Clinical performance of a quantitative pan-genus Leishmania Real-time PCR assay for diagnosis of cutaneous and visceral leishmaniasis.

Leishmaniasis is a complex vector-borne disease caused by various Leishmania species, affecting humans and animals. Current diagnostic methods have limitations, leading to potential misdiagnosis. Therefore, there is an urgent need for specific and sensitive diagnostic tools. We evaluated the sensitivity of a quantitative real-time PCR (qPCR) assay targeting the 18S gene in diverse clinical sample matrices. The assay showed a wide dynamic range and a limit of detection (LoD) of 1 parasite equivalent per milliliter (eq-p/mL) for all tested species. It exhibited high specificity for Leishmania DNA, with no amplification against other microorganisms. When applied to samples from patients with visceral and cutaneous leishmaniasis, the qPCR assay provided results that matched the reference methods and allowed estimation of parasite burdens. This assay holds promise for diagnosing and monitoring leishmaniasis by offering high sensitivity, specificity, and the ability to estimate parasitemia. Further studies are needed to enhance Leishmania molecular diagnostics and expand their coverage for improved clinical impact.

An overview of the trypanosomatid (Kinetoplastida: Trypanosomatidae) parasites infecting several mammal species in Colombia.

BACKGROUND: Trypanosomatids are among the most critical parasites for public health due to their impact on human, animal, and plant health. Diseases associated with these pathogens manifest mainly in poor and vulnerable populations, where social, environmental, and biological factors modulate the case incidence and geographical distribution. METHODS: We used Sanger and amplicon-based next-generation sequencing (NGS) in samples from different mammals to identify trypanosomatid infections in several departments in Colombia. A total of 174 DNA samples (18 humans, 83 dogs, and 73 wild mammals) were analyzed by conventional PCR using a fragment of the heat shock protein 70 (Hsp70) gene and Sanger sequenced the positive samples. Twenty-seven samples were sent for amplicon-based NGS using the same gene fragment. Data obtained were used to perform diversity analyses. RESULTS: One hundred and thirteen samples were positive for PCR by Hsp70 fragment; these corresponded to 22.1% Leishmania spp., 18.6% L. amazonensis, 9.7% L. braziliensis, 14.2% L. infantum, 8% L. panamensis, and 27.4% Trypanosoma cruzi. Comparison of the identified species by the two sequencing technologies used resulted in 97% concordance. Alpha and beta diversity indices were significant, mainly for dogs; there was an interesting index of coinfection events in the analyzed samples: different Leishmania species and the simultaneous presence of T. cruzi and even T. rangeli in one of the samples analyzed. Moreover, a low presence of L. braziliensis was observed in samples from wild mammals. Interestingly, to our knowledge, this is the first report of Leishmania detection in Hydrochaeris hydrochaeris (capybara) in Colombia. CONCLUSIONS: The Hsp70 fragment used in this study is an optimal molecular marker for trypanosomatid identification in many hosts and allows the identification of different species in the same sample when amplicon-based sequencing is used. However, the use of this fragment for molecular diagnosis through conventional PCR should be carefully interpreted because of this same capacity to identify several parasites. This point is of pivotal importance in highly endemic countries across South America because of the co-circulation of different genera from the Trypanosomatidae family. The findings show an interesting starting point for One Health approaches in which coevolution and vector-host interactions can be studied.

Amplicon-based next-generation sequencing reveals the co-existence of multiple Leishmania species in patients with visceral leishmaniasis.

Visceral leishmaniasis (VL) is a mammalian protozoal disease propagated in the Americas by female phlebotomine sandflies, mainly caused by Leishmania infantum. However, in recent years, cases of VL caused by different Leishmania species, such as L. amazonensis and L. colombiensis, have been reported in the continent. This study used an amplicon-based next-generation sequencing approach to identify VL aetiologic species using high-depth sequencing targeting a region on the Heat Shock Protein 70 gene. In this first approach, six samples from five patients diagnosed with VL were selected and analysed to identify DNA of Leishmania spp. All samples harboured DNA of L. infantum; five samples were found to be co-infected with other Leishmania spp. or with Trypanosoma cruzi, and just one sample was mono-infected with L. infantum. This study demonstrates the usefulness of this methodology to identify trypanosomatid co-infections in clinical samples, which presents an interesting study panorama considering their biological, clinical and epidemiological implications.

Development of an Amplicon-Based Next-Generation Sequencing Protocol to Identify Leishmania Species and Other Trypanosomatids in Leishmaniasis Endemic Areas.

Trypanosomatid infections are an important public health threat affecting many low-income countries across the tropics, particularly in the Americas. Trypanosomatids can infect many vertebrate, invertebrate, and plant species and play an important role as human pathogens. Among these clinically relevant pathogens are species from the genera Leishmania and Trypanosoma. Mixed trypanosomatid infections remain a largely unexplored phenomenon. Herein, we describe the application of an amplicon-based next-generation sequencing (NGS) assay to detect and identify trypanosomatid species in mammalian reservoirs, human patients, and sand fly vectors throughout regions of Leishmania endemicity. Sixty-five samples from different departments of Colombia, including two samples from Venezuela, were analyzed: 49 samples from cutaneous leishmaniasis (CL) patients, 8 from sand flies, 2 from domestic reservoirs (Canis familiaris), and 6 from wild reservoirs (Phyllostomus hastatus). DNA from each sample served to identify the presence of trypanosomatids through conventional PCR using heat shock protein 70 (HSP70) gene as the target. PCR products underwent sequencing by Sanger sequencing and NGS, and trypanosomatid species were identified by using BLASTn against a reference database built from trypanosomatid-derived HSP70 sequences. The alpha and beta diversity indexes of amplicon sequence variants were calculated for each group. The results revealed the presence of mixed infections with more than two Leishmania species in 34% of CL samples analyzed. Trypanosoma cruzi was identified in samples from wild reservoirs, as well as in sand fly vectors. Coinfection events with three different Leishmania species were identified in domestic reservoirs. These findings depose the traditional paradigm of leishmaniasis as being a single-species-driven infection and redraw the choreography of host-pathogen interaction in the context of multiparasitism. Further research is needed to decipher how coinfections may influence disease progression. This knowledge is key to developing an integrated approach for diagnosis and treatment. IMPORTANCE Traditionally, there has been a frequent, yet incorrect assumption that phlebotomine vectors, animal reservoirs, and human hosts are susceptible to Leishmania infection by a single parasite species. However, current evidence supports that these new vector-parasite-reservoir associations lend vectors and reservoirs greater permissiveness to certain Leishmania species, thus promoting the appearance of coinfection events, particularly in disease-endemic regions. The present study describes the application of an amplicon-based next-generation sequencing (NGS) assay to detect and identify trypanosomatid species in mammalian reservoirs, human patients, and sand fly vectors from regions of endemicity for leishmaniasis. This changes our understanding of the clinical course of leishmaniasis in areas of endemicity.

Spatial and Temporal Variability of Visceral Leishmaniasis in Colombia, 2007 to 2018.

Visceral leishmaniasis (VL) is a neglected tropical disease associated with poverty and is endemic in 56 countries worldwide. Brazil, Venezuela, and Colombia are the most affected countries in South America. In Colombia, the National Public Health Surveillance System (SIVIGILA) consolidates epidemiological information and monitors all VL cases nationwide. However, to date, no studies have investigated the occurrence of VL in Colombia using metadata analysis. We studied the demographic data, the spatial and temporal distribution of VL cases, and the association with vector distribution of Leishmania species in Colombia from 2007 to 2018. We found 306 VL cases reported to SIVIGILA for this period, with a coverage of 25.5 cases/year, and a mortality of 2.28% (seven deaths). The highest number of confirmed cases (N = 52) occurred in 2007; the lowest (N = 9) occurred in 2012. The cases were reported mainly in children (< 7 years) affiliated with the subsidized health regimen. Regarding the geographic distribution, the cases were reported by 42 municipalities distributed in 10 departments. The occurrence of VL cases toward the northeast of Colombia, and the distribution of vectors, such as Lutzomyia longipalpis and Lu. evansi, may be changing the panorama of VL in the country. We conclude that VL, mainly in recent years, shows a temporal and spatial variability associated with the occurrence of cases in new settings. Our findings increase our understanding and knowledge of this disease, and suggest the need to monitor and prioritize areas with changes in geographic expansion to improve prevention and control actions in the country.

Evaluation of four rapid diagnostic tests for canine and human visceral Leishmaniasis in Colombia.

BACKGROUND: Leishmaniasis caused by different species of Leishmania affect 98 countries worldwide. Visceral Leishmaniasis (VL) is the mortal clinical presentation of the disease that causes the dead to more than 90% of the patients who suffer it. The diagnosis of VL is made by the direct observation of the parasite in bone marrow, spleen and/or liver aspirates that requires complex proceedings. Therefore, serum samples are submitted to Indirect Immunofluorescence to identify the presence of anti-Leishmania antibodies. Despite the variability in the diagnostic performance of the Immunochromatographic Tests (ICTs), there are many evidences that suggest that ICTs can be used for epidemiological screening. However, in Colombia there are not any evidence about the performance of the ICTs for VL diagnosis, both for human and canine serum samples. Therefore, this study evaluated the diagnostic performance of 4 ICTs for VL (2 ICTs in human sera and 2 ICTs in canine sera) in samples from endemic areas of Colombia. METHODS: We selected a total of 156 human serum samples (82 positive and 74 negative for VL) and 126 canine serum samples (71 positive and 54 negative) diagnosed by in house Indirect Immunofluorescence (IIF). The samples were submitted to the ICTs following the manufacturers' instructions. Statistical analysis was performed to evaluate the diagnostic performance of each ICT in comparison with the IIF. PCR for HSP70 gene and sanger sequencing was performed in samples with negative results for both ICTs. RESULTS: The sensitivity (S) of both ICTs for human samples (Ad-bio Leishmania IgG/IgM Combo Rapid Test and Kalazar Detect™) was 91.5% and specificity (E) were 93.2 and 89.2% respectively, while for the ICTs tested on canine samples (Kalazar Detect™ Rapid Test, Canine and DPP® CVL rapid test) we found S values between 82.9 and 85.7% and E values between 79.6 and 92.6%. We found L. infantum by PCR and sequencing in 2 human samples, and L. braziliensis and L. amazonensis in canine serum samples that were negative by both ICTs. CONCLUSIONS: We conclude that both tests evaluated on human samples have a similar diagnostic performance, while the Kalazar Detect™ Rapid Test, Canine showed a better diagnostic performance than the DPP® CVL rapid test evaluated on canine samples. Also, we suggest that it is necessary to design tests with antigens of the circulating strains to increase its diagnostic utility.

Description of Leishmania species among dogs and humans in Colombian Visceral Leishmaniasis outbreaks.

We report the species detected in dogs and humans from outbreaks of visceral leishmaniasis in Colombia. In this study, 91 sera from patients (n = 38) and dogs (n = 53) diagnosed with visceral leishmaniasis using IFAT were analyzed to determine the causative species. DNA extraction, PCR amplification, DNA sequencing and species identification was performed. Results were obtained with 13 of the sera. A phylogenetic tree and a network of haplotypes were constructed. Leishmania infantum chagasi (11/13), Leishmania braziliensis (1/13) and Leishmania amazonensis (1/13) were identified as the circulating species and genetic variability in one of the L. infantum chagasi strains was demonstrated. This is the first study describing Leishmania species in outbreaks of visceral leishmaniasis in Colombia.

Analytical Performance of a Loop-Mediated Isothermal Amplification Assay for Leishmania DNA Detection in Sandflies and Direct Smears of Patients with Cutaneous Leishmaniasis.

Loop-mediated isothermal amplification (LAMP) is ideal for the detection of Leishmania DNA as it is a quick and easy-to-perform test that does not require complex or sophisticated equipment or infrastructure. However, the application of this technique in the detection of Leishmania DNA has not been comprehensively analyzed to date (analytical validation). Our objective was to evaluate the sensitivity and analytical specificity (anticipated reportable range [ARR], the limit of detection [LoD], and accuracy) of LAMP targeting the 18S rRNA gene in the diagnosis of six New World Leishmania species. We then applied the validated LAMP assay across 50 samples of sandflies and 50 direct smears from a recent outbreak of cutaneous leishmaniasis in Colombia to determine its diagnostic performance. The LAMP assay exclusively amplified the DNA of Leishmania spp., and an ARR of between 1 × 104 and 1 × 10-2 equivalent parasites/mL was determined. An LoD of 1 × 10-2 equivalent parasites/mL was established and there was no statistically significant variation in terms of accuracy. Finally, a sensitivity of 100% in direct smears and sandflies samples was calculated and a specificity of 90.9% for direct smears using microscopy as reference and 96.8% for sandflies using real-time polymerase chain reaction as reference were determined. To our knowledge, this is the first attempt to analytically validate a LAMP test to detect Leishmania DNA, which showed good diagnostic potential from sandflies and direct smear samples.

Analytical Performance of Four Polymerase Chain Reaction (PCR) and Real Time PCR (qPCR) Assays for the Detection of Six Leishmania Species DNA in Colombia.

Leishmaniasis comprises a spectrum of parasitic diseases caused by protozoans of the genus Leishmania. Molecular tools have been widely employed for the detection of Leishmania due to its high sensitivity and specificity. However, the analytical performance of molecular platforms as PCR and real time PCR (qPCR) including a wide variety of molecular markers has never been evaluated. Herein, the aim was to evaluate the analytical performance of 4 PCR-based assays (designed on four different targets) and applied on conventional and real-time PCR platforms. We evaluated the analytical performance of conventional PCR and real time PCR, determining exclusivity and inclusivity, Anticipated Reportable Range (ARR), limit of detection (LoD) and accuracy using primers directed to kDNA, HSP70, 18S and ITS-1 targets. We observed that the kDNA was the most sensitive but does not meet the criterion of exclusivity. The HSP70 presented a higher LoD in conventional PCR and qPCR in comparison with the other markers (1 × 101 and 1 × 10-1 equivalent parasites/mL respectively) and had a higher coefficient of variation in qPCR. No statistically significant differences were found between the days of the test with the four molecular markers. The present study revealed that the 18S marker presented the best performance in terms of analytical sensitivity and specificity for the qPCR in the species tested (species circulating in Colombia). Therefore, we recommend to explore the analytical and diagnostic performance in future studies using a broader number of species across America.

Evaluation of a Multilocus Sequence Typing (MLST) scheme for Leishmania (Viannia) braziliensis and Leishmania (Viannia) panamensis in Colombia.

BACKGROUND: Leishmaniases are parasitic vector-borne diseases affecting more than 12 million people in 98 countries. In Colombia, leishmaniasis is widespread and the most common clinical manifestation is cutaneous, mainly caused by L. panamensis and L. braziliensis. Currently, the genetic diversity of these species in Colombia is unknown. To address this, we applied molecular techniques for their characterization, using multilocus sequence typing (MLST) to explore the genetic variability and phylodynamics of the disease. METHODS: Seven previously described genetic markers were selected highlighting the implementation of a mitochondrial marker. Markers were applied to 163 samples from isolates obtained between 1980 and 2001. RESULTS: The identification of the samples showed an excellent correlation with typing tests previously applied (MLEE, monoclonal antibodies). Isolates of L. braziliensis showed greater genetic diversity than L. panamensis, and a greater number of diploid sequence types (DSTs). In addition, the geographical distribution of DSTs for each species were obtained through georeferencing maps. CONCLUSIONS: To our knowldge, this study represents the first description of the genetic variability of L. panamensis in Colombia and South America, and is the first to propose a scheme of MLST for epidemiological surveillance of leishmaniasis in the country.

Taxonomy, diversity, temporal and geographical distribution of Cutaneous Leishmaniasis in Colombia: A retrospective study.

Leishmaniases are tropical zoonotic diseases, caused by kinetoplastid parasites from the genus Leishmania. New World (NW) species are related to sylvatic cycles although urbanization processes have been reported in some South American Countries such as Colombia. Currently, few studies show the relative distribution of Leishmania species related to cutaneous Leishmaniasis (CL) in South America due to the lack of accurate surveillance and public health systems. Herein, we conducted a systematic estimation of the Leishmania species causing CL in Colombia from 1980 to 2001 via molecular typing and isoenzymes. A total of 327 Leishmania isolates from humans, sandflies and reservoirs were typed as L. panamensis 61.3% (201), L. braziliensis 27.1% (88), L. lainsoni 0.6% (2), L. guyanensis 0.9% (3), L. infantum chagasi 4% (12), L. equatoriensis 0.6% (2), L. mexicana 2.1% (8), L. amazonensis 2.8% (9) and L. colombiensis 0.6% (2). This is the first report of two new Leishmania species circulating in Colombia and suggests the need to convince the Colombian government about the need to deploy and standardize tools for the species identification to provide adequate management to individuals suffering this pathology.