weights and post-stratification to allow for differences in non-response rates based on age group, sex, and census-tract income. Using results for both tests, we calculated a seroprevalence range maximising either specificity (positive for both tests) or sensitivity (positive for either test).

Findings: Seroprevalence was 5·0% (95% CI 4·7-5·4) by the point-of-care test and 4·6% (4·3-5·0) by immunoassay, with a specificity-sensitivity range of 3·7% (3·3-4·0; both tests positive) to 6·2% (5·8-6·6; either test positive), with no differences by sex and lower seroprevalence in children younger than 10 years (<3·1% by the point-of-care test). There was substantial geographical variability, with higher prevalence around Madrid (>10%) and lower in coastal areas (<3%). Seroprevalence among 195 participants with positive PCR more than 14 days before the study visit ranged from 87·6% (81·1-92·1; both tests positive) to 91·8% (86·3-95·3; either test positive). In 7273 individuals with anosmia or at least three symptoms, seroprevalence ranged from 15·3% (13·8-16·8) to 19·3% (17·7-21·0). Around a third of seropositive participants were asymptomatic, ranging from 21·9% (19·1-24·9) to 35·8% (33·1-38·5). Only 19·5% (16·3-23·2) of symptomatic participants who were seropositive by both the point-of-care test and immunoassay reported a previous PCR test.

Interpretation: The majority of the Spanish population is seronegative to SARS-CoV-2 infection, even in hotspot areas. Most PCR-confirmed cases have detectable antibodies, but a substantial proportion of people with symptoms compatible with COVID-19 did not have a PCR test and at least a third of infections determined by serology were asymptomatic. These results emphasise the need for maintaining public health measures to avoid a new epidemic wave.

Funding: Spanish Ministry of Health, Institute of Health Carlos III, and Spanish National Health System.
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http://dx.doi.org/10.1016/S0140-6736(20)31483-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7336131PMC
August 2020

Latest trends in Leishmania infantum infection in dogs in Spain, Part I: mapped seroprevalence and sand fly distributions.

Parasit Vectors 2020 Apr 21;13(1):204. Epub 2020 Apr 21.

Grupo de Investigación Epicontrol-Carnívoros, Departamento de Sanidad Animal, Facultad de Veterinaria, Universidad Complutense de Madrid, Madrid, Spain.

Background: This report describes L. infantum infection seroprevalence in dogs in Spain through data obtained from peer-reviewed literature and a cross-sectional serological survey assessing epidemiological and habitat variables as risk factors for infection. The study also provides preliminary sand fly species distribution data and indicates factors affecting their distribution and density.

Methods: Three different studies were conducted in Spain: (i) a peer-reviewed literature seroprevalence survey (1985-2019); (ii) a cross-sectional serological survey (2011-2016); and (iii) a preliminary entomological survey (2013-2014). In the cross-sectional serological survey, 1739 dogs from 74 different locations including 25 Spanish provinces were tested for L. infantum by indirect immunofluorescence antibody test (IFAT) (antibody titre ≥ 1:100). Seroprevalence of L. infantum infection was analysed by province and bioclimatic zone. Statistics were used to analyse relationships between several dog- and environment-related variables and L. infantum seroprevalence. In parallel, during 2013-2014, sand flies were collected across the Iberian Peninsula and the Balearic Islands using CDC light traps to examine relationships between habitat-related factors and sand fly species densities (number of sand flies per trap per hour).

Results: The literature review revealed that the provinces showing the highest seroprevalence were Balearic Islands (57.1%), Ourense (35.6%), Málaga (34.6%) and Cáceres (34.2%), and those showing the lowest seroprevalence were Vizcaya (0%), Cantabria (2.0%) and Álava (3.3%). In our survey, anti-Leishmania IgG antibodies were detected in 176 of the 1739 dogs rendering a seroprevalence of 10.12%. Percentage seroprevalence distributions significantly varied among bioclimatic belts. Seropositivity for L. infantum was related to size (large breed dogs versus small) and were significantly higher in younger dogs (≤ 1 years-old). In the entomological survey, 676 sand flies of five species were captured: 562 (83.13%) Phlebotomus perniciosus; 64 (9.47%) Sergentomyia minuta; 38 (5.62%) P. ariasi: 6 (0.89%) P. sergenti; and 6 (0.89%) P. papatasi. Phlebotomus perniciosus showed a greater density in the thermo-Mediterranean than in the meso-Mediterranean zone. Densities of S. minuta and P. ariasi were significantly higher in rural habitats.

Conclusions: This updated seroprevalence map of L. infantum infection in dogs in Spain defines non-endemic, hypoendemic, endemic and hyperendemic areas, and confirms P. perniciosus as the most abundant sand fly vector in Spain.
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http://dx.doi.org/10.1186/s13071-020-04081-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7171843PMC
April 2020

Leishmaniasis immunopathology-impact on design and use of vaccines, diagnostics and drugs.

Semin Immunopathol 2020 06 9;42(3):247-264. Epub 2020 Mar 9.

Faculty of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London, UK.

Leishmaniasis is a disease complex caused by 20 species of protozoan parasites belonging to the genus Leishmania. In humans, it has two main clinical forms, visceral leishmaniasis (VL) and cutaneous or tegumentary leishmaniasis (CL), as well as several other cutaneous manifestations in a minority of cases. In the mammalian host Leishmania parasites infect different populations of macrophages where they multiply and survive in the phagolysosomal compartment. The progression of both VL and CL depends on the maintenance of a parasite-specific immunosuppressive state based around this host macrophage infection. The complexity and variation of immune responses and immunopathology in humans and the different host interactions of the different Leishmania species has an impact upon the effectiveness of vaccines, diagnostics and drugs.
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http://dx.doi.org/10.1007/s00281-020-00788-yDOI Listing
June 2020

Surveillance for Leishmania asymptomatic infection in endemic foci of cutaneous leishmaniasis in Venezuela: a combination of leishmanin skin test and PCR using blood clots improves detection and enables identification of species.

Trans R Soc Trop Med Hyg 2020 06;114(6):433-439

Centro Nacional de Referencia de Flebotomos y otros Vectores (CNRFV), Instituto de Investigaciones Biomedicas "Dr. Francisco J.Triana-Alonso" (BIOMED), Facultad de Ciencias de la Salud, Universidad de Carabobo, Maracay, Venezuela.

Background: Little is known about the prevalence of asymptomatic leishmaniasis in Venezuela. The objective of this study was to quantify Leishmania asymptomatic infection in six endemic foci of cutaneous leishmaniasis (CL) in Portuguesa State, Venezuela, where no previous data were available.

Methods: Study of the prevalence of Leishmania asymptomatic infection was carried out in 841 individuals from six endemic foci of CL in the municipalities Sucre and Ospino, Portuguesa State. We applied the leishmanin skin test (LST) and the internal transcribed spacer 1 (ITS1) PCR to DNA from sera and blood clots of all LST-positive and 20% of LST-negative patients.

Results: Of 841 inhabitants tested by LST, 197 returned a positive reaction (23.42%); all of the LST-positives (197) and 121 negatives were screened by nested PCR using serum and blood clots. Among the LST-positive group, 2.54% were PCR-positive with sera, while 44.67% were positive with blood clots. In the LST-negative group, PCR was positive in 2.48% of serum samples and in 38.84% of blood clots.

Conclusions: It is recommended that LST and PCR on blood clots are used together to detect exposure and asymptomatic infection and for identification of the Leishmania species.
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http://dx.doi.org/10.1093/trstmh/trz130DOI Listing
June 2020

Target Product Profile for a point-of-care diagnostic test for dermal leishmaniases.

Parasite Epidemiol Control 2019 May 7;5:e00103. Epub 2019 Mar 7.

Foundation for Innovative New Diagnostics, Geneva, Switzerland.

Objectives: Localized cutaneous leishmaniasis and its evolving forms diffuse cutaneous leishmaniasis, mucosal leishmaniasis and cutaneous leishmaniasis recidivans, together with the visceral leishmaniasis sequelae post-kala azar dermal leishmaniasis account for about one million dermal leishmaniases cases per year worldwide. Although not lethal, the dermal leishmaniases cause chronic and disfiguring skin lesions, which are an important cause of morbidity and stigma.Microscopy remains the reference test for diagnosis of dermal leishmaniasis; however, it has low and variable sensitivity and requires well trained personnel. The technical complexity and cost of the more sensitive molecular techniques (e.g. PCR) limits their application in routine diagnosis in endemic areas. Point-of-care (POC) tests for early diagnosis are much needed in order to benefit both patients and communities, by reducing the risk of both sequelae and transmission. To this end we developed a Target Product Profile (TPP) for a POC test for dermal leishmaniases.

Methods: The TPP was defined through several rounds of discussions and by consensus with stakeholders and experts in dermal leishmaniases from different type of organizations and endemic regions.

Results And Conclusions: A rapid, simple and robust test that can be implemented in resource-limited settings, enabling decentralized diagnosis and treatment of dermal leishmaniasis should be developed. Ideally it should enable the diagnosis of all forms of dermal leishmaniasis, but the minimally accepted target would be localized cutaneous leishmaniasis. A minimum sensitivity of 95% and specificity of 90% would be required. The consensus was that the POC test should target antigens.
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http://dx.doi.org/10.1016/j.parepi.2019.e00103DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6423987PMC
May 2019

Evaluation of point of care tests for the diagnosis of cutaneous leishmaniasis in Suriname.

BMC Infect Dis 2019 Jan 7;19(1):25. Epub 2019 Jan 7.

Foundation for Innovative New Diagnostics, Geneva, Switzerland.

Background: Cutaneous leishmaniasis (CL) is a serious health problem in Suriname. To expand the diagnostic options, two newly developed diagnostic tests, i.e. the rapid diagnostic test CL Detect™ Rapid Test (CL Detect) and the Loopamp™ Leishmania Detection Kit (Loopamp) were evaluated.

Methods: Diagnostic test performance was compared to the routine diagnostic approach in place, i.e. clinical symptoms combined with microscopy, and to polymerase chain reaction (PCR), which was used as a reference standard. The study population (n = 93) was a typical representation of the CL affected population in Suriname and mainly infected with Leishmania guyanensis.

Results: CL Detect had a very low sensitivity compared to microscopy (36.7%) or PCR (35.8%), due to a high number of false negative results. The specificity of the CL Detect compared to microscopy and PCR was 85.7 and 83.3% respectively. Loopamp sensitivity was 84.8% compared to microscopy and 91.4% compared to PCR. The Loopamp test had a moderate specificity (42.9%) compared to microscopy, but a good specificity compared to PCR (91.7%).

Conclusion: The CL Detect is not likely to be a good replacement for the routine diagnostic procedure for CL in Suriname. The high sensitivity of the easy to perform Loopamp enables the implementation of sensitive molecular diagnosis in resource limited settings.
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http://dx.doi.org/10.1186/s12879-018-3634-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6323762PMC
January 2019

Molecular typing reveals the co-existence of two transmission cycles of American cutaneous leishmaniasis in the Andean Region of Venezuela with Lutzomyia migonei as the vector.

Mem Inst Oswaldo Cruz 2018 Dec 6;113(12):e180323. Epub 2018 Dec 6.

Universidad de Carabobo, Facultad de Ciencias de la Salud, Centro Nacional de Referencia de Flebotomos y otros Vectores, Instituto de Investigaciones Biomédicas Dr Francisco J Triana-Alonso, Maracay, Venezuela.

BACKGROUND The transmission routes for American cutaneous leishmaniasis (ACL) are in flux, so studies examining its transmission in humans, mammalian hosts, and sand fly vectors are urgently needed. OBJECTIVES The aim of this work was understand the epidemiological cycles of Leishmania spp., which causes ACL in the Andean Region of Venezuela, by identifying the Leishmania and the sand fly species involved in human and dog infections. METHODS Thirty-one biopsies from patients in Mérida and Táchira states with suspected ACL were studied by both parasitological tests (cultures and hamster inoculation) and a molecular test [Internal transcribed spacer 1 (ITS1) nested polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP)]. We also conducted a survey to detect Leishmania infection in dogs (Immunifluorescence antibody test and ITS1 nested PCR-RFLP) and sand flies (ITS1 nested PCR-RFLP) from El Carrizal, a highly endemic focus of ACL in Venezuela. FINDINGS Three different Leishmania species were identified in the clinical samples from humans (Leishmania braziliensis, L. guyanensis, and L. mexicana) and dogs (L. guyanensis and L. mexicana). The predominant sand fly species found were those from the Verrucarum group (infected with L. mexicana) and Lutzomyia migonei (infected with L. guyanensis and L. mexicana). MAIN CONCLUSIONS We show that Lu. migonei may be the putative vector in two ACL epidemiological cycles, involving L. guyanensis and L. mexicana. We also report for the first time the presence of L. guyanensis in domestic animals.
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http://dx.doi.org/10.1590/0074-02760180323DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6282108PMC
December 2018

Cost Effectiveness of New Diagnostic Tools for Cutaneous Leishmaniasis in Afghanistan.

Appl Health Econ Health Policy 2019 04;17(2):213-230

ISGlobal, Hospital-Clínic-Universitat de Barcelona, Carrer Rosselló 132, 08036, Barcelona, Spain.

Background And Objectives: Cutaneous leishmaniasis is responsible for chronic and disfiguring skin lesions resulting in morbidity and social stigma. The gold standard to diagnose cutaneous leishmaniasis is microscopy but has a variable sensitivity and requires trained personnel. Using four scenarios, the objective of this study is to compare the cost effectiveness of microscopy with two new tools: Loopamp™ Leishmania Detection Kit (LAMP) and CL Detect™ Rapid Test (RDT).

Methods: Data related to the cost and accuracy of these tools were collected at the clinic of the National Malaria and Leishmaniasis Control Program in Kabul, Afghanistan. The effectiveness estimates were measured based on the tools' performance but also indirectly, using the disability-adjusted life years. A decision tree was designed in TreeAge Healthcare Pro 2016, combined with a Markov model representing the natural history of cutaneous leishmaniasis. In addition to a deterministic analysis, univariate sensitivity and probabilistic analyses were performed to test the robustness of the results.

Results: If the tools are compared at the National Malaria and Leishmaniasis Control Program level in a period of low incidence, microscopy remains the preferred option. LAMP becomes more appropriate during cutaneous leishmaniasis seasons or outbreaks when its capacity to process several tests (e.g. up to 48) at a time can be maximised. RDT has a cost similar to microscopy when used at the reference clinic but as it is relatively easy to use, it could be implemented at the peripheral level, which would become cheaper than employing microscopy at the reference clinic. Moreover, combining RDT with microscopy or LAMP at the reference clinic for the negative suspects is economically more interesting than directly performing LAMP or microscopy respectively on all cutaneous leishmaniasis suspects at the reference clinic.

Conclusions: When taking advantage of their respective strengths, LAMP and RDT can prove to be cost-effective alternatives to using microscopy alone at the reference clinic.
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http://dx.doi.org/10.1007/s40258-018-0449-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6439180PMC
April 2019

Genome Dynamics during Environmental Adaptation Reveal Strain-Specific Differences in Gene Copy Number Variation, Karyotype Instability, and Telomeric Amplification.

mBio 2018 11 6;9(6). Epub 2018 Nov 6.

Unité de Parasitologiemoléculaire et Signalisation, Institut Pasteur, Paris, France

Protozoan parasites of the genus adapt to environmental change through chromosome and gene copy number variations. Only little is known about external or intrinsic factors that govern genomic adaptation. Here, by conducting longitudinal genome analyses of 10 new clinical isolates, we uncovered important differences in gene copy number among genetically highly related strains and revealed gain and loss of gene copies as potential drivers of long-term environmental adaptation in the field. In contrast, chromosome rather than gene amplification was associated with short-term environmental adaptation to culture. Karyotypic solutions were highly reproducible but unique for a given strain, suggesting that chromosome amplification is under positive selection and dependent on species- and strain-specific intrinsic factors. We revealed a progressive increase in read depth towards the chromosome ends for various isolates, which may represent a nonclassical mechanism of telomere maintenance that can preserve integrity of chromosome ends during selection for fast growth. Together our data draw a complex picture of genomic adaptation in the field and in culture, which is driven by a combination of intrinsic genetic factors that generate strain-specific phenotypic variations, which are under environmental selection and allow for fitness gain. Protozoan parasites of the genus cause severe human and veterinary diseases worldwide, termed leishmaniases. A hallmark of biology is its capacity to adapt to a variety of unpredictable fluctuations inside its human host, notably pharmacological interventions, thus, causing drug resistance. Here we investigated mechanisms of environmental adaptation using a comparative genomics approach by sequencing 10 new clinical isolates of the , , and complexes that were sampled across eight distinct geographical regions. Our data provide new evidence that parasites adapt to environmental change in the field and in culture through a combination of chromosome and gene amplification that likely causes phenotypic variation and drives parasite fitness gains in response to environmental constraints. This novel form of gene expression regulation through genomic change compensates for the absence of classical transcriptional control in these early-branching eukaryotes and opens new venues for biomarker discovery.
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http://dx.doi.org/10.1128/mBio.01399-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6222132PMC
November 2018

Evaluation of point-of-care tests for cutaneous leishmaniasis diagnosis in Kabul, Afghanistan.

EBioMedicine 2018 Nov 2;37:453-460. Epub 2018 Nov 2.

Foundation for Innovative New Diagnostics, Campus Biotech, Chemin des Mines 9, 1202 Geneva, Switzerland. Electronic address:

Background: Kabul (Afghanistan) is a major focus of cutaneous leishmaniasis (CL) caused by Leishmania tropica. Microscopy remains the reference test for diagnosis despite its low performance. We evaluated whether Loopamp™ Leishmania Detection Kit (Loopamp) and CL Detect™ Rapid Test (CL Detect), detecting Leishmania DNA and antigen, respectively could improve CL diagnosis.

Methods: A diagnostic accuracy study with prospective inclusion was conducted in a leishmaniasis reference clinic in Kabul. Slit skin samples from CL suspects were analysed by microscopy. Samples taken with a dental broach were tested with CL Detect, Loopamp, and PCR. All samples were transferred to the Academic Medical Center (AMC, the Netherlands) for PCR and Loopamp analyses. The diagnostic performance of the tests was evaluated against a reference combining microscopy and PCR.

Findings: 274 CL suspects were included in the study. In Kabul, CL Detect had a 65·4% sensitivity [95% Confidence Interval (CI): 59.2-71.2%] and a 100% specificity [95% CI: 80.5-100%], while these were 87.6% [95%CI: 82.9-91.3%] and 70.6% [95% CI: 44.0-89.7%] for Loopamp. At AMC the Loopamp's sensitivity (92.2% [95% CI: 88.2-95.2%]) and specificity (94.1% [95% CI: 71.3-99.8%]) were higher. An algorithm where CL Detect negative suspects would be tested by Loopamp yielded a 93.4% sensitivity [95% CI: 89.6-96.1%] and a 94.1% specificity [95% CI: 71.3-99.8%] when Loopamp's performance at AMC was used.

Interpretation: The high specificity of CL Detect and the performance of Loopamp allow their use in a diagnostic algorithm that would minimize the number of CL patients referred for confirmation. FUND: Federal Ministry of Education and Research, Germany.
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http://dx.doi.org/10.1016/j.ebiom.2018.10.063DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6286266PMC
November 2018

The burden of congenital Chagas disease and implementation of molecular diagnostic tools in Latin America.

BMJ Glob Health 2018 11;3(5):e001069. Epub 2018 Oct 11.

Foundation for Innovative New Diagnostics (FIND), Geneva, Switzerland.

It is estimated that between 8000 and 15 000 infected babies are born every year to infected mothers in Chagas disease endemic countries. Currently, poor access to and performance of the current diagnostic algorithm, based on microscopy at birth and serology at 8-12 months after delivery, is one of the barriers to congenital Chagas disease (CCD) control. Detection of parasite DNA using molecular diagnostic tools could be an alternative or complement to current diagnostic methods, but its implementation in endemic regions remains limited. Prompt diagnosis and treatment of CCD cases would have a positive clinical and epidemiological impact. In this paper, we analysed the burden of CCD in Latin America, and the potential use of molecular tests to improve access to early diagnosis and treatment of infected newborns.
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http://dx.doi.org/10.1136/bmjgh-2018-001069DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6195131PMC
October 2018

Evaluation of fluorimetry and direct visualization to interpret results of a loop-mediated isothermal amplification kit to detect Leishmania DNA.

Parasit Vectors 2018 04 17;11(1):250. Epub 2018 Apr 17.

WHO Collaborating Centre for Leishmaniasis, National Centre for Microbiology, Instituto de Salud Carlos III, Madrid, Spain.

Background: Nucleic acid amplification tests (NAATs) have proven to be advantageous in the diagnosis of leishmaniases, allowing sensitive diagnosis of: (i) cutaneous leishmaniasis in long duration lesions and (ii) visceral leishmaniasis using a less-invasive sample like peripheral blood, in opposition to tissue aspiration required for parasite demonstration by microscopy. Despite their benefits, the implementation of NAATs for leishmaniasis diagnosis at the point-of-care has not been achieved yet, mostly due to the complexity and logistical issues associated with PCR-based methods.

Methods: In this work, we have evaluated the performance of a ready-to-use loop-mediated isothermal amplification (LAMP) kit using two real time fluorimeters to amplify leishmanial DNA obtained by silica column-based and Boil & Spin protocols.

Results: The different approaches used to run and interpret the LAMP reactions showed a performance equivalent to PCR and real-time PCR, using spiked and clinical samples. The time to positivity obtained with real-time fluorimetry showed an excellent correlation with both Ct values and parasite load from real-time quantitative PCR.

Conclusions: The results obtained open the possibility of using a highly stable, ready-to-use LAMP kit for the accurate diagnosis of leishmaniasis at the point-of-care. Furthermore, the feasibility of relating time to positivity, determined with a portable real-time fluorimeter, with the parasite burden could have a wider application in the management of leishmaniasis, such as in treatment efficacy monitoring or as a pharmacodynamics tool in clinical trials.
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http://dx.doi.org/10.1186/s13071-018-2836-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5905109PMC
April 2018

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

PLoS Negl Trop Dis 2018 03 1;12(3):e0006277. Epub 2018 Mar 1.

WHO Collaborating Centre for Leishmaniasis, National Centre for Microbiology, Instituto de Salud Carlos III, Majadahonda, 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.
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http://dx.doi.org/10.1371/journal.pntd.0006277DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5849364PMC
March 2018

Sensitive and less invasive confirmatory diagnosis of visceral leishmaniasis in Sudan using loop-mediated isothermal amplification (LAMP).

PLoS Negl Trop Dis 2018 02 14;12(2):e0006264. Epub 2018 Feb 14.

Foundation for Innovative New Diagnostics-FIND, Geneva, Switzerland.

Background: Confirmatory diagnosis of visceral leishmaniasis (VL), as well as diagnosis of relapses and test of cure, usually requires examination by microscopy of samples collected by invasive means, such as splenic, bone marrow or lymph node aspirates. This causes discomfort to patients, with risks of bleeding and iatrogenic infections, and requires technical expertise. Molecular tests have great potential for diagnosis of VL using peripheral blood, but require well-equipped facilities and trained personnel. More user-friendly, and field-amenable options are therefore needed. One method that could meet these requirements is loop-mediated isothermal amplification (LAMP) using the Loopamp Leishmania Detection Kit, which comes as dried down reagents that can be stored at room temperature, and allows simple visualization of results.

Methodology/principal Findings: The Loopamp Leishmania Detection Kit (Eiken Chemical Co., Japan), was evaluated in the diagnosis of VL in Sudan. A total of 198 VL suspects were tested by microscopy of lymph node aspirates (the reference test), direct agglutination test-DAT (in house production) and rK28 antigen-based rapid diagnostic test (OnSite Leishmania rK39-Plus, CTK Biotech, USA). LAMP was performed on peripheral blood (whole blood and buffy coat) previously processed by: i) a direct boil and spin method, and ii) the QIAamp DNA Mini Kit (QIAgen). Ninety seven of the VL suspects were confirmed as cases by microscopy of lymph node aspirates. The sensitivity and specificity for each of the tests were: rK28 RDT 98.81% and 100%; DAT 88.10% and 78.22%; LAMP-boil and spin 97.65% and 99.01%; LAMP-QIAgen 100% and 99.01%.

Conclusions/significance: Due to its simplicity and high sensitivity, rK28 RDT can be used first in the diagnostic algorithm for primary VL diagnosis, the excellent performance of LAMP using peripheral blood indicates that it can be also included in the algorithm for diagnosis of VL as a simple test when parasitological confirmatory diagnosis is required in settings that are lower than the reference laboratory, avoiding the need for invasive lymph node aspiration.
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http://dx.doi.org/10.1371/journal.pntd.0006264DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5828521PMC
February 2018

Analytical sensitivity and specificity of a loop-mediated isothermal amplification (LAMP) kit prototype for detection of Trypanosoma cruzi DNA in human blood samples.

PLoS Negl Trop Dis 2017 Jul 20;11(7):e0005779. Epub 2017 Jul 20.

Laboratorio de Biología Molecular de la Enfermedad de Chagas, Instituto de Investigaciones en Ingeniería Genética y Biología Molecular - Consejo Nacional de Investigaciones Científicas y Tecnológicas (INGEBI-CONICET), Buenos Aires Argentina.

This study aimed to assess analytical parameters of a prototype LAMP kit that was designed for detection of Trypanosoma cruzi DNA in human blood. The prototype is based on the amplification of the highly repetitive satellite sequence of T.cruzi in microtubes containing dried reagents on the inside of the caps. The reaction is carried out at 65°C during 40 minutes. Calcein allows direct detection of amplified products with the naked eye. Inclusivity and selectivity were tested in purified DNA from Trypanosoma cruzi stocks belonging to the six discrete typing units (DTUs), in DNA from other protozoan parasites and in human DNA. Analytical sensitivity was estimated in serial dilutions of DNA samples from Sylvio X10 (Tc I) and CL Brener (Tc VI) stocks, as well as from EDTA-treated or heparinized blood samples spiked with known amounts of cultured epimastigotes (CL Brener). LAMP sensitivity was compared after DNA extraction using commercial fiberglass columns or after "Boil & Spin" rapid preparation. Moreover, the same DNA and EDTA-blood spiked samples were subjected to standardized qPCR based on the satellite DNA sequence for comparative purposes. A panel of peripheral blood specimens belonging to Chagas disease patients, including acute, congenital, chronic and reactivated cases (N = 23), as well as seronegative controls (N = 10) were evaluated by LAMP in comparison to qPCR. LAMP was able to amplify DNAs from T. cruzi stocks representative of the six DTUs, whereas it did not amplify DNAs from Leishmania sp, T. brucei sp, T. rangeli KPN+ and KPN-, P. falciparum and non-infected human DNA. Analytical sensitivity was 1x10-2 fg/μL of both CL Brener and Sylvio X10 DNAs, whereas qPCR detected up to 1x 10-1 fg/μL of CL Brener DNA and 1 fg/μl of Sylvio X10 DNA. LAMP detected 1x10-2 parasite equivalents/mL in spiked EDTA blood and 1x10-1 par.eq/mL in spiked heparinized blood using fiberglass columns for DNA extraction, whereas qPCR detected 1x10-2 par.eq./mL in EDTA blood. Boil & Spin extraction allowed detection of 1x10-2 par.eq /mL in spiked EDTA blood and 1 par.eq/ml in heparinized blood. LAMP was able to detect T.cruzi infection in peripheral blood samples collected from well-characterised seropositive patients, including acute, congenital, chronic and reactivated Chagas disease. To our knowledge, this is the first report of a prototype LAMP kit with appropriate analytical sensitivity for diagnosis of Chagas disease patients, and potentially useful for monitoring treatment response.
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http://dx.doi.org/10.1371/journal.pntd.0005779DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5544240PMC
July 2017

LEISHMANIA INFANTUM INFECTION IN BENNETT'S WALLABIES (MACROPUS RUFOGRISEUS RUFOGRISEUS) IN A SPANISH WILDLIFE PARK.

J Zoo Wildl Med 2016 Jun;47(2):586-93

Although dogs are the main reservoir for human Leishmania infantum infection, the disease has also been reported in other domestic and wild mammals. In 2011, a fatal case of naturally acquired leishmaniosis was described for the first time in a Bennett's wallaby (Macropus rufogriseus rufogriseus) kept in a wildlife park in Madrid (Spain). This study was designed to assess the infection status of twelve Bennett's wallabies in the same park one year after this incident. Phlebotomus perniciosus, the main vector of L. infantum in Spain, was screened for using sticky and Centers for Disease Control miniature light traps. L. infantum infection was confirmed by molecular diagnosis in four animals, but only one wallaby returned a positive serology result. The presence of the sand fly vector was also confirmed in this habitat. These results suggest that the first case of L. infantum in a wallaby in this park was not an isolated incident and stress the need for further work to determine the role of this parasite in the morbidity and mortality of these macropods. Madrid was recently the scene of an outbreak of human cutaneous and visceral leishmaniosis. Epidemiological studies have so far revealed the widespread presence of L. infantum infection in animals other than the dog. Our ongoing work suggests a risk of L. infantum infection not only among captive animals in Madrid, but also among threatened species or even species that are already extinct in the wild.
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http://dx.doi.org/10.1638/2014-0216.1DOI Listing
June 2016

DNA sequence analysis suggests that cytb-nd1 PCR-RFLP may not be applicable to sandfly species identification throughout the Mediterranean region.

Parasitol Res 2016 Mar 12;115(3):1287-95. Epub 2016 Jan 12.

Neglected Tropical Diseases Programme, Foundation for Innovative New Diagnostics-FIND, Chemin des Mines 9, Campus Biotech, 1202, Geneva, Switzerland.

Molecular methods are increasingly used for both species identification of sandflies and assessment of their population structure. In general, they are based on DNA sequence analysis of targets previously amplified by PCR. However, this approach requires access to DNA sequence facilities, and in some circumstances, it is time-consuming. Though DNA sequencing provides the most reliable information, other downstream PCR applications are explored to assist in species identification. Thus, it has been recently proposed that the amplification of a DNA region encompassing partially both the cytochrome-B (cytb) and the NADH dehydrogenase 1 (nd1) genes followed by RFLP analysis with the restriction enzyme Ase I allows the rapid identification of the most prevalent species of phlebotomine sandflies in the Mediterranean region. In order to confirm the suitability of this method, we collected, processed, and molecularly analyzed a total of 155 sandflies belonging to four species including Phlebotomus ariasi, P. papatasi, P. perniciosus, and Sergentomyia minuta from different regions in Spain. This data set was completed with DNA sequences available at the GenBank for species prevalent in the Mediterranean basin and the Middle East. Additionally, DNA sequences from 13 different phlebotomine species (P. ariasi, P. balcanicus, P. caucasicus, P. chabaudi, P. chadlii, P. longicuspis, P. neglectus, P. papatasi, P. perfiliewi, P. perniciosus, P. riouxi, P. sergenti, and S. minuta), from 19 countries, were added to the data set. Overall, our molecular data revealed that this PCR-RFLP method does not provide a unique and specific profile for each phlebotomine species tested. Intraspecific variability and similar RFLP patterns were frequently observed among the species tested. Our data suggest that this method may not be applicable throughout the Mediterranean region as previously proposed. Other molecular approaches like DNA barcoding or phylogenetic analyses would allow a more precise molecular species identification.
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http://dx.doi.org/10.1007/s00436-015-4865-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4759228PMC
March 2016

Mixed infection of Leishmania infantum and Leishmania braziliensis in rodents from endemic urban area of the New World.

BMC Vet Res 2015 Mar 20;11:71. Epub 2015 Mar 20.

Grupo de Estudos em Leishmanioses, Centro de Pesquisas René Rachou, Fundação Oswaldo Cruz, Belo Horizonte, MG, Brasil.

Background: In Brazil Leishmania braziliensis and L. infantum are the principal species responsible for cutaneous and visceral leishmaniases, respectively. Domestic dogs are the main reservoirs of visceral leishmaniasis, while rodents and marsupials are the main reservoirs for cutaneous leishmaniasis. It has also been suggested that dogs could play a role in transmission of cutaneous leishmaniasis. The identification of the species of Leishmania, the reservoirs, and the vectors involved in each particular transmission cycle is critical for the establishment of control activities. Belo Horizonte has emerged as an endemic region for leishmaniases, however, epidemiological studies assessing the contribution of wild reservoirs to transmission are scarce in the area. The aim of this study was to investigate Leishmania spp. infection in possible reservoirs of an urbanized area.

Results: A high rate of infection was found in small mammals (64.9%) and dogs (DG1 30.4% and DG2 48.6%). The presence of L. infantum and L. braziliensis was detected in small mammals and dogs, and mixed infections by both species were detected in rodents which, to the best of our knowledge, is the first description of this phenomenon in an urban area. Additionally, L. amazonensis was detected in the canine samples.

Conclusion: The possible role of these animals as a source of infection of the vector of each species of Leishmania identified should not be overlooked and should be taken into account in future control activities. The results of mixed infection by L. braziliensis and L. infantum in cosmopolitan rodents as M. musculus and R. rattus, may have important implications in the context of the control of leishmaniasis in urban areas, especially when considering that these rodents live in close relationship with human dwellings, especially those in more precarious conditions.
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http://dx.doi.org/10.1186/s12917-015-0392-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4374209PMC
March 2015

Visceral leishmaniasis and HIV coinfection in the Mediterranean region.

PLoS Negl Trop Dis 2014 Aug 21;8(8):e3021. Epub 2014 Aug 21.

Tropical Medicine & Clinical Parasitology, Infectious Diseases Department, Ramón y Cajal Hospital, Madrid, Spain.

Visceral leishmaniasis is hypoendemic in Mediterranean countries, where it is caused by the flagellate protozoan Leishmania infantum. VL cases in this area account for 5%-6% of the global burden. Cases of Leishmania/HIV coinfection have been reported in the Mediterranean region, mainly in France, Italy, Portugal, and Spain. Since highly active antiretroviral therapy was introduced in 1997, a marked decrease in the number of coinfected cases in this region has been reported. The development of new diagnostic methods to accurately identify level of parasitemia and the risk of relapse is one of the main challenges in improving the treatment of coinfected patients. Clinical trials in the Mediterranean region are needed to determine the most adequate therapeutic options for Leishmania/HIV patients as well as the indications and regimes for secondary prophylaxis. This article reviews the epidemiological, diagnostic, clinical, and therapeutic aspects of Leishmania/HIV coinfection in the Mediterranean region.
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http://dx.doi.org/10.1371/journal.pntd.0003021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4140663PMC
August 2014