Martha S. Ayala

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.

Identification of Six New World Leishmania species through the implementation of a High-Resolution Melting (HRM) genotyping assay

BackgroundLeishmaniases are tropical zoonotic diseases, caused by 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. This eco-epidemiological complexity imposes a challenge to the detection of circulating parasite species, not only related to human cases but also infecting vectors and reservoirs. Currently, no harmonized methods have been deployed to discriminate the NW Leishmania species.FindingsHerein, we conducted a systematic and mechanistic High-Resolution Melting (HRM) assay targeted to HSP70 and ITS1. Specific primers were designed that coupled with a HRM analyses permitted to discriminate six NW Leishmania species. In order to validate the herein described algorithm, we included 35 natural isolates obtained from human cases, insect vectors and mammals. Our genotyping assay allowed the correct assignment of the six NW Leishmania species (L. mexicana, L. infantum (chagasi), L. amazonensis, L. panamensis, L. guyanensis and L. braziliensis) based on reference strains. When the algorithm was applied to a set of well-characterized strains by means of PCR-RFLP, MLEE and monoclonal antibodies (MA) we observed a tailored concordance between the HRM and PCR-RFLP/MLEE/MA (KI = 1.0). Additionally, we tested the limit of detection for the HRM method showing that this is able to detect at least 10 equivalent-parasites per mL.ConclusionsThis is a rapid and reliable method to conduct molecular epidemiology and host-parasite association studies in endemic areas.

Experimental infection of Leishmania (L.) chagasi in a cell line derived from Lutzomyia longipalpis (Diptera:Psychodidae)

The present work describes the in vitro infection of a cell line Lulo, derived from Lutzomyia longipalpis embryonic tissue, by Leishmania chagasi promastigotes. This infection process is compared with a parallel one developed using the J774 cell line. The L. chagasi MH/CO/84/CI-044B strain was used for experimental infection in two cell lines. The cells were seeded on glass coverslips in 24-well plates to reach a final number of 2 x 10(5) cells/well. Parasites were added to the adhered Lulo and J774 cells in a 10:1 ratio and were incubated at 28 and 37 masculineC respectively. After 2, 4, 6, 8, and 10 days post-infection, the cells were extensively washed with PBS, fixed with methanol, and stained with Giemsa. The number of internalized parasites was determined by counting at least 400 cultured cells on each coverslip. The results showed continuous interaction between L. chagasi promastigotes with the cell lines. Some ultrastructural characteristics of the amastigote forms were observed using transmission electron microscopy. The highest percentage of infection in Lulo cells was registered on day 6 post-infection (29.6%) and on day 4 in the J774 cells (51%). This work shows similarities and differences in the L. chagasi experimental infection process in the two cell lines. However, Lulo cells emerge as a new model to study the life-cycle of this parasite.

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

BackgroundLeishmaniases 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.MethodsSeven 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.ResultsThe 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.ConclusionsTo 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.

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.

Orally transmitted acute Chagas disease in domestic travelers in Colombia

Chagas disease still represents a significant threat in endemic, but also in non-endemic areas due to travel and migration [1,2]. In this setting, during last decade, oral transmission and its associated acute forms, have been recognized as significant concerns in endemic areas, where this would lead to severe life-threatening and even fatal consequences [3,4]. Colombia has been one of the Latin American countries with a considerable number of acute Chagas disease outbreaks where oral transmission of Trypanosoma cruzi has been evidenced [5]. Nevertheless, its diagnosis, in the setting of multiple endemic tropical infectious diseases, such as tuberculosis, malaria, dengue, chikungunya, Zika, among many other, even in coinfections [6,7], represents a real challenge. In those patients with history of travel to endemic areas of Chagas disease, there should be a high suspicion of this disease, which should be included in the differential diagnosis. Here, we report two cases, in which patients traveled to the department of Casanare (Fig. 1A), where between March and April 2014, an outbreak of oral Chagas disease was reported locally. Case 1. A 33-year-old male, coming from Paz de Ariporo and Gaitán, Casanare, Colombia (Fig. 1), presenting fever, headache, arthralgia, myalgia, bilateral palpebral edema and exanthema with 20 days of evolution was received at our institution in Sincelejo, Sucre (Fig. 1A) (Clínica Santa María). At physical examination patient presented cervical lymphadenopathy, splitted first heart sound, with pericardial rub, the liver was enlarged, being palpable at 2 cm below the subcostal margin. Also presented a distended abdomen with skin rash. An X-ray and CT-scan revealed cardiomegaly and pericarditis. At the ECG an AV first degree block was evidenced. A blood smear revealed the presence of circulating trypomastigotes of Trypanosoma cruzi. In addition, an ELISA p t t

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.

Comparación entre metodologías para el diagnóstico microscópico de malaria

Introduction: As part of the pre-elimination plan for malaria in Colombia, it has been proposed to develop activities within the line of work: “Improve access and quality of malaria diagnosis”. Objective: To compare the methodology recommended by PAHO/WHO with that used in Colombia for the diagnosis of malaria. Materials and methods: Samples were collected and 88 slides were prepared for malaria diagnosis, under different scenarios according to the parameters to be evaluated. After duplicate mycroscopic reading, the respective variance calculations were performed for all possible staining comparisons with the two methods used (thick smear, combined thick smear), according to the staining (modified Romanowsky or Giemsa), with the result variable being the parasite density (500, 1,000, 5,000 and 10,000 parasites/μl of blood). Results: A Cohen kappa index of inter-rater agreement of 0.923 (95% CI: 0.768-1.078) was obtained. None of the factors (A: stain, B: methodology) or interactions (AB) had a statistically significant effect on the results with a 95% confidence level. Conclusion: Based on the results of the study, the preparation of two thick smears in the same slide stained with the modified Romanowsky stain is a suitable methodology for the diagnosis of malaria in Colombia, due to its technical characteristics, of storage, low cost, use and care.Introduction: As part of the pre-elimination plan for malaria in Colombia, it has been proposed to develop activities within the line of work: “Improve access and quality of malaria diagnosis”. Objective: To compare the methodology recommended by PAHO/WHO with that used in Colombia for the diagnosis of malaria. Materials and methods: Samples were collected and 88 slides were prepared for malaria diagnosis, under different scenarios according to the parameters to be evaluated. After duplicate mycroscopic reading, the respective variance calculations were performed for all possible staining comparisons with the two methods used (thick smear, combined thick smear), according to the staining (modified Romanowsky or Giemsa), with the result variable being the parasite density (500, 1,000, 5,000 and 10,000 parasites/μl of blood). Results: A Cohen kappa index of inter-rater agreement of 0.923 (95% CI: 0.768-1.078) was obtained. None of the factors (A: stain, B: methodology) or interactions (AB) had a statistically significant effect on the results with a 95% confidence level. Conclusion: Based on the results of the study, the preparation of two thick smears in the same slide stained with the modified Romanowsky stain is a suitable methodology for the diagnosis of malaria in Colombia, due to its technical characteristics, of storage, low cost, use and care.

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.