Carolina Flórez

Geographic distribution of human Blastocystis subtypes in South America.

Blastocystis is a cosmopolitan enteric protist colonizing probably more than 1 billion people. This protozoan exhibits genetic diversity and is subdivided into subtypes (STs). The aim of this study was to determine the distribution of Blastocystis STs in symptomatic and asymptomatic human samples from different countries of South America. A total of 346 fecal samples were genotyped by SSU rDNA showing ST1 (28.3%), ST2 (22.2%), ST3 (36.7%), ST4 (2%), ST5 (2.3%), ST6 (2%), ST7 (2.3%), ST8 (0.6%), ST12 (0.9%) and a novel ST (2.7%). These findings update the epidemiology of Blastocystis in South America and expand our knowledge of the phylogeographic differences exhibited by this stramenopile.

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.

High-Resolution Molecular Typing of Trypanosoma cruzi in 2 Large Outbreaks of Acute Chagas Disease in Colombia

Oral transmission of Trypanosoma cruzi has gained relevance because of its association with high morbidity and lethality rates. This transmission route is responsible for maintaining the infection of the parasite in sylvatic cycles, and human cases have been associated mainly with the consumption of food contaminated with triatomine feces or didelphid secretions. Several ecological changes allow the intrusion of sylvatic reservoirs and triatomines to the domestic environments with subsequent food contamination. Here, high-resolution molecular tools were used to detect and genotype T. cruzi across humans, reservoirs, and insect vectors in 2 acute outbreaks of presumptive oral transmission in eastern Colombia.

Blastocystis subtyping and its association with intestinal parasites in children from different geographical regions of Colombia

Blastocystis is a common enteric protist colonizing probably more than 1 billion people with a large variety of non-human hosts. Remarkable genetic diversity has been observed, leading to the subdivision of the genus into multiple subtypes (ST), some of which are exclusively found in non-human hosts. The aim of this study was to determine the distribution of Blastocystis STs/18S alleles in symptomatic (abdominal pain, anal pruritus, diarrhea, headache, nauseas and/or vomit) and asymptomatic children from nine geographical regions of Colombia. A total of 2026 fecal samples were collected as part of a national survey to estimate the frequency of intestinal parasites in children. A set of 256 samples that were Blastocystis positive was finally selected. The samples were submitted to DNA extraction, Real Time PCR and sequencing using Blastocystis-specific primers targeting the small subunit rRNA gene for ST identification. DNA of Ascaris lumbricoides (16.4%), Trichuris trichiura (8.2%), hookworms (Necator americanus/Ancylostoma duodenale) (7.3%), Giardia duodenalis (23.1%), Entamoeba complex (82%), Entamoeba coli (55%), Hymenolepis nana (0.8%), Endolimax nana (33.2%) and Neobalantidium coli (2.7%) was detected in the Blastocystis-positive samples. We detected ST1 (21.4%), ST2 (19.5%), ST3 (55.5%), ST4 (0.8%), ST6 (2%) and ST7 (0.8%); alleles 1, 2, 4, 81, 82 and 83 for ST1; alleles 9, 11, 12, 15, 67, 71 and 73 for ST2; alleles 34, 36, 38, 45, 49, 55, 134 and 128 for ST3; allele 42 for ST4; allele 122 for ST6, and allele 142 for ST7. Further studies implementing high-resolution molecular markers are necessary to understand the dynamics of Blastocystis transmission and the role of this Stramenopila in health and disease.

Comparative study of the biological properties of Trypanosoma cruzi I genotypes in a murine experimental model

Chagas disease is an endemic zoonosis in Latin America and caused by the parasite Trypanosoma cruzi. This kinetoplastid displays remarkable genetic variability, allowing its classification into six Discrete Typing Units (DTUs) from TcI to TcVI. T. cruzi I presents the broadest geographical distribution in the continent and has been associated to severe forms of cardiomyopathies. Recently, a particular genotype associated to human infections has been reported and named as TcIDOM (previously named TcIa-b). This genotype shows to be clonal and adapted to the domestic cycle but so far no studies have determined the biological properties of domestic (TcIDOM) and sylvatic TcI strains (previously named TcIc-e). Hence, the aim of this study was to untangle the biological features of these genotypes in murine models. We infected ICR-CD1 mice with five TcI strains (two domestic, two sylvatic and one natural mixture) and determined the course of infection during 91 days (acute and chronic phase of the disease) in terms of parasitemia, tissue tropism, immune response (IgG titers) and tissue invasion by means of histopathology studies. Statistically significant differences were observed in terms of parasitemia curves and prepatent period between domestic (TcIDOM) and sylvatic strains. There were no differences in terms of IgG antibodies response across the mice infected with the five strains. Regarding the histopathology, our results indicate that domestic strains present higher parasitemias and low levels of histopathological damage. In contrast, sylvatic strains showed lower parasitemias and high levels of histopathological damage. These results highlight the sympatric and behavioral differences of domestic and sylvatic TcI strains; the clinical and epidemiological implications are herein discussed.

Risk factors for treatment interruption and severe adverse effects to benznidazole in adult patients with Chagas disease

Background Etiological treatment of Chagas disease in chronic asymptomatic patients is still in debate and the adverse effects of traditional drugs are one of the main concerns in clinical practice. This study evaluated retrospectively the safety profile of benznidazole (BZN) and identified predictive factors for definite treatment interruption and development of severe reactions in adult patients treated with BZN in Colombia. Methods Retrospective follow-up study conducted by review of medical records of adults with chronic Chagas disease treated with BZN in Colombia. A parametric survival analysis based on a generalized gamma distribution was used for assessing risk factors for treatment interruption. A multinomial logistic regression model was used to estimate the probability of severe adverse drug reactions (ADRs). Statistical associations were expressed as time ratios (TR) and adjusted odds ratios (aOR) respectively. Results In total 224 adults patients treated with BZN were included; 172 (76.8%) completed the standard therapy (60 days of treatment), 205 (91.5%) presented ADRs and 52 cases (23.2%) required treatment interruption. The predominant symptoms were: rash (37.9%), itching (33.7%), epigastric pain (26.4%), abdominal bloating (24.2%) and nausea (22.1%). ADRs were mild (57.4%), moderate (35.5%) and severe (7.3%). Time to treatment interruption was significantly shorter when using doses of BZN ≥ 6 mg/kg/day (TR 0.55; 95% CI 0.39–0.76), presenting severe ADRs (TR 0.12; 95% CI: 0.07–0.19) and eosinophilia (TR 0.68; 95% CI: 0.49–0.94). Female sex (aOR 3.98; 95% CI 1.56–10.16), dose of BZN ≥ 6 mg/kg/day (aOR 1.41; 95% CI 1.17–1.70) and presence of > 3 ADRs (aOR 6.47; 95% CI 1.24–34.34) were considered as risk factors for developing severe ADRs. Conclusions Dose, severity of ADRs, eosinophilia and female sex were the main predictors for treatment interruption or severe ADRs. The potential implications of these findings are discussed.

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.

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.