Involvement of the Inflammasome and Th17 Cells in Skin Lesions of Human Cutaneous Leishmaniasis Caused by Leishmania (Viannia) panamensis.

Localized cutaneous leishmaniasis (LCL) caused by Leishmania (Viannia) panamensis is an endemic disease in Panama. This condition causes ulcerated skin lesions characterized by a mixed Th1/Th2 immune response that is responsible for disease pathology. However, the maintenance of the in situ inflammatory process involves other elements, such as Th17 and inflammasome responses. Although these processes are associated with parasite elimination, their role in the increase in disease pathology cannot be discarded. Thus, the role in Leishmania infection is still unclear. In this sense, the present study aimed at characterizing the Th17 and inflammasome responses in the skin lesions of patients with LCL caused by L. (V.) panamensis to help elucidate the pathogenesis of this disease in Panama. Th17 and inflammasome responses were evaluated by immunohistochemistry (IHQ) in 46 skin biopsies from patients with LCL caused by L. (V.) panamensis. The Th17 immune response was assessed using CD3, CD4, RoRγt, IL-17, IL-6, IL-23, and TGF-ß1 antibodies, and the inflammasome response was assessed by IL-1ß, IL-18, and caspase-1 antibodies. The presence of the Th17 and inflammasome responses was evidenced by a positive reaction for all immunological markers in the skin lesions. An inverse correlation between the density of amastigotes and the density of RoRγt+, IL-17+, IL-1ß +, and caspase-1+ cells was observed, but no correlation between Th17 and the inflammasome response with evolutionary disease pathology was reported. These data showed the participation of Th17 cells and the inflammasome in the inflammatory response of the skin lesions of LCL caused by L. (V.) panamensis infection. These results suggest a role in the control of tissue parasitism of IL-17 and the activation of the NLRP3 inflammasome dependent on IL-1ß but cannot exclude their role in the development of disease pathology.

Diversity, Co-Occurrence, and Nestedness Patterns of Sand Fly Species (Diptera: Psychodidae) in Two Rural Areas of Western Panamá.

Cutaneous Leishmaniasis transmission in the New World is observed in areas with rich sand fly species' faunas. The diversity and composition of sand fly species can change in response to seasonal weather and land use changes. Here, we present results from a two-year-long study where we collected, using Centers for Disease Control (CDC) light traps, sand flies from two rural areas, Las Pavas (LP) and Trinidad de las Minas (T) in western Panamá. Over 710 trap-nights, we collected 16,156 sand flies from 15 genera and 35 species. We identified 34 species in T, and the most abundant species collected was Nyssomyia trapidoi (Fairchild and Hertig, 1952) (n = 2278, 37%), followed by Psychodopygus panamensis (Shannon, 1926) (n = 1112, 18%), and Trichopygomyia triramula (Fairchild and Hertig, 1952) (n = 1063, 17%). In LP, we identified 26 species, and the most abundant species collected were Ty. triramula (n = 4729, 48%), and Ps. panamensis (n = 3444, 35%). We estimated a higher species' richness in T (Chao2 ± S.E.: 36.58 ± 3.84) than in LP (27.49 ± 2.28). In T, species' richness was significantly higher in the rainy season, but no seasonal differences were observed in LP. Species' assemblages were nested in the two areas. Phlebotomine sand fly species' abundance increased at the two sites during the rainy season. Our data suggest that seasonality is more important than land use as a factor driving sand fly species' diversity at the studied sites.

Lack of association between IL-6-174G/C gene polymorphism and Chagas disease.

The aim of this study was to investigate the role of the IL-6-174G/C gene polymorphism in susceptibility/resistance to Trypanosoma cruzi infection in two independent cohorts from Colombia and Peru. We determined the IL-6-174G/C genotypes in a sample of 399 seronegative individuals and 317 serologically positive patients from Colombia and Peru. All individuals are from regions where T. cruzi infection is endemic. No statistically significant differences in the frequency of IL-6-174G/C gene polymorphism between chagasic patients and controls or between asymptomatic and individuals with cardiomyopathy were observed. Our results do not support an evidence for a major role contribution of this IL-6 gene polymorphism in the susceptibility to or clinical manifestations of Chagas disease in these studied cohorts.

Prevalence and subtype distribution of Blastocystis sp. infecting children from a rural community in Panama.

Blastocystis sp. is a commonly reported intestinal parasite with a worldwide distribution. Phylogenetic analyses describe at least 17 subtypes for this parasite, and nine of them have been found in humans. However, the prevalence and some epidemiological characteristics of this parasitic infection in rural communities are not well known. The objective of this cross-sectional study was to evaluate the prevalence, subtypes, and epidemiological factors related to Blastocystis sp. Infection in children from of a small rural community in the central area of Panama. For this, 66 fecal samples from children (1 to 12 years old), were initially analyzed for the presence of parasites by a formalin-ethyl acetate/concentration method. Molecular detection and identification of Blastocystis sp. subtypes were carried out by amplification and sequencing of a partial fragment of the small-subunit ribosomal RNA gene. Using data from a questionnaire, analyses of epidemiological conditions potentially associated with Blastocystis sp. transmission were also conducted. Microscopic diagnostics showed that 33.3% (22/66) of the analyzed samples presented entero-parasites. Among them, Blastocystis sp. was the most prevalent, with 21.2% (14/66), followed by the E. histolytica/dispar/moshkovskii complex 4.5% (3/66), Giardia lamblia 1.5% (1/66) and Strongyloides stercoralis 1.5% (1/66). PCR-based analyses detected a prevalence of Blastocystis sp. infection of 74.2% (49/66) in apparently healthy children. Phylogenetic analysis revealed two different subtypes of this parasite: ST1 with 42.2% (28/66) infected, and ST3 with 31.8% (21/66) infected. In addition, recent diarrhea was significantly associated with Blastocystis sp. infection. None of the other risk factors evaluated was statistically associated with infection. These results highlight the need to further investigate clinical, epidemiological, and genetic characteristics of Blastocystis sp. infections in this community.

Performance of immunohistochemistry as a useful tool for the diagnosis of cutaneous leishmaniasis in Panama, Central America.

Cutaneous leishmaniasis (CL) is one of the most frequent parasitic zoonoses in Panama. Currently, conventional, molecular and histopathological tests are performed to diagnose CL. Immunohistochemistry (IHC) has proven to be a valuable tool to facilitate the diagnosis of leishmaniasis and to study the cellular immune response developed during the infection. Therefore, considering the absence of IHC in the diagnostic routine in Panama, the objective of this study is to demonstrate the usefulness of this test as a complementary diagnostic tool for improving the sensitivity of histopathology (HP) and helping to study the cellular immune response of patients. Samples from patients with suspected CL were analysed by intradermal reaction of Montenegro (IDRM), smears, culture, PCR (Viannia, Hsp-70), HP and IHC. According to the diagnostic criteria, 95.8% of patients were positive for Leishmania sp., that was characterized as Leishmania (V.) panamensis by PCR-HSP70/RFLP. From positive samples, Leishmania was detected by the tested diagnostic methods in the following degrees: 100% by IDRM, 60% by smears, 93.3% by culture, 100% by kDNA PCR, 78.3% by PCR Hsp-70, 50% by HP and 73.9% by IHC. Although IHC had a poor correlation (k = 0.191) with the diagnostic criteria, the sensitivities of both HP (76.1%) and smears (89.1%) were improved by combining them with IHC. IHC considerably improved the detection of the Leishmania parasites in the histopathological sections, supporting the need to implement this diagnostic tool in Panama. In addition, immunohistochemistry allows evaluation of the patient's immune response and thus provides new guidelines for the treatment and control of CL in Panama.

Evaluation of PCR for cutaneous leishmaniasis diagnosis and species identification using filter paper samples in Panama, Central America.

Cutaneous leishmaniasis (CL) is a major vectorborne disease in Panama. In this study, the diagnostic performance and usefulness of two DNA extraction procedures from skin scraping samples collected on FTA filter paper for subsequent PCR diagnosis of CL was evaluated. A positive CL laboratory diagnosis was based on a positive parasitological test (Giemsa-stained smears or in vitro culture) and/or positive PCR test performed from skin scrapings collected in TE buffer (PCR-TE). Of 100 patients with skin lesions suggestive of CL, 82 (82%) were confirmed as CL positive. The sensitivity was calculated for each of the PCR approaches from samples collected on filter paper. The highest sensitivity was achieved by PCR-FTA processed by Chelex 100 (PCR-Chelex) (0.94). PCR-FTA extracted using the FTA purification reagent presented a lower sensitivity (0.60). Good concordance between routine PCR-TE and PCR-Chelex was observed (percent agreement=0.88, κ index=0.65). In conclusion, use of FTA filter paper for skin scraping collection combined with PCR is a reliable and convenient method for CL diagnosis in Panama, with comparable performance to the routine PCR method and with improved sensitivity compared with those of conventional parasitological methods.

Prevalence of trypanosome infections in dogs from Chagas disease endemic regions in Panama, Central America.

The prevalence of canine trypanosomosis was investigated in two Chagas disease endemic rural communities located in the central region of Panama. Serologic tests for Trypanosoma cruzi infection revealed a prevalence of 11.1%. Hemocultures coupled with PCR analysis demonstrated a Trypanosoma rangeli infection rate of 5.1%. An overall trypanosome infection index of 16.2% (16/99) was detected in this canine population. One dog had a mixed infection of T. cruzi and T. rangeli. Six of the trypanosome-infected dogs belong to people who were diagnosed of Chagas disease. We conclude that dogs from this rural area of Panama are frequently infected with trypanosomes transmitted by the sylvatic vector, Rhodnius pallescens, and suggest that dogs are important in the peridomestic transmission cycle of trypanosomes as reservoirs and hosts. The epidemiological implications of these findings are discussed.

Chemokine receptor CCR5 polymorphisms and Chagas' disease cardiomyopathy.

In this study we investigated the possible role of two CCR5 gene polymorphisms, CCR5Delta32 deletion and CCR5 59029 A-->G promoter point mutation, in determining the susceptibility to Trypanosoma cruzi infection as well as in the development of chagasic heart disease. These CCR5 polymorphisms were assessed in 85 seropositive (asymptomatic, n=53; cardiomyopathic, n=32) and 87 seronegative individuals. The extremely low frequency (0.009) of the CCR5Delta32 allele in our population did not allow us to analyse its possible influence on T. cruzi infection. We found no differences in the distribution of CCR5 59029 promoter genotype or phenotype frequencies between total chagasic patients and controls. However, we observed that the CCR5 59029-A/G genotype was significantly increased in asymptomatic with respect to cardiomyopathic patients (P=0.02; OR=0.33, 95% CI 0.10-0.94). In addition, the presence of the CCR5 59029-G allele was also increased in asymptomatics when compared with cardiomyopathics (P=0.02; OR=0.35, 95% CI 0.12-0.96). Our data suggest that the CCR5 59029 promoter polymorphism may be involved in a differential susceptibility to chagasic cardiomyopathy.

Lack of association between NRAMP1 gene polymorphisms and Trypanosoma cruzi infection.

Genetic analysis in mice and humans have established the key role of the human natural resistance-associated macrophage protein 1 (NRAMP1) in resistance to intracellular infections. In the present study we investigated whether four NRAMP1 polymorphisms (5'(GT)n, -236 C-->T, D543N, and 3'UTR deletion) were important in determining the susceptibility to Trypanosoma cruzi infections as well as in the development of chagasic cardiac disease. Genotyping for these variants was assessed in 83 seropositive (asymptomatic, n=51, cardiomyopathic, n=32) and 85 seronegative individuals from a Peruvian population where T. cruzi is endemic. No statistically significant differences either between patients and controls or between asymptomatic and cardiomyopathic individuals were observed with respect to NRAMP1 variants. Our data suggest that the NRAMP1 genetic polymorphism analysed do not play a major role in the pathogenesis of T. cruzi infection in this Peruvian sample.

No evidence for association of the inducible nitric oxide synthase promoter polymorphism with Trypanosoma cruzi infection.

The purpose of the present study was to address the possible contribution of the (CCTTT)n microsatellite polymorphism in the NOS2 promoter region to the susceptibility to chronic Trypanosoma cruzi infection and to Chagas' disease related cardiomyopathy. We determined the (CCTTT)n genotypes in a sample of 76 serologically positive chagasic individuals and in 78 healthy controls. No statistically significant differences were observed between total chagasic patients and healthy controls with regard to frequency of the (CCTTT)n microsatellite repeat of any given length. Likewise, we found no differences in the distribution of the (CCTTT)n microsatellite repeats between seropositives without manifestations of the disease and those with chagasic cardiomyopathy. Our data suggest that the NOS2 promoter pentanucleotide microsatellite polymorphisms analyzed do not play a major role in the pathogenesis of chronic T. cruzi infection in this Peruvian sample.

HLA class I and class II allele distribution in the Peruvian population.

The distribution of HLA-A, -B, -C, -DRB1 and -DQB1 alleles in the Peruvian population was studied and compared with those of other populations in order to provide further information about their anthropological origin. Our data are consistent with the Mestizo character of this population. In terms of genetic distance Peruvians are closest to Bolivians, which is in agreement with the geographical location and the cultural and anthropological background of the two human groups. Several HLA-B alleles originally described in genetically isolated Amerindian tribes are also present in the sample studied here. This fact and the reported finding of these alleles in several Amerindian groups suggests that they were present in the first wave of humans that populated South America (Paleoindians) before they split to give rise to the different South American tribes.