Publications by authors named "Gert Van der Auwera"

51 Publications

Surveillance of leishmaniasis cases from 15 European centres, 2014 to 2019: a retrospective analysis.

Euro Surveill 2022 Jan;27(4)

The members of the network are listed under Investigators.

BackgroundSurveillance of human leishmaniasis in Europe is mostly limited to country-specific information from autochthonous infections in the southern part. As at the end of 2021, no integrated analysis has been performed for cases seen across centres in different European countries.AimTo provide a broad perspective on autochthonous and imported leishmaniasis cases in endemic and non-endemic countries in Europe.MethodsWe retrospectively collected records from cutaneous, mucosal and visceral leishmaniasis cases diagnosed in 15 centres between 2014 and 2019. Centres were located in 11 countries: Belgium, France, Germany, Italy, the Netherlands, Norway, Portugal, Spain, Sweden, Switzerland and the United Kingdom. Data on country of infection, reason for travelling, infecting species, age and sex were analysed.ResultsWe obtained diagnostic files from 1,142 cases, of which 76%, 21% and 3% had cutaneous, visceral, and mucosal disease, respectively. Of these, 68% were men, and 32% women, with the median age of 37 years (range: 0-90) at diagnosis. Visceral leishmaniasis was mainly acquired in Europe (88%; 167/190), while cutaneous leishmaniasis was primarily imported from outside Europe (77%; 575/749). Sixty-two percent of cutaneous leishmaniasis cases from outside Europe were from the Old World, and 38% from the New World. Geographic species distribution largely confirmed known epidemiology, with notable exceptions.ConclusionsOur study confirms previous reports regarding geographic origin, species, and traveller subgroups importing leishmaniasis into Europe. We demonstrate the importance of pooling species typing data from many centres, even from areas where the aetiology is presumably known, to monitor changing epidemiology.
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http://dx.doi.org/10.2807/1560-7917.ES.2022.27.4.2002028DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8796293PMC
January 2022

Clinical diversity and treatment results in Tegumentary Leishmaniasis: A European clinical report in 459 patients.

PLoS Negl Trop Dis 2021 10 13;15(10):e0009863. Epub 2021 Oct 13.

Université de Paris, UMRs-1134, Inserm, Centre Médical de l'Institut Pasteur, Institut Pasteur, Paris, France.

Background: Cutaneous leishmaniasis (CL) is frequent in travellers and can involve oro-nasal mucosae. Clinical presentation impacts therapeutic management.

Methodology: Demographic and clinical data from 459 travellers infected in 47 different countries were collected by members of the European LeishMan consortium. The infecting Leishmania species was identified in 198 patients.

Principal Findings: Compared to Old World CL, New World CL was more frequently ulcerative (75% vs 47%), larger (3 vs 2cm), less frequently facial (17% vs 38%) and less frequently associated with mucosal involvement (2.7% vs 5.3%). Patients with mucosal lesions were older (58 vs 30 years) and more frequently immunocompromised (37% vs 3.5%) compared to patients with only skin lesions. Young adults infected in Latin America with L. braziliensis or L. guyanensis complex typically had an ulcer of the lower limbs with mucosal involvement in 5.8% of cases. Typically, infections with L. major and L. tropica acquired in Africa or the Middle East were not associated with mucosal lesions, while infections with L. infantum, acquired in Southern Europe resulted in slowly evolving facial lesions with mucosal involvement in 22% of cases. Local or systemic treatments were used in patients with different clinical presentations but resulted in similarly high cure rates (89% vs 86%).

Conclusion/significance: CL acquired in L. infantum-endemic European and Mediterranean areas displays unexpected high rates of mucosal involvement comparable to those of CL acquired in Latin America, especially in immunocompromised patients. When used as per recommendations, local therapy is associated with high cure rates.
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http://dx.doi.org/10.1371/journal.pntd.0009863DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8544871PMC
October 2021

Assessing Skin Parasite Load: A Proof of Concept Study of a Microbiopsy Device in an Indian Setting.

Front Cell Infect Microbiol 2021 11;11:645121. Epub 2021 Mar 11.

Department of Public Health, Institute of Tropical Medicine, Antwerp, Belgium.

Background: In the endgame of the elimination initiative of visceral leishmaniasis (VL) on the Indian subcontinent, one of the main questions remaining is whether asymptomatically infected individuals also contribute to transmission. We piloted a minimally invasive microbiopsy device that could help answer this question. While the potential of this device has been previously illustrated in Ethiopia, no such information is available for the setting of the Indian subcontinent. In this proof of concept study we aimed to assess 1) to what extent skin parasite load obtained with the new microbiopsy device correlates with disease status, 2) to what extent skin parasite load correlates with blood parasite load in the same subject, and 3) to what extent the skin parasite load obtained from different sampling sites on the body correlates with one another.

Methods: We performed a pilot study in Bihar, India, including 29 VL patients, 28 PKDL patients, 94 asymptomatically infected individuals, 22 endemic controls (EC), and 28 non-endemic controls (NEC). Presence of infection with in the blood was assessed using Direct Agglutination Test, rK39 ELISA, Whole Blood Analysis measuring IFN-γ and qPCR. A skin sample was collected with the microbiopsy device on two different locations on the body. PKDL patients provided a third skin sample from the edge of a PKDL lesion. Parasite load in the skin was measured by qPCR.

Findings: We found a clear correlation between the skin parasite load obtained with the microbiopsy device and disease status, with both higher skin parasite loads and higher proportions of positive skin samples in VL and PKDL patients compared to asymptomatics, EC, and NEC. No clear correlation between skin parasite load and blood parasite load was found, but a moderate correlation was present between the skin parasite load in arm and neck samples. In addition, we found four positive skin samples among asymptomatic individuals, and 85% of PKDL lesions tested positive using this microbiopsy device.

Conclusions: In line with previous pilot studies, our results from an Indian setting suggest that the microbiopsy device provides a promising tool to measure skin parasite load, and - if validated by xenodiagnosis studies - could facilitate much needed larger scale studies on infectiousness of human subgroups. In addition, we advocate further evaluation of this device as a diagnostic tool for PKDL.
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http://dx.doi.org/10.3389/fcimb.2021.645121DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8006290PMC
July 2021

Epidemiology, clinical pattern and impact of species-specific molecular diagnosis on management of leishmaniasis in Belgium, 2010-2018: A retrospective study.

Travel Med Infect Dis 2020 Nov - Dec;38:101885. Epub 2020 Sep 22.

Department of Clinical Sciences, Institute of Tropical Medicine, Antwerp, Belgium.

Background: Species-directed therapy of leishmaniasis has been recommended for travelers since 2014, but little is known about species distribution and treatment practices in non-endemic countries. We aimed to describe leishmaniasis cases in Belgium since species typing became available and evaluate its impact on patient management.

Method: Retrospective analysis of all patients diagnosed by PCR at our national reference laboratory from 2010 to 2018. Species were typed by Hsp-70 sequencing.

Results: We identified 18 visceral leishmaniasis (VL) and 147 (muco)cutaneous leishmaniasis ((M)CL) cases. VL was exclusively due to L. infantum and consistently treated with liposomal amphotericin B, with four observed failures. (M)CL was caused by ten different species. Of 62 cases diagnosed and species typed after 2014 with timing information, 28 (45.2%) were treated before the species result was available. Therapy was not species-directed in 10/32(28.1%) of those treated after species identification. Patients treated according to the guidelines tended to have a favorable outcome more often than those who were not (36/44, 81.8% versus 8/19, 57.9%; p = 0.045).

Conclusions: In contrast to VL, various species caused (M)CL in our setting and species result was often not considered for treatment. Outcome tended to be better however when therapy was species-directed.
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http://dx.doi.org/10.1016/j.tmaid.2020.101885DOI Listing
July 2021

Visceral Leishmaniasis, Northern Somalia, 2013-2019.

Emerg Infect Dis 2020 01;26(1):153-154

We identified visceral leishmaniasis caused by Leishmania donovani in a previously unknown focus in northern Somalia. Clinical and epidemiologic characteristics of 118 cases during 2013-2019 in Bosaso, the region's commercial capital, have raised suspicion of visceral leishmaniasis endemicity status there.
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http://dx.doi.org/10.3201/eid2601.181851DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6924893PMC
January 2020

Longitudinal evaluation of asymptomatic Leishmania infection in HIV-infected individuals in North-West Ethiopia: A pilot study.

PLoS Negl Trop Dis 2019 10 8;13(10):e0007765. Epub 2019 Oct 8.

Medical College, University of Gondar, Gondar, Ethiopia.

Background: In endemic regions, asymptomatic Leishmania infection is common. In HIV patients, detection of asymptomatic Leishmania infection could potentially identify those at risk of visceral leishmaniasis (VL). However, data on the prevalence, incidence, and determinants of asymptomatic infection, and the risk of VL are lacking.

Methods: We conducted a cross-sectional survey at a single ART centre, followed by a prospective cohort study amongst HIV-infected adults in HIV care in a district hospital in a VL-endemic area in North-West Ethiopia (9/2015-8/2016). Asymptomatic Leishmania infection was detected using the direct agglutination test (DAT), rK39-rapid diagnostic test (RDT)), PCR on peripheral blood and the KAtex urine antigen test, and defined as positivity on any Leishmania marker. All individuals were followed longitudinally (irrespective of the Leishmania test results). Risk factors for asymptomatic Leishmania infection were determined using logistic regression.

Results: A total of 534 HIV-infected individuals enrolled in HIV care were included in the study. After excluding 13 patients with a history of VL and an 10 patients with incomplete baseline Leishmania tests, 511 were included in analysis. The median age was 38 years (interquartile range (IQR) 30-45), 62.6% were male. The median follow-up time was 12 months (IQR 9-12). No deaths were reported during the study period. Most (95.5%) were on antiretroviral treatment at enrolment, for a median of 52 months (IQR 27-79). The median CD4 count at enrolment was 377 cells/mm3 (IQR 250-518). The baseline prevalence of Leishmania infection was 12.8% in males and 4.2% in females. Overall, 7.4% tested positive for rK39, 4.3% for DAT, 0.2% for PCR and 0.2% for KAtex. Independent risk factors for a prevalent infection were male sex (odds ratio (OR) 3.2; 95% confidence intervals (CI) 14-7.0) and concurrent malaria infection (OR 6.1; 95% CI 1.9-18.9). Amongst the 49 prevalent (baseline) infections with further follow-up, the cumulative incidence of losing the Leishmania markers by one year was 40.1%. There were 36 incident infections during the course of the study, with a cumulative one-year risk of 9.5%. Only one case of VL was detected during follow-up.

Conclusions: We found a high prevalence of asymptomatic Leishmania infection, persisting in most cases. The incidence was more modest and overt VL was rare. Larger and longer studies with more complete follow-up may help to decide whether a test and treat strategy would be justified in this context.

Trial Registration: ClinicalTrials.gov NCT02839603.
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http://dx.doi.org/10.1371/journal.pntd.0007765DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6799935PMC
October 2019

Ecology and seasonality of sandflies and potential reservoirs of cutaneous leishmaniasis in Ochollo, a hotspot in southern Ethiopia.

PLoS Negl Trop Dis 2019 08 19;13(8):e0007667. Epub 2019 Aug 19.

Evolutionary Ecology group, University of Antwerp, Antwerp, Belgium.

Background: Ochollo is a village in southern Ethiopia burdened with cutaneous leishmaniasis (CL), where Phlebotomus pedifer is the only vector for Leishmania aethiopica and hyraxes are confirmed reservoir hosts. A detailed description of the different players of transmission, and the ecology and seasonality of the vector needs to be established in order to accomplish efficient control programs.

Methods And Findings: Between March 2017 and February 2018, a monthly sandfly collection was carried out in different habitats and records of temperature and humidity were taken. Rodents and hyraxes were trapped in the dry and wet season. All samples were screened for Leishmania kinetoplast DNA (kDNA). Positive samples were further processed for determination of the Leishmania species and the species of the sandfly/small mammal that was found infected. Additionally, the species of 400 sandfly specimens from different habitats and seasons was identified. 17,190 Sergentomyia and Phlebotomus sandflies were caught and showed an overall kDNA prevalence of 2.6%, all were L. aethiopica infections only found in P. pedifer. The overall sandfly and P. pedifer abundance peaked in the dry season and was negatively correlated with the %RH. The kDNA prevalence varied over the months and was negatively correlated with the temperature. Total sandfly abundance did not differ between the sampled habitats, but P. pedifer was the distinct predominant species only in caves. Moreover, significantly more infected sandflies were found in caves. Only 1/192 rodents were kDNA positive, while 20.0% (5/25) of Heterohyrax brucei were found infected.

Conclusions: This study suggests that caves may be a source of multiplication of the infection. If an outdoor control program would be considered, it would be useful to focus on caves in the wet season, when the sandfly abundance is lowest. The captured rodent species appear not important for transmission and the contribution of hyraxes in transmission should be further investigated.
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http://dx.doi.org/10.1371/journal.pntd.0007667DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6715250PMC
August 2019

Cutaneous Leishmaniasis Due to .

EClinicalMedicine 2018 Dec 8;6:69-81. Epub 2019 Jan 8.

Unit of HIV and Neglected Tropical Diseases, Department of Clinical Sciences, Institute of Tropical Medicine, Antwerp, Belgium.

is the main causative species for cutaneous leishmaniasis (CL) in Ethiopia. Despite its considerable burden, has been one of the most neglected species. In this review, published evidence on history, geography, vector, reservoir, epidemiology, parasitology, and immunology is discussed and knowledge gaps are outlined. endemic regions are limited to the highland areas, although nationwide studies on CL prevalence are lacking. and are the sandfly vectors and hyraxes are considered to be the main reservoir, but the role of other sandfly species and other potential reservoirs requires further investigation. Where and how transmission occurs exactly are also still unknown. Most CL patients in Ethiopia are children and young adults. Lesions are most commonly on the face, in contrast to CL caused by other species which may more frequently affect other body parts. CL lesions caused by seem atypical and more severe in their presentation as compared to other species. Mucocutaneous leishmaniasis and diffuse cutaneous leishmaniasis are relatively common, and healing of lesions caused by seems to take longer than that of other species. A thorough documentation of the natural evolution of as well as in depth studies into the immunological and parasitological characteristics that underpin the atypical and severe clinical presentation are needed. Better understanding of CL caused by this parasite species will contribute to interventions related to transmission, prevention, and treatment.
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http://dx.doi.org/10.1016/j.eclinm.2018.12.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6537575PMC
December 2018

Accuracy of a Rapid Diagnostic Test Based on Antigen Detection for the Diagnosis of Cutaneous Leishmaniasis in Patients with Suggestive Skin Lesions in Morocco.

Am J Trop Med Hyg 2018 09 5;99(3):716-722. Epub 2018 Jul 5.

Department of Public Health, Institute of Tropical Medicine, Antwerp, Belgium.

In rural areas in Morocco, diagnosing cutaneous leishmaniasis (CL) can be challenging. We evaluated the accuracy of a rapid diagnostic test (RDT) based on antigen detection, CL Detect Rapid Test (Inbios International Inc., Seattle, WA), in this setting. We consecutively recruited patients with new skin ulcers in nine primary health centers. We took a dental broach sample for the RDT and two other tissue samples by scraping the border and center of the lesion with a scalpel and smearing it on a slide. We duplicated each smear by pressing a clean slide against it and processed the slides by microscopy, polymerase chain reaction (PCR) internal transcribed spacer 1, and kDNA minicircle PCR. In a subgroup with positive PCR, the species was identified using PCR-restriction fragment length polymorphism and PCR-sequencing of genes. A participant with positive microscopy and/or PCR was considered a confirmed CL case. We computed sensitivity (Se) and specificity (Sp) of the RDT compared with this reference standard (ClinicalTrials.gov registration: NCT02979002). Between December 2016 and July 2017, we included 219 patients, 50% of them were under 18 years old. Rapid diagnostic test Se was 68% [95% confidence interval (CI): 61-74], Sp 94% [95% CI: 91-97], positive predictive value 95% [95% CI: 92-98], and negative predictive value 64% [95% CI: 58-70]. Despite its low Se, this novel RDT is a useful addition to clinical management of CL in Morocco, especially in isolated localities. Rapid diagnostic test-positive lesions can be treated as CL; but when RDT negative, microscopy should be done in a second step. The Se of the RDT can probably be optimized by improving the sampling procedure.
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http://dx.doi.org/10.4269/ajtmh.18-0066DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6169188PMC
September 2018

Recurrence of visceral and muco-cutaneous leishmaniasis in a patient under immunosuppressive therapy.

BMC Infect Dis 2017 07 7;17(1):478. Epub 2017 Jul 7.

Centre Hospitalier Universitaire (CHU) de Liège, Liège, Belgium.

Background: Leishmaniasis is a protozoan disease caused by parasites of the genus Leishmania, transmitted to humans by sandflies. The diagnosis of leishmaniasis is often challenging as it mimics many other infectious or malignant diseases. The disease can present in three ways: cutaneous, mucocutaneous, or visceral leishmaniasis, which rarely occur together or consecutively.

Case Presentation: The patient was a 52 years old immunosuppressed Belgian woman with a long history of severe rheumatoid arthritis. She underwent bone marrow biopsy to explore thrombocytopenia. Diagnosis of visceral leishmaniasis was made by identification of Leishman Donovan (LD) bodies in macrophages. Treatment with liposomal amphotericin B was successful. She later developed cutaneous leishmaniasis treated with amphotericin B lipid complex. She next presented with relapsing cutaneous lesions followed by rapidly progressing lymphadenopathies. Biopsy confirmed the diagnosis of leishmaniasis. Treatments by miltefosine, amphotericin B, N-methyl-glucamine antimoniate were subsequently initiated. She later presented a recurrent bone marrow involvement treated with intramuscular paromomycin and miltefosine. She died two years later from leukemia. At the time of death, she presented with a mucosal destruction of the nose. A Leishmania-specific PCR (Polymerase Chain Reaction) identified L. infantum as etiological agent.

Conclusions: Clinicians should be aware of the potential concomitant or sequential involvement of multiple anatomic localizations of Leishmania in immunosuppressed patients.
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http://dx.doi.org/10.1186/s12879-017-2571-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5501116PMC
July 2017

Detection and identification of Leishmania spp.: application of two hsp70-based PCR-RFLP protocols to clinical samples from the New World.

Parasitol Res 2017 Jul 1;116(7):1843-1848. Epub 2017 Jun 1.

PECET (Program of Study and Control of Tropical Diseases) University of Antioquia, Medellín, Colombia.

Leishmaniasis is highly prevalent in New World countries, where several methods are available for detection and identification of Leishmania spp. Two hsp70-based PCR protocols (PCR-N and PCR-F) and their corresponding restriction fragment length polymorphisms (RFLP) were applied for detection and identification of Leishmania spp. in clinical samples recruited in Colombia, Guatemala, and Honduras. A total of 93 cases were studied. The samples were classified into positive or suspected of leishmaniasis according to parasitological criteria. Molecular amplification of two different hsp70 gene fragments and further RFLP analysis for identification of Leishmania species was done. The detection in parasitologically positive samples was higher using PCR-N than PCR-F. In the total of samples studied, the main species identified were Leishmania panamensis, Leishmania braziliensis, and Leishmania infantum (chagasi). Although RFLP-N was more efficient for the identification, RFLP-F is necessary for discrimination between L. panamensis and Leishmania guyanesis, of great importance in Colombia. Unexpectedly, one sample from this country revealed an RFLP pattern corresponding to Leishmania naiffi. Both molecular variants are applicable for the study of clinical samples originated in Colombia, Honduras, and Guatemala. Choosing the better tool for each setting depends on the species circulating. More studies are needed to confirm the presence of L. naiffi in Colombian territory.
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http://dx.doi.org/10.1007/s00436-017-5454-6DOI Listing
July 2017

Single locus genotyping to track Leishmania donovani in the Indian subcontinent: Application in Nepal.

PLoS Negl Trop Dis 2017 03 1;11(3):e0005420. Epub 2017 Mar 1.

Department of Biomedical Sciences, Institute of Tropical Medicine, Antwerp, Belgium.

Background: We designed a straightforward method for discriminating circulating Leishmania populations in the Indian subcontinent (ISC). Research on transmission dynamics of visceral leishmaniasis (VL, or Kala-azar) was recently identified as one of the key research priorities for elimination of the disease in the ISC. VL in Bangladesh, India, and Nepal is caused by genetically homogeneous populations of Leishmania donovani parasites, transmitted by female sandflies. Classical methods to study diversity of these protozoa in other regions of the world, such as microsatellite typing, have proven of little use in the area, as they are not able to discriminate most genotypes. Recently, whole genome sequencing (WGS) so far identified 10 different populations termed ISC001-ISC010.

Methodology / Principle Findings: As an alternative to WGS for epidemiological or clinical studies, we designed assays based on PCR amplification followed by dideoxynucleotide sequencing for identification of the non-recombinant genotypes ISC001 up to ISC007. These assays were applied on 106 parasite isolates collected in Nepal between 2011 and 2014. Combined with data from WGS on strains collected in the period 2002-2011, we provide a proof-of-principle for the application of genotyping to study treatment outcome, and differential geographic distribution.

Conclusions / Significance: Our method can aid in epidemiological follow-up of visceral leishmaniasis in the Indian subcontinent, a necessity in the frame of the Kala-azar elimination initiative in the region.
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http://dx.doi.org/10.1371/journal.pntd.0005420DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5348045PMC
March 2017

Comparison of Leishmania typing results obtained from 16 European clinical laboratories in 2014.

Euro Surveill 2016 Dec;21(49)

Hospital for Tropical Diseases, London, United Kingdom.

Leishmaniasis is endemic in southern Europe, and in other European countries cases are diagnosed in travellers who have visited affected areas both within the continent and beyond. Prompt and accurate diagnosis poses a challenge in clinical practice in Europe. Different methods exist for identification of the infecting Leishmania species. Sixteen clinical laboratories in 10 European countries, plus Israel and Turkey, conducted a study to assess their genotyping performance. DNA from 21 promastigote cultures of 13 species was analysed blindly by the routinely used typing method. Five different molecular targets were used, which were analysed with PCR-based methods. Different levels of identification were achieved, and either the Leishmania subgenus, species complex, or actual species were reported. The overall error rate of strains placed in the wrong complex or species was 8.5%. Various reasons for incorrect typing were identified. The study shows there is considerable room for improvement and standardisation of Leishmania typing. The use of well validated standard operating procedures is recommended, covering testing, interpretation, and reporting guidelines. Application of the internal transcribed spacer 1 of the rDNA array should be restricted to Old World samples, while the heat-shock protein 70 gene and the mini-exon can be applied globally.
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http://dx.doi.org/10.2807/1560-7917.ES.2016.21.49.30418DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5291127PMC
December 2016

Visceral Leishmaniasis Patients Display Altered Composition and Maturity of Neutrophils as well as Impaired Neutrophil Effector Functions.

Front Immunol 2016 29;7:517. Epub 2016 Nov 29.

Department of Medicine, Imperial College London , London , UK.

Immunologically, active visceral leishmaniasis (VL) is characterized by profound immunosuppression, severe systemic inflammatory responses, and an impaired capacity to control parasite replication. Neutrophils are highly versatile cells, which play a crucial role in the induction as well as the resolution of inflammation, the control of pathogen replication, and the regulation of immune responses. Neutrophil functions have been investigated in human cutaneous leishmaniasis; however, their role in human VL is poorly understood. In the present study we evaluated the activation status and effector functions of neutrophils in patients with active VL and after successful anti-leishmanial treatment. Our results show that neutrophils are highly activated and have degranulated; high levels of arginase, myeloperoxidase, and elastase, all contained in neutrophils' granules, were found in the plasma of VL patients. In addition, we show that a large proportion of these cells are immature. We also analyzed effector functions of neutrophils that are essential for pathogen clearance and show that neutrophils have an impaired capacity to release neutrophil extracellular traps, produce reactive oxygen species, and phagocytose bacterial particles, but not parasites. Our results suggest that impaired effector functions, increased activation, and immaturity of neutrophils play a key role in the pathogenesis of VL.
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http://dx.doi.org/10.3389/fimmu.2016.00517DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5126105PMC
November 2016

Molecular identification of Leishmania spp. clinical isolates from Colombia based on hsp70 gene.

Biomedica 2016 Feb 23;36(0):37-44. Epub 2016 Feb 23.

Departamento de Parasitología, Instituto de Medicina Tropical Pedro Kourí, La Habana, Cuba.

Introduction: Leishmaniasis is highly prevalent in Colombia, where at least six different species can cause disease of varying clinical presentations in humans. The identification of the infecting species is quite important for prognosis, therapeutics and epidemiology. Different techniques with variable discriminatory power have been used for the identification. 

Objective: To carry out the molecular identification of Leishmania species through the amplification of a fragment of the hsp70 gene. 

Materials And Methods: Molecular amplification of the hsp70 gene fragment (PCR-hsp70) followed by restriction fragment length polymorphism analysis (RFLP) was done for identification purposes using DNA from 81 clinical isolates of Leishmania. 

Results: A single amplicon was obtained for all samples analyzed. The enzymatic restrictions of the 81 PCR products identified 70 with a banding pattern corresponding to L. braziliensis with two different patterns (62 and eight isolates, respectively), nine isolates compatible with L. panamensis and two with L. guyanensis. The geographical origin of the isolates is consistent with previous reports about the distribution of the corresponding species in Colombia. 

Conclusions: The PCR-hsp70/RFLP technique used is a valid tool for the identification of Leishmania species isolated from clinical samples of patients in Colombia, which may also be applicable to the study of strains obtained from vectors and reservoirs with epidemiological significance.
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http://dx.doi.org/10.7705/biomedica.v36i2.2688DOI Listing
February 2016

Phylogenetic analysis of the Trypanosoma genus based on the heat-shock protein 70 gene.

Infect Genet Evol 2016 09 13;43:165-72. Epub 2016 May 13.

Department of Biomedical Sciences, Institute of Tropical Medicine, Antwerp, Belgium. Electronic address:

Trypanosome evolution was so far essentially studied on the basis of phylogenetic analyses of small subunit ribosomal RNA (SSU-rRNA) and glycosomal glyceraldehyde-3-phosphate dehydrogenase (gGAPDH) genes. We used for the first time the 70kDa heat-shock protein gene (hsp70) to investigate the phylogenetic relationships among 11 Trypanosoma species on the basis of 1380 nucleotides from 76 sequences corresponding to 65 strains. We also constructed a phylogeny based on combined datasets of SSU-rDNA, gGAPDH and hsp70 sequences. The obtained clusters can be correlated with the sections and subgenus classifications of mammal-infecting trypanosomes except for Trypanosoma theileri and Trypanosoma rangeli. Our analysis supports the classification of Trypanosoma species into clades rather than in sections and subgenera, some of which being polyphyletic. Nine clades were recognized: Trypanosoma carassi, Trypanosoma congolense, Trypanosoma cruzi, Trypanosoma grayi, Trypanosoma lewisi, T. rangeli, T. theileri, Trypanosoma vivax and Trypanozoon. These results are consistent with existing knowledge of the genus' phylogeny. Within the T. cruzi clade, three groups of T. cruzi discrete typing units could be clearly distinguished, corresponding to TcI, TcIII, and TcII+V+VI, while support for TcIV was lacking. Phylogenetic analyses based on hsp70 demonstrated that this molecular marker can be applied for discriminating most of the Trypanosoma species and clades.
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http://dx.doi.org/10.1016/j.meegid.2016.05.016DOI Listing
September 2016

Transmission of Leishmania donovani in the Hills of Eastern Nepal, an Outbreak Investigation in Okhaldhunga and Bhojpur Districts.

PLoS Negl Trop Dis 2015 7;9(8):e0003966. Epub 2015 Aug 7.

Department of Internal Medicine, B.P. Koirala Institute of Health Sciences, Dharan, Nepal.

Background: In the Indian subcontinent, Visceral leishmaniasis is endemic in a geographical area coinciding with the Lower Gangetic Plain, at low altitude. VL occurring in residents of hill districts is therefore often considered the result of Leishmania donovani infection during travel. Early 2014 we conducted an outbreak investigation in Okhaldhunga and Bhojpur districts in the Nepal hills where increasing number of VL cases have been reported.

Methodology/principal Findings: A house-to-house survey in six villages documented retrospectively 35 cases of Visceral Leishmaniasis (VL). Anti-Leishmania antibodies were found in 22/23 past-VL cases, in 40/416 (9.6%) persons without VL and in 12/155 (7.7%) domestic animals. An age- and sex- matched case-control study showed that exposure to known VL-endemic regions was no risk factor for VL, but having a VL case in the neighbourhood was. SSU-rDNA PCR for Leishmania sp. was positive in 24 (5%) of the human, in 18 (12%) of the animal samples and in 16 (14%) bloodfed female Phlebotomus argentipes sand flies. L. donovani was confirmed in two asymptomatic individuals and in one sand fly through hsp70-based sequencing.

Conclusions/significance: This is epidemiological and entomological evidence for ongoing local transmission of L. donovani in villages at an altitude above 600 meters in Nepal, in districts considered hitherto non-endemic for VL. The VL Elimination Initiative in Nepal should therefore consider extending its surveillance and control activities in order to assure VL elimination, and the risk map for VL should be redesigned.
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http://dx.doi.org/10.1371/journal.pntd.0003966DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4529159PMC
April 2016

Species typing in dermal leishmaniasis.

Clin Microbiol Rev 2015 Apr;28(2):265-94

Institute of Tropical Medicine, Department of Biomedical Sciences, Antwerp, Belgium Antwerp University, Department of Biomedical Sciences, Antwerp, Belgium.

Leishmania is an infectious protozoan parasite related to African and American trypanosomes. All Leishmania species that are pathogenic to humans can cause dermal disease. When one is confronted with cutaneous leishmaniasis, identification of the causative species is relevant in both clinical and epidemiological studies, case management, and control. This review gives an overview of the currently existing and most used assays for species discrimination, with a critical appraisal of the limitations of each technique. The consensus taxonomy for the genus is outlined, including debatable species designations. Finally, a numerical literature analysis is presented that describes which methods are most used in various countries and regions in the world, and for which purposes.
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http://dx.doi.org/10.1128/CMR.00104-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4402951PMC
April 2015

[Detection of Leishmania spp. based on the gene encoding HSP20].

Rev Peru Med Exp Salud Publica 2014 Oct-Dec;31(4):635-43

Centro de Biología Molecular Severo Ochoa, Madrid, España.

Objectives: Explore a new target for molecular diagnosis of Leishmania.

Materials And Methods: We evaluated the utility of the gene that encodes the heat shock protein 20-kDa (Hsp20) for detecting Leishmania by polymerase chain reaction (PCR). PCR was normalized and analytical parameters were determined, as well as the validity and diagnostic accuracy, and concordance with the PCR - 18S. PCR-Hsp20 with DNA was obtained from a group of clinical samples from different sources.

Results: The analytical parameters were adequate. The sensitivity obtained was 86% and the specificity was 100%. The concordance with the reference method was good (κ = 0.731), which supports its potential use for diagnosis. The possibility of subsequent identification of the species by sequencing the amplified product gives an additional advantage.

Conclusions: The usefulness of this gene as a new target for the detection of Leishmania was demonstrated. Because of its potential, it is recommended to improve the sensitivity of the method and to evaluate it in different endemic regions.
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January 2017

Direct Leishmania species typing in Old World clinical samples: evaluation of 3 sensitive methods based on the heat-shock protein 70 gene.

Diagn Microbiol Infect Dis 2014 Sep 21;80(1):35-9. Epub 2014 May 21.

Department of BioMedical Sciences, Institute of Tropical Medicine, Antwerp, Belgium. Electronic address:

In the diagnosis of leishmaniasis, identification of the causative Leishmania species is relevant for treatment, prognosis, and epidemiology. Three new hsp70-based PCR variants were developed and recently validated on clinical samples from Peru, without the need for culturing. We evaluated their performance on 133 clinical samples (bone marrow, blood, buffy coat, lymph node aspirates, lesion biopsies) from 42 cutaneous and 56 visceral leishmaniasis patients and 35 negative cases, all from Old World countries (Italy, Sudan, Israel, and Tunisia). The 3 new PCRs were significantly more sensitive than those previously described for hsp70, and their respective restriction fragment analyses were more efficient for species identification. In 79% of the parasitologically confirmed positive samples, the species could be identified directly from sample DNA. This evaluation demonstrated that these new tools are globally applicable in different geographical, clinical, and sampling contexts, and they could become the reference method for identification of Leishmania species in clinical specimens.
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http://dx.doi.org/10.1016/j.diagmicrobio.2014.05.012DOI Listing
September 2014

Easy identification of leishmania species by mass spectrometry.

PLoS Negl Trop Dis 2014 Jun 5;8(6):e2841. Epub 2014 Jun 5.

AP-HP, Groupe Hospitalier Pitié-Salpêtrière, Service Parasitologie-Mycologie, Paris, France; Université Pierre et Marie Curie-Paris6, UMR S945 Paris, France; Institut National de la Santé et de la Recherche Médicale U945, Paris, France.

Background: Cutaneous leishmaniasis is caused by several Leishmania species that are associated with variable outcomes before and after therapy. Optimal treatment decision is based on an accurate identification of the infecting species but current methods to type Leishmania isolates are relatively complex and/or slow. Therefore, the initial treatment decision is generally presumptive, the infecting species being suspected on epidemiological and clinical grounds. A simple method to type cultured isolates would facilitate disease management.

Methodology: We analyzed MALDI-TOF spectra of promastigote pellets from 46 strains cultured in monophasic medium, including 20 short-term cultured isolates from French travelers (19 with CL, 1 with VL). As per routine procedure, clinical isolates were analyzed in parallel with Multilocus Sequence Typing (MLST) at the National Reference Center for Leishmania.

Principal Findings: Automatic dendrogram analysis generated a classification of isolates consistent with reference determination of species based on MLST or hsp70 sequencing. A minute analysis of spectra based on a very simple, database-independent analysis of spectra based on the algorithm showed that the mutually exclusive presence of two pairs of peaks discriminated isolates considered by reference methods to belong either to the Viannia or Leishmania subgenus, and that within each subgenus presence or absence of a few peaks allowed discrimination to species complexes level.

Conclusions/significance: Analysis of cultured Leishmania isolates using mass spectrometry allows a rapid and simple classification to the species complex level consistent with reference methods, a potentially useful method to guide treatment decision in patients with cutaneous leishmaniasis.
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http://dx.doi.org/10.1371/journal.pntd.0002841DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4046964PMC
June 2014

Evaluation of four single-locus markers for Leishmania species discrimination by sequencing.

J Clin Microbiol 2014 Apr 22;52(4):1098-104. Epub 2014 Jan 22.

Institute of Tropical Medicine, Antwerp, Belgium.

Several genetic markers have been described for discriminating Leishmania species. In most reported cases, one or a few polymorphisms are the basis of species identification, and the methods were validated on a limited number of strains from a particular geographical region. Therefore, most techniques may underestimate the global intraspecies variability and are applicable only in certain areas. In addition, interlaboratory standardization is mostly absent, complicating comparisons among different studies. Here, we compared species typing results from all sequence polymorphisms found in four popular markers that can be applied directly on clinical samples: the miniexon or spliced leader, the internal transcribed spacer of the ribosomal DNA array, the 7SL RNA gene, and the heat shock protein 70 gene. Clustering was evaluated among 74 Leishmania strains, selected to represent a wide geographic distribution and genetic variability of the medically relevant species of the genus. Results were compared with a multilocus sequence typing (MLST) approach using 7 single-copy household genes and with multilocus enzyme electrophoresis (MLEE), still considered the gold standard by some. We show that strain groupings are highly congruent across the four different single-locus markers, MLST, and MLEE. Overall, the heat shock protein 70 gene and the miniexon presented the best resolutions for separating medically relevant species. As gene sequence analysis is validated here on a global scale, it is advocated as the method of choice for use in genetic, clinical, and epidemiological studies and for managing patients with unknown origins of infection, especially in Western infectious disease clinics dealing with imported leishmaniasis.
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http://dx.doi.org/10.1128/JCM.02936-13DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3993476PMC
April 2014

Differentiation between Trypanosoma cruzi and Trypanosoma rangeli using heat-shock protein 70 polymorphisms.

Trop Med Int Health 2014 Feb 13;19(2):195-206. Epub 2013 Nov 13.

Parasitology Department, Institute of Tropical Medicine Pedro Kouri, La Havana, Cuba.

Objective: Differential diagnosis of infection with Trypanosoma cruzi or T. rangeli is relevant for epidemiological studies and clinical practice as both species infect humans, but only T. cruzi causes Chagas' disease. Their common antigen determinants complicate the distinction between both species, while current PCR assays used for differentiation show some drawbacks. We developed and validated a generic PCR discriminating the species by restriction fragment length polymorphism (RFLP) analysis and a duplex PCR specifically amplifying a differently sized fragment of both species.

Methods: The assays are based upon a partial region of the heat-shock protein 70 gene (hsp70). The analytical sensitivity and specificity were determined for both PCRs. The assays were analytically evaluated on a panel of six T. cruzi, one T. cruzi marinkellei and four T. rangeli strains, various other infectious pathogens, a panel of spiked samples of T. cruzi, and artificially mixed infections of T. cruzi and T. rangeli. Finally, the tools were applied on 36 additional isolates of Trypanosoma species.

Results: The detection limit of the PCRs was between 0.05 and 0.5 parasite genomes, and 1-10 parasites spiked in 200 μl blood. In artificial mixtures, PCR-RFLP picked up both species in ratios up to 10(2) and duplex PCR up to 10(4) . In the 36 isolates tested, both single and mixed infections were identified. All assays were shown to be specific.

Conclusion: Our PCRs show high potential for the differential diagnosis of T. cruzi and T. rangeli, which in view of their sensitivity can aid in the confirmation of infection with these parasites in vectors, reservoirs and clinical samples.
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http://dx.doi.org/10.1111/tmi.12222DOI Listing
February 2014

HindII and SduI digests of heat-shock protein 70 PCR for Leishmania typing.

Diagn Microbiol Infect Dis 2013 Nov 17;77(3):245-7. Epub 2013 Sep 17.

Departamento de Parasitología, Instituto de Medicina Tropical "Pedro Kourí", Havana, Cuba.

Restriction fragment length polymorphisms of the heat-shock protein 70 gene have been used for discriminating Leishmania species. Here, we validated HindII as a much cheaper alternative to EcoRII and SduI for discriminating Leishmania (Viannia) braziliensis from Leishmania (Viannia) naiffi and an atypical Leishmania (V.) braziliensis group, which was previously not possible.
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http://dx.doi.org/10.1016/j.diagmicrobio.2013.07.023DOI Listing
November 2013

Evolution and species discrimination according to the Leishmania heat-shock protein 20 gene.

Infect Genet Evol 2013 Aug 28;18:229-37. Epub 2013 May 28.

Departamento de Parasitología, Instituto de Medicina Tropical "Pedro Kourí", Habana, Cuba.

The Leishmania genus comprises up to 35 species, of which 20 are responsible for human disease. However, the taxonomic status for many of them is under discussion. The small Heat Shock Proteins (sHSPs) are physiologically relevant, protecting cellular proteins from aggregation and maintaining cellular viability under intensive stress conditions. In Leishmania, a protein of this class was previously described, the 20-kDa heat-shock protein (HSP20), which is encoded by a single gene. In the present study, we used this target, alone or in combination with hsp70 gene, to investigate the phylogenetic relationships among Leishmania species. Using a pair of degenerate primers it was possible amplifying a 370bp fragment of the hsp20 coding region in 39 strains of very different geographic origins, representing in total 16 Leishmania species (14 if L. chagasi and L. archibaldi are considered synonymous names of L. infantum and L. donovani, respectively). Nucleotide sequences were readily obtained by direct sequencing of the amplification products. Both phylogenetic trees and networks based on either hsp20 sequences or combined datasets of hsp20 and hsp70 sequences were constructed. These phylogenic analyses supported the division of the Leishmania genus into nine species: L. (L.) donovani, L. (L.) major, L. (L.) tropica, L. (L.) aethiopica, L. (L.) mexicana, L. (V.) lainsoni, L. (V.) naiffi, L. (V.) guyanensis and L. (V.) braziliensis. Additionally, by network analysis, the subspecies L. (L.) donovani infantum and L. (V.) braziliensis peruviana were recognized within the L. (L.) donovani and L. (V.) braziliensis species, respectively. Therefore, hsp20 gene was found to be a suitable molecular marker for Leishmania typing and classification purposes. In addition, this study represents a solid contribution to the objective of establishing a more reliable taxonomy for the genus Leishmania.
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http://dx.doi.org/10.1016/j.meegid.2013.05.020DOI Listing
August 2013

Real-time PCR assay for detection and quantification of Leishmania (Viannia) organisms in skin and mucosal lesions: exploratory study of parasite load and clinical parameters.

J Clin Microbiol 2013 Jun 3;51(6):1826-33. Epub 2013 Apr 3.

Instituto de Medicina Tropical Alexander von Humboldt, Universidad Peruana Cayetano Heredia, Lima, Peru.

Earlier histopathology studies suggest that parasite loads may differ between cutaneous leishmaniasis (CL) and mucosal leishmaniasis (ML) lesions and between acute and chronic CL. Formal demonstration requires highly sensitive detection and accurate quantification of Leishmania in human lesional tissue. In this study, we developed a quantitative real-time PCR (qPCR) assay targeting minicircle kinetoplast DNA (kDNA) to detect and quantify Leishmania (Viannia) parasites. We evaluated a total of 156 lesion biopsy specimens from CL or ML suspected cases and compared the quantitative performance of our kDNA qPCR assay with that of a previously validated qPCR assay based on the glucose-6-phosphate dehydrogenase (G6PD) gene. We also examined the relationship between parasite load and clinical parameters. The kDNA qPCR sensitivity for Leishmania detection was 97.9%, and its specificity was 87.5%. The parasite loads quantified by kDNA qPCR and G6PD qPCR assays were highly correlated (r = 0.87; P < 0.0001), but the former showed higher sensitivity (P = 0.000). CL lesions had 10-fold-higher parasite loads than ML lesions (P = 0.009). Among CL patients, the parasite load was inversely correlated with disease duration (P = 0.004), but there was no difference in parasite load according to the parasite species, the patient's age, and number or area of lesions. Our findings confirm that CL and recent onset of disease (<3 months) are associated with a high parasite load. Our kDNA qPCR assay proved highly sensitive and accurate for the detection and quantification of Leishmania (Viannia) spp. in lesion biopsy specimens. It has potential application as a diagnostic and follow-up tool in American tegumentary leishmaniasis.
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http://dx.doi.org/10.1128/JCM.00208-13DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3716068PMC
June 2013

(Post-) Genomic approaches to tackle drug resistance in Leishmania.

Parasitology 2013 Oct 12;140(12):1492-505. Epub 2013 Mar 12.

Molecular Parasitology Unit, Department of Biomedical Sciences, Institute of Tropical Medicine, Nationalestraat 155, B-2000 Antwerp, Belgium.

Leishmaniasis, like other neglected diseases is characterized by a small arsenal of drugs for its control. To safeguard the efficacy of current drugs and guide the development of new ones it is thus of utmost importance to acquire a deep understanding of the phenomenon of drug resistance and its link with treatment outcome. We discuss here how (post-)genomic approaches may contribute to this purpose. We highlight the need for a clear definition of the phenotypes under consideration: innate and acquired resistance versus treatment failure. We provide a recent update of our knowledge on the Leishmania genome structure and dynamics, and compare the contribution of targeted and untargeted methods for the understanding of drug resistance and show their limits. We also present the main assays allowing the experimental validation of the genes putatively involved in drug resistance. The importance of analysing information downstream of the genome is stressed and further illustrated by recent metabolomics findings. Finally, the attention is called onto the challenges for implementing the acquired knowledge to the benefit of the patients and the population at risk.
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http://dx.doi.org/10.1017/S0031182013000140DOI Listing
October 2013

Molecular and serological markers of Leishmania donovani infection in healthy individuals from endemic areas of Bihar, India.

Trop Med Int Health 2013 May 7;18(5):548-54. Epub 2013 Mar 7.

Infectious Disease Research Laboratory, Banaras Hindu University, Varanasi, India.

Objectives: Recent epidemiological reports indicate that asymptomatic human infections with Leishmania donovani, the causative agent of visceral leishmaniasis or Kala-azar (KA), occur frequently in India. We explored markers of infection.

Methods: Blood samples were collected from 286 healthy subjects from 16 villages in the Muzaffarpur district of Bihar. These individuals were classified into three groups: (i) persons with no history of KA and living in a house where no KA cases were previously reported, (ii) persons with no history of KA but living in a house where KA cases were diagnosed at the time of sampling or in the past, and (iii) successfully treated KA patients. Each sample was tested using a Leishmania-specific PCR to detect Leishmania DNA, and two serological tests to demonstrate anti-Leishmania antibodies: the Direct Agglutination Test and rK39 ELISA.

Results: PCR positivity was similar among the three groups (20-25%). In contrast, among treated patients, the percentage of serologically positive individuals was roughly five times that of healthy individuals with no KA history, as measured with either test. Living in a house where KA had been reported did not affect seropositivity.

Conclusion: A significant proportion of asymptomatic infections of Leishmania exist in endemic regions. Using a combination of molecular and serological tests increases the capacity to detect infections at different stages. Further work is required to understand the kinetics of the markers.
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http://dx.doi.org/10.1111/tmi.12085DOI Listing
May 2013

Identification of Leishmania tropica from micro-foci of cutaneous leishmaniasis in the Kenyan Rift Valley.

Pathog Glob Health 2012 Jul;106(3):159-65

Centre for Clinical Research, Kenya Medical Research Institute, Nairobi, Kenya.

We performed diagnosis and species identification of parasites in lesion samples from suspected cutaneous leishmaniasis patients in four villages, three of which are in a known Leishmania tropica endemic region in Kenya. Samples were analyzed both by microscopy and PCR for Leishmania, and typed by an assay using four ribosomal DNA-based species-identification PCRs. The lesions were demonstrated to be caused by L. tropica, which confirms the re-emergence of cutaneous leishmaniasis from this species after a period of reduced incidence in the endemic zone. Our report highlights the importance of an intervention and sustained Leishmania control program.
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http://dx.doi.org/10.1179/2047773212Y.0000000015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4001575PMC
July 2012
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