Detection of Leishmania infantum in animals and their ectoparasites by conventional PCR and real time PCR

Visceral leishmaniosis (VL) is a parasitic disease caused by Leishmania infantum, which is primarily transmitted by phlebotomine sandflies. However, there has been much speculation on the role of other arthropods in the transmission of VL. Thus, the aim of this study was to assess the presence of L. infantum in cats, dogs and their ectoparasites in a VL-endemic area in northeastern Brazil. DNA was extracted from blood samples and ectoparasites, tested by conventional PCR (cPCR) and quantitative real time PCR (qPCR) targeting the L. infantum kinetoplast DNA. A total of 280 blood samples (from five cats and 275 dogs) and 117 ectoparasites from dogs were collected. Animals were apparently healthy and not previously tested by serological or molecular diagnostic methods. Overall, 213 (76.1 %) animals and 51 (43.6 %) ectoparasites were positive to L. infantum, with mean parasite loads of 795.2, 31.9 and 9.1 fg in dogs, cats and ectoparasites, respectively. Concerning the positivity between dogs and their ectoparasites, 32 (15.3 %) positive dogs were parasitized by positive ectoparasites. The overall concordance between the PCR protocols used was 59.2 %, with qPCR being more efficient than cPCR; 34.1 % of all positive samples were exclusively positive by qPCR. The high number of positive animals and ectoparasites also indicates that they could serve as sentinels or indicators of the circulation of L. infantum in risk areas.     

Compared with conventional PCR assay,qPCR assay greatly improves the detection efficiency of predation

Studies of predation can contribute greatly to understanding predator–prey relationships and can also provide integral knowledge concerning food webs and multi‐trophic level interactions. Both conventional polymerase chain reaction (cPCR) and quantitative PCR (qPCR) have been employed to detect predation in the field because of their sensitivity and reproducibility. However, to date, few studies have been used to comprehensively demonstrate which method is more sensitive and reproducible in studies of predation. We used a Drosophila melanogaster‐specific DNA fragment (99 bp) to construct a tenfold gradient dilution of standards. Additionally, we obtained DNA samples from Pardosa pseudoannulata individuals that fed on D. melanogaster at various time since feeding. Finally, we compared the sensitivity and reproducibility between cPCR and qPCR assays for detecting DNA samples from feeding trials and standards. The results showed that the cPCR and qPCR assays could detect as few as 1.62 × 10³ and 1.62 × 10¹ copies of the target DNA fragment, respectively. The cPCR assay could detect as few as 48 hr post‐feeding of the target DNA fragment. But the qPCR assay showed that all spiders were positive after consuming prey at various time intervals (0, 24, 48, 72, and 96 hr). A smaller proportion of the technical replicates were positive using cPCR, and some bands on the agarose gel were absent or gray, while some were white and bright for the same DNA samples after amplification by cPCR. By contrast, a larger proportion of the technical replicates were positive using qPCR and the coefficients of variation of the Ct value for the three technical replicates of each DNA sample were less than 5%. These data showed that qPCR was more sensitive and highly reproducible in detecting such degraded DNA from predator's gut. The present study provides an example of the use of cPCR and qPCR to detect the target DNA fragment of prey remains in predator's gut.     

Comparison of multiplex PCR and standard bacteriological methods of detecting Salmonella on chicken skin

A multiplex PCR technique was compared with standard bacteriological techniques for the detection of Salmonella , and Salm. enteritidis in particular, in samples of chicken skin obtained from retail outlets. Five of 68 samples were positive by both systems (7.4%), 58 of 68 were negative by both systems (85%), five were positive by PCR only (7.4%) but none were positive by culture only. The PCR was quicker and more sensitive than the bacteriological methods, finding 15% of samples positive for Salmonella compared to 7.4%, and taking less than 24 h to reach a result as opposed to 2–3 d.     

Molecular diagnosis of strongyloidiasis in tropical areas: a comparison of conventional and real-time polymerase chain reaction with parasitological methods

This study aimed to evaluate the use of conventional polymerase chain reaction (cPCR) and real-time quantitative PCR (qPCR) in the diagnosis of human strongyloidiasis from stool samples in tropical areas. Stool samples were collected from individuals and were determined to be positive for Strongyloides stercoralis (group I), negative for S. stercoralis (group II) and positive for other enteroparasite species (group III). DNA specific to S. stercoralis was found in 76.7% of group I samples by cPCR and in 90% of group I samples by qPCR. The results show that molecular methods can be used as alternative tools for detecting S. stercoralis in human stool samples in tropical areas.     

Redefining relativity: quantitative PCR at low template concentrations for industrial and environmental microbiology

The application of PCR techniques in environmental and industrial microbiology is complicated by innumerable organic and inorganic contaminants and enzyme inhibitors that copurify with nucleic acids. These complications are compounded in quantitative PCR (qPCR) methods, which are predicated upon subtle yet significant assumptions of amplification efficiency and the representativeness of the sample with respect to the environment or industrial process from which it was obtained. In low-biomass and/or low-template situations, additional concerns related to target gene spatial heterogeneity in the sample, differential DNA (or RNA) extraction efficiency, molecular sampling error, attenuation of PCR inhibitors and amplification bias can quickly undermine fundamental assumptions of conventional competitive PCR (cPCR) and most-probable-number PCR (MPN-PCR) formats. A critical evaluation of cPCR and MPN-PCR assumptions is therefore presented within the context of environmental microbiology and low-template enumerations. Fundamental conclusions from the analysis of qPCR assumptions are that: (a) environmental qPCR enumerations are invariably estimates, not absolute enumerations, which are relative to the PCR standard; (b) traditional cPCR assays are ill-suited for environmental applications, especially in low-biomass situations; and (c) both cPCR and traditional MPN-PCR practices insufficiently account for field-scale, process-level or experimental variations that arise and become amplified in PCR enumerations. Thus, sample representativeness and errors related to sample replication are frequently more important than errors related to the qPCR assay itself. Based upon this critique of qPCR assumptions, an alternate qPCR method for routine environmental application is described which is based upon replicative limiting dilution analysis and the pragmatic tradeoffs between analytical sensitivity and practical utility. Received 9 February 1998/ Accepted in revised form 26 June 1998     

Evaluation of MAT, IgM ELISA and PCR methods for the diagnosis of human leptospirosis

Serum samples from 125 patients with clinical suspicion of leptospirosis were tested by the microscopic agglutination test (MAT), IgM ELISA and PCR for the diagnosis of the disease. Most patients were adult males and 74.1% (p<0.001) of the patients exposed to water and 77.6% (p<0.001) of those exposed to animals, were respectively considered confirmed or probable cases by MAT. The clinical symptoms mainly observed among the patients considered confirmed or probable cases were fever (95.6%), jaundice and headache (79.4%), myalgia (77.9%), nausea and vomiting (64.7%). About 63% of the confirmed or probable cases were patients that lived in Belo Horizonte, a big city of the Minas Gerais state, Brazil, showing the occurrence of urban leptospirosis. Among the 47 confirmed cases of leptospirosis diagnosed by MAT, 44 (94%) serum samples were positive by IgM ELISA and 17 (36%) were PCR positive. Among the 33 probable cases, 10 (30%) samples showed positive amplification by PCR. By considering MAT as the standard test, the sensitivity and specificity of IgM ELISA was 96.6% and 93.3%, respectively. A relevant finding in our study was the number of positive cases verified by PCR (13-29%) and IgM ELISA (3-7%) among the 45 unconfirmed cases by MAT, demonstrating the value of PCR in the early diagnosis of human leptospirosis.     

Detection of Brucella canis from inguinal lymph nodes of naturally infected dogs by PCR

The aim of this study was to standardize and evaluate a PCR assay for the detection of Brucella canis (B. canis) in lymph node samples of naturally infected dogs. The performance of the PCR was compared with the results of bacteriological culture as reference method. Forty-eight inguinal lymph node samples were collected from 48 dogs (18 males and 30 females) that died in the city's pound in the years 2007-2008 and were examined by microbiological culture and the PCR assay. B. canis was isolated from 4 (8.3%) of 48 lymph node samples. Forty-four (91.7%) of the samples were bacteriological culture negative. B. canis DNA was directly detected from all culture positive lymph node samples (n = 4) by PCR. All of the culture negative samples were confirmed as negative by PCR. When the culture method was used as a gold standard, sensitivity and specificity of the PCR assay were found to be 100%. The limit of PCR detection of B. canis DNA was 1.4 × 10¹ CFU/g at least. In conclusion, the PCR assay has been shown to have a diagnostic performance equal to bacteriological culture for detection of B. canis. By a non-hazardous protocol for laboratory workers, the assay can be performed in one day.     

Highly sensitive direct detection and quantification of Burkholderia pseudomallei bacteria in environmental soil samples by using real-time PCR

The soil bacterium and potential biothreat agent Burkholderia pseudomallei causes the infectious disease melioidosis, which is naturally acquired through environmental contact with the bacterium. Environmental detection of B. pseudomallei represents the basis for the development of a geographical risk map for humans and livestock. The aim of the present study was to develop a highly sensitive, culture-independent, DNA-based method that allows direct quantification of B. pseudomallei from soil. We established a protocol for B. pseudomallei soil DNA isolation, purification, and quantification by quantitative PCR (qPCR) targeting a type three secretion system 1 single-copy gene. This assay was validated using 40 soil samples from Northeast Thailand that underwent parallel bacteriological culture. All 26 samples that were B. pseudomallei positive by direct culture were B. pseudomallei qPCR positive, with a median of 1.84 × 10(4) genome equivalents (range, 3.65 × 10(2) to 7.85 × 10(5)) per gram of soil, assuming complete recovery of DNA. This was 10.6-fold (geometric mean; range, 1.1- to 151.3-fold) higher than the bacterial count defined by direct culture. Moreover, the qPCR detected B. pseudomallei in seven samples (median, 36.9 genome equivalents per g of soil; range, 9.4 to 47.3) which were negative by direct culture. These seven positive results were reproduced using a nested PCR targeting a second, independent B. pseudomallei-specific sequence. Two samples were direct culture and qPCR negative but nested PCR positive. Five samples were negative by both PCR methods and culture. In conclusion, our PCR-based system provides a highly specific and sensitive tool for the quantitative environmental surveillance of B. pseudomallei.     

Detection of Leptospira spp. in semen and vaginal fluids of goats and sheep by polymerase chain reaction

Thirteen goat herds and seven sheep flocks in the state of Rio de Janeiro, Brazil were screened for leptospirosis. From the three herds and three flocks with greatest seroreactivity, 19 goats (16 females and three bucks) and 40 sheep (26 ewes and 14 rams) that were seropositive (specific anti-Leptospira titres > or =400, based on a microscopic agglutination test), were selected for more detailed studies. From those animals, samples of vaginal fluids or semen were collected for bacteriological and molecular assays. For both species of animals, the most prevalent reactions were to serovars Hardjo, Shermani, and Grippotyphosa. Although leptospires were detected by darkfield microscopy in three vaginal fluid samples (from two goats and one ewe), pure isolates were not obtained by bacteriological culture of vaginal fluids or semen. However, seven vaginal fluid samples (from four goats and three ewes) and six semen samples (all from rams) were positive on polymerase chain reaction (PCR). Based on these findings, in addition to analogous findings in cattle, we inferred that there is potential for venereal transmission of leptospirosis in small ruminants.     

Detection and quantification of Legionella pneumophila in water samples using competitive PCR

The presence of high levels of Legionella pneumophila in man-made aquatic systems correlates with the incidence of nosocomial Legionnaires' disease. This requires a rapid, reliable, and sensitive quantification of L. pneumophila concentrations in suspected water systems. In this research, a homologous competitor was developed and evaluated in a L. pneumophila competitive polymerase chain reaction (cPCR) to quantify this human pathogen in a quick, cost-effective, and reliable way. Accuracy of cPCR was evaluated by analyzing cooling tower and tap water samples spiked with known concentrations of L. pneumophila bacteria, in parallel with the standard culture method. Legionella pneumophila amounts detected and calculated from cPCR and culture correlated very well: r = 0.998, P = 0.002 for tap water and r = 0.990, P = 0.009 for cooling tower water. Nevertheless, for both kinds of water samples, mean numbers of L. pneumophila calculated from cPCR results were always higher than those obtained by culture. This study makes it clear that the rapid, sensitive, and cost-effective L. pneumophila cPCR is a promising alternative to the standard time-consuming culture method and expensive real-time PCR to enumerate L. pneumophila bacteria in environmental water samples.     

Comparison of culture and PCR for the detection of Brucella melitensis in blood and lymphoid tissues of serologically positive and negative slaughtered sheep

Aims: To compare the culture and PCR methods for detection of Brucella melitensis in blood and lymphoid tissue samples obtained from slaughtered sheep (n = 162) testing positive/negative in serological tests (Rose Bengal test and serum agglutination test). Methods and Results: Of 162 sheep examined, 45 were positive and 117 negative in serological tests. A PCR assay based on a pair of Br. melitensis‐specific primers was used to detect DNA in blood and lymphoid tissue. Brucella melitensis was isolated from 1·2% (2/162) and 17·2% (28/162) of the blood and lymphoid tissue samples respectively. Positive PCR products with a molecular size of 731 bp were obtained from 27·7% (45/162) of blood and 29·0% (47/162) of lymphoid tissue samples. Conclusions: The species‐specific PCR assay detected a higher number of Br. melitensis DNA both from serologically positive (P < 0·01 in blood PCR, P < 0·001 in tissue PCR) and serologically negative (P < 0·001 in both blood PCR and tissue PCR) sheep compared with classical bacteriological culture methods. Significance and Impact of the Study: The results emphasize the importance of using more than one type of diagnostic technique for the detection of animals positive for brucellosis, especially with epidemiological purposes.     

Culture and PCR analysis of joint fluid in the diagnosis of prosthetic joint infection

This prospective study compared PCR and culture techniques in the diagnosis of prosthetic joint infection (PJI). We obtained joint fluid samples (JFS; n=115) from patients who had failed total joint arthroplasty between January 2003 and June 2005; 49 were positive for PJI according to established strict criteria. JFS were analyzed by PCR (n=35; control n=66) or culture (n=46, control n=48). PCR was positive in 71% of PJI cases, resulting in sensitivity, specificity, accuracy, positive predictive value, negative predictive value, and likelihood ratio for positive results as follows: 0.71; 0.97; 0.88; 0.93; 0.87 and 23.6, respectively. Culture was positive in 44% of PJI samples. Corresponding statistics were 0.44; 0.94; 0.69; 0.87; 0.63 and 7.0, respectively. Significantly higher sensitivity, accuracy and negative predictive values were calculated for PCR versus culture, and there was 83% concordance between the results of intraoperative culture and PCR detection of causative bacteria. Therefore, we conclude that PCR analysis of synovial fluid increases the utility of pre-operative aspiration for patients who require revision total joint surgery.     

Detection and quantification of Leptospira interrogans in hamster and rat kidney samples: immunofluorescent imprints versus real-time PCR

A major limitation in the clinical management and experimental research of leptospirosis is the poor performance of the available methods for the direct detection of leptospires. In this study, we compared real-time PCR (qPCR), targeting the lipL32 gene, with the immunofluorescent imprint method (IM) for the detection and quantification of leptospires in kidney samples from the rat and hamster experimental models of leptospirosis. Using a virulent strain of Leptospira interrogans serovar Copenhageni, a chronic infection was established in the rat model, which were euthanized 28 days post-infection, while the hamster model simulated an acute infection and the hamsters were euthanized eight days after inoculation. Leptospires in the kidney samples were detected using culture isolation, qPCR and the IM, and quantified using qPCR and the IM. In both the acute and chronic infection models, the correlation between quantification by qPCR and the IM was found to be positive and statistically significant (P<0.05). Therefore, this study demonstrates that the IM is a viable alternative for not only the detection but also the quantification of leptospires, particularly when the use of qPCR is not feasible.     

Detection of the oyster herpesvirus in commercial bivalve in northern California, USA: conventional and quantitative PCR

The ostreid herpesvirus (OsHV-1) and related oyster herpesviruses (OsHV) are associated with world-wide mortalities of larval and juvenile bivalves. To quantify OsHV viral loads in mollusc tissues, we developed a SYBR Green quantitative PCR (qPCR) based on the A-region of the OsHV-1 genome. Reaction efficiency and precision were demonstrated using a plasmid standard curve. The analytical sensitivity is 1 copy per reaction. We collected Crassostrea gigas, C. sikamea, C. virginica, Ostrea edulis, O. lurida, Mytilus galloprovincialis, and Venerupis phillipinarum from Tomales Bay (TB), and C. gigas from Drakes Estero (DE), California, U.S.A., and initially used conventional PCR (cPCR) to test for presence of OsHV DNA. Subsequently, viral loads were quantified in selected samples of all tested bivalves except O. lurida. Copy numbers were low in each species tested but were significantly greater in C. gigas (p < 0.0001) compared to all other species, suggesting a higher level of infection. OsHV DNA was detected with cPCR and/or qPCR and confirmed by sequencing in C. gigas, C. sikamea, C. virginica, O. edulis, M. galloprovincialis, and V phillipinarum from TB and C. gigas from DE. These data indicate that multiple bivalve species may act as reservoirs for OsHV in TB. A lack of histological abnormalities in potential reservoirs requires alternative methods for their identification. Further investigation is needed to determine the host-parasite relationship for each potential reservoir, including characterization of viral loads and their relationship with infection (via in situ hybridization), assessments of mortality, and host responses.     

Detection of Salmonella Enteritidis in asymptomatic carrier animals: comparison of quantitative real-time PCR and bacteriological culture methods

Quantification of Salmonella in asymptomatic carrier animals can be used to assess microbial risk and monitor the level of contamination in domestic animals used for food production. We examined the sensitivity, specificity and accuracy of real-time qPCR, without pre-enrichment or selective enrichment stages, for the quantification of S. enterica serovar Enteritidis in resistant mice, as a model of asymptomatic carrier animal. The results were compared with those obtained by traditional bacteriological culture methods, the gold standard test. Two hundred and forty-three samples, including spleen, liver, mesenteric lymph nodes, portions of intestine, intestinal content of the ileocecal portion, and feces, were collected from a group of 27 C57BL/6 mice, that had been intragastrically inoculated with high doses of S. enterica serovar Enteritidis. The real-time qPCR assay presented a consistent linearity of the standard curve (r(2) = 0.999), with very low differences between melting temperatures, and low coefficients of variation in intra- (< 1%) and interassay (< 2%) comparisons. The primers were highly specific; there was no amplification with other Salmonella serovars or with DNA from uninfected tissues and feces from mice. The detection limit of the technique was defined as 32 copies of S. enterica serovar Enteritidis. A sensitivity of 90%, a specificity of 77% and an accuracy of 79% were obtained. The higher sensitivity of PCR was reflected in a kappa coefficient of 0.41, showing moderate agreement between tests. We conclude that real-time qPCR is a good alternative for diagnostic scanning in asymptomatic carrier animals, due to its high sensitivity and rapidity.     

Aseptic meningitis caused by Leptospira spp diagnosed by polymerase chain reaction

Leptospirosis is a zoonotic disease caused by the pathogenic Leptospira spp. The clinical presentations are diverse, ranging from undifferentiated fever to fulminant disease including meningeal forms. The neurological leptospirosis forms are usually neglected. The aim of this study was to investigate leptospirosis as the cause of aseptic meningitis using different diagnostic techniques including the polymerase chain reaction (PCR). Thirty-nine cerebrospinal fluid (CSF) samples from patients presenting with meningeal abnormalities, predominance of lymphocytes and negative results by traditional microbiological tests were processed by leptospiral culture, anti-leptospiral antibody response and PCR. Leptospira spp DNA was detected in 23 (58.97%) of the CSF samples. Anti-leptospiral antibodies were found in 13 (33.33%) CSF samples. Twelve CSF samples were positive by PCR assay and negative by microscopic agglutination test (MAT) assay. Two CSF samples were positive by MAT and negative by PCR. The positive and negative agreement between both tests was 11 and 14, respectively. CSF samples from six cases of unknown diagnosis were positive by PCR assay. Eight cases showed positive results using PCR and MAT. Leptospirosis could be detected by PCR assay from the 3rd-26th day after illness onset. The sensitivity of the PCR was assessed with confirmed cases of leptospirosis (by MAT) and found to be 89.5%. All CSFs were negative by culture. PCR was found to be a powerful tool for diagnosing meningitis cases of leptospirosis. We recommend that it may be used as a supplementary diagnostic tool, especially in the early stages of the disease, when other diagnostic techniques such as serology are not sensitive.     

A study on the determination of risk factors associated with babesiosis and prevalence of Babesia sp., by PCR amplification, in small ruminants from Southern Punjab (Pakistan)

Babesiosis is a parasitic infection due to the multiplication of tick borne parasite, Babesia sp., in erythrocytes of host, which includes a wide variety of vertebrates including small ruminants causing decreased livestock output and hence economic losses. The objective of the present study was to establish a PCR based method for the detection of Babesia sp. in small ruminant population in Southern Punjab and to determine the risk factors involve in the spread of babesiosis. A total of 107 blood samples were collected from 40 sheep and 67 goats in seven districts of Southern Punjab from randomly selected herds. Data on the characteristics of the animals and the herd were collected through questionnaires. 36 blood samples (34% of total) produced the DNA fragment specific for 18S rRNA gene of Babesia sp., by PCR amplification, of which 20 were sheep and 16 were goats. Samples from all seven district contained Babesia positive samples and prevalence varied between 18 to 68%. It was observed that male animals (P = 0.009) and young animals under one year of age (P = 0.01) were more prone to the parasite. It was observed that herds consist of more than 15 animals (P = 0.007), composed of mixed species of small ruminants (P = 0.022), associated with dogs (P = 0.003) and dogs having ticks on their bodies (P = 0.011) were among the major risk factors for the spread of babesiosis in small ruminants.     

Development and validation of multiplex real-time PCR assays for rapid detection of cytomegalovirus,Epstein-Barr virus,and polyomavirus BK in whole blood from transplant candidates

Transplant recipients are more susceptible to bacterial and viral infections. Cytomegalovirus (CMV), Epstein-Barr virus (EBV), and polyomavirus BK (BK) are risk factors for graft dysfunction. All three of them are latent viruses that can cause serious disease in immunocompromised patients. Mainly qualitative PCR tests are required for diagnosis and quantitative monitoring, which are used to follow the response to transplantation. We developed a multiplex real-time PCR (qPCR) method to detect these viruses during blood screenings of transplant recipients. We also validated analytical and clinical performance tests using the developed multiplex qPCR. The limit of detection (LOD) was 100, 125, and 183 copies/ml for CMV, EBV, and BK, respectively. These results had high linearity (R² = 0.997) and reproducibility (CV range, 0.95–2.38%, 0.52–3.32%, and 0.31–2.45%, respectively). Among 183 samples, we detected 8 samples that were positive for CMV, while only 6 were positive for EBV, and 3 were positive for BK. Therefore, the viral infection prevalence in transplant candidates was 4.40% for CMV, 3.29% for EBV, and 1.64% for BK. This multiplex qPCR method should be used widely for diagnosing and monitoring latent viral infections in transplant recipients.     

Nasal,Oral and Ear Swabs for Canine Visceral Leishmaniasis Diagnosis: New Practical Approaches for Detection of Leishmania infantum DNA

Background

The aim of this study was to evaluate the potential use of nasal, oral, and ear swabs for molecular diagnosis of canine visceral leishmaniasis (CVL) in an endemic urban area in Brazil.

Methodology/Principal Findings

Sixty-two naturally infected and ten healthy dogs were enrolled in this study. Bone marrow aspirates, peripheral blood, skin biopsy, and conjunctival, nasal, oral, and ear swabs were collected. All samples, except blood, were submitted to conventional PCR (cPCR) and quantitative real time PCR (qPCR) to detect and quantify Leishmania infantum DNA, respectively. All dogs were submitted to thorough clinical analysis and were included based on a combination of serological (ELISA immunoassay and immunofluorescent antibody test) and parasitological methods. The cPCR positivity obtained from nasal swab samples was 87% (54/62), equivalent to those from other samples (P>0.05). Positive results were obtained for 79% (22/28) in oral swabs and 43% (12/28) in ear swab samples. A significant difference was observed between these data (P = 0.013), and the frequency of positive results from oral swab was equivalent to those from other samples (P>0.05). The use of ear swab samples for cPCR assays is promising because its result was equivalent to skin biopsy data (P>0.05). The qPCR data revealed that parasite loads in mucosal tissues were similar (P>0.05), but significantly lower than the parasite burden observed in bone marrow and skin samples (P<0.05).

Conclusions

Nasal and oral swab samples showed a high potential for the qualitative molecular diagnosis of CVL because their results were equivalent to those observed in samples collected invasively. Considering that mucosae swab collections are painless, noninvasive, fast and practical, the combination of these samples would be useful in massive screening of dogs. This work highlights the potential of practical approaches for molecular diagnosis of CVL and human leishmaniasis infections.