Pediatric visceral leishmaniasis: a retrospective study to propose the diagnostic tests algorithm in southern Iran.

Leishmania infantum is the most common cause of visceral leishmaniasis (VL) in Iran, where mainly the patients are children under the age of 5 years. Timely, less invasive, and accurate diagnosis and proper treatment of the disease are necessary. This retrospective study aimed to search for a less invasive but robust algorithm on VL diagnostic tests in children. Four hundred and fifteen patients with clinical suspicion of VL, 50 healthy children from VL endemic areas, 46 healthy individuals from non-endemic VL areas, and 47 non-VL diseases were tested using three diagnostic tests: indirect immunofluorescent antibody test (IFAT), rK39-rapid diagnostic test (rK39-RDT), and quantitative PCR (qPCR). One hundred and two suspected VL cases were positive in at least one test and were cured after receiving appropriate treatment. Of these 102 VL patients, 94 were positive in qPCR, 84 in IFAT, and 79 in rK39-RDT. None of the tests detected all the patients, but overall, qPCR is capable of detecting more VL patients than serological tests, i.e., 92.2%, compared to IFAT, 82.4%, and rK39, 77.5%. There was only a significant difference between the sensitivity of qPCR and rK39-RDT (p = 0.024). The specificity was 100% for qPCR and IFAT (≥128) and 98.6% for rK39-RDT. qPCR alone is capable of detecting most of the VL-suspected children. Serological tests like IFAT and rk39-RDT are recommended to increase the overall sensitivity of detection in patients with a negative molecular test. Combining qPCR with a serological test (IFAT or rK39-RDT) can help diagnose 98% of VL. In laboratories without molecular facilities, we recommend testing with the combination of rK39-RDT and IFAT yielding a combined sensitivity of 93.1% equivalent to that of qPCR in our study.

Detection of Leishmania infantum DNA by real-time PCR in saliva of dogs.

This study developed a real-time quantitative PCR (qPCR) assay to detect L. infantum kinetoplast DNA (kDNA) in canine saliva. The qPCR showed an efficiency of 93.8%, a coefficient of correlation of 0.996 and a detection limit of 0.5 fg/reaction (0.005 parasites), although it detected until 0.25 fg/reaction (0.0025 parasites). When samples from 12 dogs experimentally infected with L. infantum were collected, L. infantum kDNA was detected at 16-weeks post-infection (wpi) in 41.7% and 91.7% of saliva and bone marrow samples, respectively, and at 47-wpi in 75% of both samples. L. infantum kDNA can be detected by qPCR in canine saliva, with lower sensitivity in the early stages of infection and a lower parasite load estimation compared to bone marrow. However, saliva had similar sensitivities to bone marrow in the later stages of the infection and could be used to detect L. infantum kDNA being aware of its limitations.

High molecular prevalence of Leishmania in phlebotomine sand flies fed on chicken blood in Brazil.

Leishmaniases are endemic in Brazil, where Leishmania infantum has been detected in humans, dogs, cats, and phlebotomine vectors. Monitoring synanthropic vector populations is critical for leishmaniasis control-surveillance in such transmission-prone areas. Here, a suite of molecular approaches were used to assess Leishmania infection prevalence and to identify blood-meal sources in a large sample of sand flies collected in anthropic environments of a Leishmania-transmission area in Mato Grosso do Sul State (Rio Verde de Mato Grosso municipality), Central-West Brazil. We sampled sand flies monthly (January-June 2014 and 2016) in one peri-domestic site within each of six neighborhoods with recent records of human visceral and/or tegumentary leishmaniasis. kDNA-qPCR plus rDNA ITS-sequencing were used to detect and identify Leishmania in pooled female sand flies. Individual engorged females (n = 58) were used for blood-meal analyses through High-Resolution Melting (HRM) targeting the mtDNA cytb gene. Overall, 90.5% of 420 CDC trap-nights yielded vectors, for a total catch of 24,989 sand flies. We sub-sampled and identified 3088 sand flies of 12 species, including 2775 Lutzomyia longipalpis (the most abundant species at all sampling sites) and 297 Nyssomyia whitmani. Female sand flies (n = 1261) were grouped in 159 pools, of which 92 Lu. longipalpis (minimum infection rate [MIR] 8%) and 7 Ny. whitmani pools (MIR 7%) were Leishmania kDNA-positive. Most positive Lu. longipalpis were collected in the 2016 rainy season. Sequencing confirmed L. infantum in Lu. longipalpis samples. HRM analyses identified chicken DNA in 57 sand flies (98.3%), 37 of which were Leishmania DNA-positive (64.9%); human blood was found in just one (Leishmania-negative) female. Our data show ongoing risk of L. infantum transmission to humans in the study area, where Leishmania-infected sandfly vectors are common and heavily rely on chicken blood in the peri-domestic environment.

Molecular identification of Leishmania infection in the most relevant sand fly species and in patient skin samples from a cutaneous leishmaniasis focus, in Morocco.

BACKGROUND: Cutaneous leishmaniasis (CL) is an infectious disease caused by various species of Leishmania and transmitted by several species of sand flies. CL is among the most neglected tropical diseases, and it has represented a major health threat over the past 20 years in Morocco. The main objectives of this study were to identify relevant sand fly species and detect Leishmania infection in the most prevalent species and patient skin samples in Taza, a focus of CL in North-eastern Morocco. METHODOLOGY AND FINDING: A total of 3672 sand flies were collected by CDC miniature light traps. Morphological identification permitted the identification of 13 species, namely 10 Phlebotomus species and 3 Sergentomyia species. P. longicuspis was the most abundant species, comprising 64.08% of the total collected sand flies, followed by P. sergenti (20.1%) and P. perniciosus (8.45%). Using nested-kDNA PCR, seven pools of P. sergenti were positive to Leishmania tropica DNA, whereas 23 pools of P. longicuspis and 4 pools of P. perniciosus tested positive for Leishmania infantum DNA. The rates of P. longicuspis and P. perniciosus Leishmania infection were 2.51% (23/915) and 7.27% (4/55), respectively, whereas the infection prevalence of P. sergenti was 3.24%. We also extracted DNA from lesion smears of 12 patients suspected of CL, among them nine patients were positive with enzymatic digestion of ITS1 by HaeIII revealing two profiles. The most abundant profile, present in eight patients, was identical to L. infantum, whereas L. tropica was found in one patient. The results of RFLP were confirmed by sequencing of the ITS1 DNA region. CONCLUSION: This is the first molecular detection of L. tropica and L. infantum in P. sergenti and P. longicuspis, respectively, in this CL focus. Infection of P. perniciosus by L. infantum was identified for the first time in Morocco. This study also underlined the predominance of L. infantum and its vector in this region, in which L. tropica has been considered the causative agent of CL for more than 20 years.

High-throughput sequencing of kDNA amplicons for the analysis of Leishmania minicircles and identification of Neotropical species.

Leishmania kinetoplast DNA contains thousands of small circular molecules referred to as kinetoplast DNA (kDNA) minicercles. kDNA minicircles are the preferred targets for sensitive Leishmania detection, because they are present in high copy number and contain conserved sequence blocks in which polymerase chain reaction (PCR) primers can be designed. On the other hand, the heterogenic nature of minicircle networks has hampered the use of this peculiar genomic region for strain typing. The characterization of Leishmania minicirculomes used to require isolation and cloning steps prior to sequencing. Here, we show that high-throughput sequencing of single minicircle PCR products allows bypassing these laborious laboratory tasks. The 120 bp long minicircle conserved region was amplified by PCR from 18 Leishmania strains representative of the major species complexes found in the Neotropics. High-throughput sequencing of PCR products enabled recovering significant numbers of distinct minicircle sequences from each strain, reflecting minicircle class diversity. Minicircle sequence analysis revealed patterns that are congruent with current hypothesis of Leishmania relationships. Then, we show that a barcoding-like approach based on minicircle sequence comparisons may allow reliable identifications of Leishmania spp. This work opens up promising perspectives for the study of kDNA minicercles and a variety of applications in Leishmania research.

Detection of Leishmania infantum DNA in conjunctival swabs of cats by quantitative real-time PCR.

Although some studies have investigated the potential role of cats as a reservoir for Leishmania, their role in the epidemiology of visceral leishmaniasis (VL) is still poorly understood. Molecular diagnostic techniques are an important tool in VL diagnosis, and PCR shows high sensitivity and specificity for Leishmania spp. detection. Quantitative real-time PCR (qPCR) is a method that permits quantitative analysis of a large number of samples, resulting in more sensitive, accurate, and reproducible measurements of specific DNA present in the sample. This study compared real-time PCR (qPCR) and conventional PCR (cPCR) for detection of Leishmania spp. in blood and conjunctival swab (CS) samples of healthy cats from a non-endemic area in the state of São Paulo, Brazil. Of all CS samples, 1.85% (2/108) were positive for Leishmania spp. by both cPCR as qPCR (kappa index = 1), indicating excellent agreement between the two methods. The DNA from the two CS-cPCR- and CS-qPCR-positive samples was further tested with a PCR test amplifying the Leishmania spp. discriminative rRNA internal transcribed spacer 1 (ITS 1), of which one sample generated a 300-350-bp DNA fragment whose size varies according to the Leishmania species. Following sequencing, the fragment showed 100% similarity to a GenBank L. infantum sequence obtained from a cat in Italy. In conclusion, the association of qPCR and CS proved to be effective for detection of Leishmania in cats. Conjunctival swab samples were shown to be a practical and better alternative to blood samples and may be useful in the diagnosis and studies of feline leishmaniasis.

Evaluation of urine for Leishmania infantum DNA detection by real-time quantitative PCR.

The availability of some sorts of biological samples which require noninvasive collection methods has led to an even greater interest in applying molecular biology on visceral leishmaniasis (VL) diagnosis, since these samples increase the safety and comfort of both patients and health professionals. In this context, this work aimed to evaluate the suitability of the urine as a specimen for Leishmania infantum kinetoplast DNA detection by real-time quantitative PCR (qPCR). Subsequent to the reproducibility analysis, the detection limit of the qPCR assay was set at 5fg (~0.025 parasites) per µL of urine. From the comparative analysis performed with a set of diagnostic criteria (serological and molecular reference tests), concordance value of 96.08% was obtained (VL-suspected and HIV/AIDS patients, n=51) (P>0.05). Kappa coefficient (95% CI) indicated a good agreement between the test and the set of diagnostic criteria (k=0.778±0.151). The detection of Leishmania DNA in urine by qPCR was possible in untreated individuals, and in those with or without suggestive renal impairment. Fast depletion of the parasite's DNA in urine after treatment (from one dose of meglumine antimoniate) was suggested by negative qPCR results, thus indicating it as a potential alternative specimen to follow up the efficacy of therapeutic approaches. Even when evaluated in a clinically heterogeneous set of patients, the urine showed good prospect as sample for VL diagnosis by qPCR, also indicating a good negative predictive value for untreated suspected patients.

Cutaneous Leishmaniasis in Khyber Pakhtunkhwa Province of Pakistan: Clinical Diversity and Species-Level Diagnosis.

This study primarily aimed to identify the causative species of cutaneous leishmaniasis (CL) in the Khyber Pakhtunkhwa Province of Pakistan and to distinguish any species-specific variation in clinical manifestation of CL. Diagnostic performance of different techniques for identifying CL was assessed. Isolates of Leishmania spp. were detected by in vitro culture, polymerase chain reaction (PCR) on DNA extracted from dried filter papers and microscopic examination of direct lesion smears from patients visiting three major primary care hospitals in Peshawar. A total of 125 CL patients were evaluated. Many acquired the disease from Peshawar and the neighboring tribal area of Khyber Agency. Military personnel acquired CL while deployed in north and south Waziristan. Leishmania tropica was identified as the predominant infecting organism in this study (89.2%) followed by Leishmania major (6.8%) and, unexpectedly, Leishmania infantum (4.1%). These were the first reported cases of CL caused by L. infantum in Pakistan. PCR diagnosis targeting kinetoplast DNA was the most sensitive diagnostic method, identifying 86.5% of all samples found positive by any other method. Other methods were as follows: ribosomal DNA PCR (78.4%), internal transcribed spacer 2 region PCR (70.3%), culture (67.1%), and microscopy (60.5%). Clinical examination reported 14 atypical forms of CL. Atypical lesions were not significantly associated with the infecting Leishmania species, nor with "dry" or "wet" appearance of lesions. Findings from this study provide a platform for species typing of CL patients in Pakistan, utilizing a combination of in vitro culture and molecular diagnostics. Moreover, the clinical diversity described herein can benefit clinicians in devising differential diagnosis of the disease.

Molecular and Serological Evidence of Leishmania Infection in Stray Dogs from Visceral Leishmaniasis-Endemic Areas of Bangladesh.

Visceral leishmaniasis (VL), or kala-azar, is mainly caused by two closely related Leishmania species, Leishmania infantum and Leishmania donovani Leishmania infantum is responsible for zoonotic VL, with dogs as the main reservoir host in the Mediterranean, the Middle East, Asia, and South America. In the Indian subcontinent, VL is caused by L. donovani and is considered anthroponotic, although the only known vector, the sand fly, is zoophilic in nature. The role of domestic and stray dogs in VL transmission is still unclear in this area. We screened 50 stray dogs from VL-endemic areas of Bangladesh for serological and molecular evidence of Leishmania infection. We detected anti-Leishmania antibodies in six (12%) dog serum samples using rK39 immunochromatographic tests. We observed Leishmania kinetoplast DNA in 10 (20%) buffy coat DNA samples by real-time polymerase chain reaction (PCR), five of which were positive based on internal transcribed spacer 1-PCR. A sequencing analysis of the amplified products confirmed that the parasitic DNA was derived from L. donovani Our findings support the hypothesis that stray dogs are an animal reservoir for L. donovani in this endemic region. Further studies are required to determine the precise role of dogs in the epidemiology of VL in Bangladesh.

Seroprevalence of Leishmania infection and molecular detection of Leishmania tropica and Leishmania infantum in stray cats of Izmir, Turkey.

Leishmaniasis caused by more than 20 species of genus Leishmania is transmitted by the bite of infected phlebotomine sand flies. The studies on Leishmania infection in cats is very few in Turkey and therefore we aimed to screen stray cats living in city of Izmir located in western Turkey using nested PCR targeting kinetoplast DNA and serological techniques (ELISA and IFA). Leishmania DNA positive samples were also studied by ITS1 real time PCR. Whole blood and serum samples were obtained from stray cats (n: 1101) living in different counties of Izmir. In serological assays, a serum sample was considered positive in 1:40 dilution in IFA and for ELISA a serum sample was accepted positive when the absorbance value (AV) exceeded the mean AV + Standard Deviation (SD) of the negative control serum samples. According to the results, the seropositivity rates were 10.8% (119/1101) and 15.2% (167/1101) by in house ELISA and IFA, respectively. Among serology coherent samples, the seropositivity rate was 11.1% (116/1047) as detected by both assays after discordant samples (n: 54) were discarded. Of the 1101 stray cats, six (0.54%) were positive by nested PCR while only one of these six samples was positive by ITS1 real time PCR. During PCR, three controls designated as Leishmania infantum, Leishmania tropica, and Leishmania major were used for species identification. According to nested PCR results, L. tropica was identified in two cats (no.76 and 95). In another cat (no. 269), there were two bands in which one of them was well-matched with L. infantum and the other band had ∼850 bp size which does not match with any controls. Remaining three cats (no. 86, 514, and 622) also had the ∼850 bp atypical band size. ITS1 real time PCR detected L. tropica in only one cat (no. 622) which showed an atypical band size in nested PCR. These results indicated that three cats with only one atypical band (no. 86, 514, and 622) and the cat with mixed infection (no. 269) were infected with L. tropica. Altogether, L. tropica was detected in all six DNA positive cats and L. infantum was detected in one cat with mixed infection. In conclusion, although the reservoir role of cats in nature is still unclear the high seroprevalence rate against Leishmania parasites and detecting parasite DNA in stray cats in Izmir indicates that the stray cats are frequently bitten by infected sand flies. Further research activities are required to reveal the frequency of leishmaniasis in cats in different regions of Turkey where Leishmania species are endemic.

Gold nanoparticle-based lateral flow biosensor for rapid visual detection of Leishmania-specific DNA amplification products.

Leishmaniasis is a disease, caused by Leishmania parasites, which infect humans and animals, posing a major social and economic burden worldwide. The need for accurate and sensitive disease diagnosis led to the widespread adoption of PCR amplification. Detection of the amplification products (i.e. gel electrophoresis) require time-consuming protocols performed by trained personnel, with high cost. Aim of the present study was the simplification of PCR product detection, using a nucleic acid lateral flow, combined with functionalized gold nanoparticles. Amplification reactions targeting kinetoplastid DNA of Leishmania spp were performed on canine blood samples and a positive signal was formed as a red test zone. The visual detection was completed in 20min. Extensive optimization enabled the detection of 100fmol of target DNA. Clinical samples of infected dog blood were analyzed with high specificity. Overall, the proposed lateral flow biosensor can be considered an appealing alternative platform for Leishmania-specific amplification products detection with low cost and attractive simplicity.

A Novel Molecular Test to Diagnose Canine Visceral Leishmaniasis at the Point of Care.

Dogs are the principal reservoir hosts of zoonotic visceral leishmaniasis (VL) but current serological methods are not sensitive enough to detect all subclinically infected animals, which is crucial to VL control programs. Polymerase chain reaction (PCR) methods have greater sensitivity but require expensive equipment and trained personnel, impairing its implementation in endemic areas. We developed a diagnostic test that uses isothermal recombinase polymerase amplification (RPA) to detect Leishmania infantum. This method was coupled with lateral flow (LF) reading with the naked eye to be adapted as a point-of-care test. The L. infantum RPA-LF had an analytical sensitivity similar to real time-PCR, detecting DNA of 0.1 parasites spiked in dog blood, which was equivalent to 40 parasites/mL. There was no cross amplification with dog or human DNA or with Leishmania braziliensis, Leishmania amazonensis, or Trypanosoma cruzi. The test also amplified Leishmania donovani strains (N = 7). In a group of clinically normal dogs (N = 30), RPA-LF detected more subclinical infections than rK39 strip test, a standard serological method (50% versus 13.3% positivity, respectively; P = 0.005). Also, RPA-LF detected L. infantum in noninvasive mucosal samples of dogs with a sensitivity comparable to blood samples. This novel molecular test may have a positive impact in leishmaniasis control programs.

[Comparison of direct microscopy, culture and polymerase chain reaction methods for the diagnosis of cutaneous leishmaniasis]. / Kutanöz leysmanyazis tanisinda direkt mikroskopi, kültür ve polimeraz zincir reaksiyonu yöntemlerinin karsilastirilmasi

Cutaneous leishmaniasis (CL) is an endemic disease especially in Southeastern Anatolia of Turkey and recently shows a trend for spread to other regions of the country including the Aegean region. The diagnosis of CL is based on combined evaluation of epidemiological data with the clinical symptoms and laboratory findings. Direct microscopic examination and culture methods are mainly used in the routine diagnosis of CL, while molecular methods are mainly used for research. The aim of this study was to detect the presence of Leishmania spp. in samples obtained from CL-suspected patients by using direct microscopy, culture and polymerase chain reaction (PCR) methods and to compare the results. A total of 55 patients who were admitted to Parasitology Laboratory of Adnan Menderes University Hospital, Aydin (located at Aegean region in Turkey), between 2012-2014 were included in the study. Smear preparations from the skin lesions of cases were fixed and stained with Giemsa, and the presence of amastigote forms were evaluated by direct microscopy. NNN medium was used for the cultivation of samples. Total genomic DNA of Leishmania from the samples were extracted with a commercial kit (NucleoSpin Tissue(®) Kit, Macherey-Nagel, Germany) and PCR was performed by using 13A and 13B primers to amplify the 116 base pair fragment of Leishmania spp. specific kinetoplast DNA. Amastigotes were observed in 29 (53%) of the 55 samples by direct microscopy, promastigotes were detected among 34 (62%) samples in culture, and parasite-specific amplicons were revealed in 30 (55%) samples by PCR. All assays were positive in 24 patients while in 18 patients all of the tests yielded negative results. Thirty-seven (67%) out of 55 cases were diagnosed as CL when reactivity in at least one of these three methods were considered as positive. Accordingly the positivity rates of the methods were 78.4% (29/37) for direct microscopy, 92% (34/37) for culture and 81.1% (30/37) for PCR in CL-diagnosed patients, indicating culture as the most sensitive method. Regarding the culture method as gold standard, the sensitivity and specificity of direct microscopy were calculated as 76.4% and 86%, respectively, while PCR presented with 85.3% sensitivity and 95% specificity. In conclusion, it was thought that the usage of more than one method for CL diagnosis leads to increase the sensitivity and specificity which enables the diagnosis of a wide range of patients.

PCR associated with molecular hybridization detects Leishmania (Viannia) braziliensis in healthy skin in canine tegumentary leishmaniasis.

Tegumentary leishmaniasis (TL) is a zoonotic disease that affects humans and domestic dogs. In Brazil, TL is considered endemic, and Leishmania (Viannia) braziliensis is the prevalent species causing this disease. There is debate about the role of dogs (Canis familiaris) as domestic reservoirs in the transmission cycle of TL. To date, classical parasitological techniques, including parasite isolation in culture media, have been able to detect parasites only from cutaneous lesions. In this study, we detected L. (V.) braziliensis DNA in intact skin fragments collected from 3 naturally infected dogs from the state of Rio de Janeiro, Brazil, with the use of PCR techniques associated with molecular hybridization. The detection of parasitic DNA in this anatomical site is an important finding vis-à-vis the importance of the domestic dogs in endemic areas of TL.

[Comparing two protocols of DNA extraction of Trypanosoma cruzi cultured in axenic medium]. / Comparación de dos protocolos de extracción de ADN de Trypanosoma cruzi cultivados en medio axénico.

OBJECTIVES: To compare two extraction protocols of Trypanosoma cruzi DNA for use in DNA amplification of kinetoplast minicircles (kDNA) through the technique of Polymerase Chain Reaction (PCR). MATERIALS AND METHODS: Epimastigotes of T. cruzi were cultured in axenic conditions and masses from 1.5 to 100 x 106 parasites were obtained. DNA extraction was performed using two protocols: extraction with organic solvents (phenol/chloroform), and with resin (Chelex100), from different parasitic sediments. Concentration and purity of DNA was determined by spectrophotometry, and integrity was assessed by agarose gel electrophoresis. Analysis of variance and comparisons of means were performed through Tukey's test, using the Statistix 8.0 software. RESULTS: Ten DNA extractions were done of each one of the different amounts of parasitic sediments. In the DNA extraction with Chelex100 resin, a higher performance was obtained but a lower purity and integrity compared to the extraction with organic solvents. However, it allowed a product amplification of 330 bp of T. cruzi kDNA. CONCLUSIONS: Although the technique of Chelex100 provided less purity and integrity of DNA, it allowed a successful amplification of kDNA by PCR, avoiding the use of laborious techniques and toxic organic solvents.

Comparación de dos protocolos de extracción de ADN de Trypanosoma cruzi cultivados en medio axénico / Comparing two protocols of DNA extraction of Trypanosoma cruzi cultured in axenic medium

Objetivos. Comparar dos protocolos de extracción de ADN de Trypanosoma cruzi para su uso en la amplificación de ADN de minicírculos de kinetoplasto (ADNk) mediante la técnica de Reacción en Cadena de Polimerasa (PCR). Materiales y métodos. Se cultivaron epimastigotas de T. cruzi en condiciones exénicas obteniéndose masas entre 1,5 hasta 100 x 10(6) parásitos. A partir de estas se procedió a la extracción de ADN mediante dos protocolos: extracción con solventes orgánicos (fenol/cloroformo), y empleo de resina (Chelex®100), a partir de los diferentes sedimentos parasitarios. La concentración y pureza del ADN se determinó por espectrofotometría y la integridad se evaluó mediante electroforesis en geles de agarosa. Se realizó el análisis de varianza y comparaciones de medias mediante la prueba de Tukey, utilizando el software Statistix 8.0. Resultados. Se realizaron diez extracciones de ADN de cada una de las diferentes cantidades de parásitos sedimentados. En la extracción de ADN con la resina Chelex®100 se obtuvo mayor rendimiento, pero menor pureza e integridad respecto a la extracción con solventes orgánicos. Sin embargo, permitió la amplificación del producto de 330 pb de ADNk de T. cruzi. Conclusiones. Aun cuando la técnica de Chelex®100 proporcionó menor pureza e integridad del ADN, permitió la amplificación con éxito de ADNk por PCR, evitando el uso de técnicas laboriosas y solventes orgánicos tóxicos.

Objectives. To compare two extraction protocols of Trypanosoma cruzi DNA for use in DNA amplification of kinetoplast minicircles (kDNA) through the technique of Polymerase Chain Reaction (PCR). Materials and methods. Epimastigotes of T. cruzi were cultured in axenic conditions and masses from 1.5 to 100 x 106 parasites were obtained. DNA extraction was performed using two protocols: extraction with organic solvents (phenol/chloroform), and with resin (Chelex®100), from different parasitic sediments. Concentration and purity of DNA was determined by spectrophotometry, and integrity was assessed by agarose gel electrophoresis. Analysis of variance and comparisons of means were performed through Tukey’s test, using the Statistix 8.0 software. Results. Ten DNA extractions were done of each one of the different amounts of parasitic sediments. In the DNA extraction with Chelex®100 resin, a higher performance was obtained but a lower purity and integrity compared to the extraction with organic solvents. However, it allowed a product amplification of 330 bp of T. cruzi kDNA. Conclusions. Although the technique of Chelex®100 provided less purity and integrity of DNA, it allowed a successful amplification of kDNA by PCR, avoiding the use of laborious techniques and toxic organic solvents.

Absence of Leishmania infantum in cave bats in an endemic area in Spain.

Though dogs have been historically considered the main reservoir of Leishmania infantum, the role of wildlife in its epidemiology is attracting increasing attention. Rodents, wild carnivores and, recently, hares (Lepus spp.) have been proposed as sylvatic reservoirs for this parasite. Bats have never been tested for L. infantum infection in Europe. Nevertheless, bats have a widespread distribution, they live in abundant colonies, and some species inhabit caves, where constant temperatures and humidity provide ideal habitat for the sand fly vector. We tested blood samples from 35 Schreibers' bats (Miniopterus schreibersii), abundant cave bats in NE Spain, which is an enzootic area of leishmaniasis. A PCR-amplifying fragment of the high copy of Leishmania donovani group kDNA minicircles was used. None of the analyzed samples were positive (maximum possible prevalence = 8.20%). Though the susceptibility of this bat to parasitization by L. infantum cannot be ruled out, our survey indicates that this species may not be a relevant sylvatic reservoir of L. infantum in the Mediterranean area. Nevertheless, even if the prevalence of infection in bats is low, such an abundant taxonomic group would still provide a significant maintenance population for the parasite.

Otimização de ferramentas moleculares baseadas em multiplex PCR para inclusão de controles de qualidade amostral no diagnóstico das leishmanioses / Development of molecular tools multiplex PCR-based for the inclusion of sample quality controls in the diagnosis of leishmaniasis

A detecção precoce das leishmanioses e a rápida instituição do tratamento são de suma importância para os indivíduos e comunidades afetadas, visto que pacientes contribuem para a manutenção do ciclo da doença. Diante das limitações apresentadas pelos métodos tradicionais de diagnóstico, a PCR tem se apresentado como ferramenta promissora para a detecção dos casos. No entanto, perdas de DNA durante o processo de purificação podem afetar mais significativamente o material genético dos parasitos, gerando resultados falso-negativos. Este estudo teve como objetivo desenvolver e avaliar dois protocolos de triplex PCR para investigar possíveis causas de negatividade no diagnóstico molecular das formas visceral (LV) e tegumentar (LT) das leishmanioses. Pares de primers para detecção de um controle interno (gene G3PD) e dois controles externos (DNA genômico de M. pachydermatis e plasmídeo comercial pUC18 foram adaptados a protocolos de PCR convencionais validados para a detecção de L. infantum e L.(V.) braziliensis e duas reações triplex foram otimizadas. Dados de sensibilidade (S), especificidade (E) e eficiência (e) dos novos sistemas foram calculados em 186 amostras de sangue coletadas de cães em áreas endêmicas, utilizando como referência protocolos de PCR e PCR em tempo real (qPCR) consagrados na literatura. A concordância entre os novos testes e os testes de referência foi determinada pelo cálculo do índice de Kappa. A tríplex PCR para o diagnóstico da LV mostrou S = 78.68 por cento, E= 85.29 por cento e e= 81.05 por cento com boa concordância (K = 0.60, p< 0.0001) com o conjunto de resultados PCR/qPCR. Para o diagnóstico da LT observou-se S = 97.29 por cento, E = 79.16 por cento, e = 90.16 por cento, com K = 0.78, (p<0.0001) indicando excelente nível de concordância entre os testes. As novas ferramentas apresentadas podem ser aplicadas para aumentar a acurácia no diagnóstico das leishmanioses, contribuindo para a rápida implementação do tratamento e, reduzindo a longo prazo os índices de mortalidade e morbidade das leishmanioses.

First detection of Leishmania infantum kinetoplast DNA in hair of wild mammals: application of qPCR method to determine potential parasite reservoirs.

The data presented in this paper describe the application of a method for a reliable and non-invasive diagnosis of leishmaniosis in wild reservoirs, based on the detection of Leishmania infantum kinetoplast DNA (kDNA) in hair samples by Real Time PCR (qPCR). The study has been performed on 68 ear/leg hair samples from 5 different wild species (Vulpes vulpes, Canis lupus, Martes foina, Rattus norvegicus and Erinaceus europaeus) from several geographic areas of West and North Spain. The presence of Leishmania kDNA was detected in 14 of the 68 analyzed samples, being the highest quantity of DNA observed in foxes. This is the first report of the presence of Leishmania in a hedgehog. The kDNA remained stable under the exposure of hair to different environmental conditions (freezing or high temperature, ultraviolet rays or treatment with tanning salts). This detection method could constitute a suitable alternative for the search of the parasite in wild hosts, due to the numerous advantages that hair samples present for collection, transport and storage processes.

Detection and chronology of parasitic kinetoplast DNA presence in hair of experimental Leishmania major infected BALB/c mice by Real Time PCR.

Hair can accumulate foreign chemical or biological substances. Recently, it has been reported that parasite DNA can also be detected in the hair of Leishmania infantum infected dogs. The aim of this work has been to find out whether parasite DNA incorporates in the hair of Leishmania major experimentally infected animals. For this purpose, a group of 4 BALB/c mice, intradermally inoculated in both ears with 1000 L. major V1 strain promastigote forms, was monitored for parameters associated to the infection during 35 days. Weekly, ear swelling was measured, and hair samples from ears and leg were collected. Blood samples were obtained before challenge and at day 35 post infection, when parasite load was measured in ear, lymph node and spleen by limit dilution. Ear swelling and other parameters observed in the infected mice were consistent with those described for this model. The presence of parasite kinetoplast DNA (kDNA) was detected by Real Time PCR in all ear and leg hair samples at the final timepoint. These data suggests that hair is a specialized tissue in the sequestration and removal of foreign DNA. Detection of DNA in hair could be, therefore, a useful tool to chronologically record the infection process during experimental mice assays.