Leishmaniasis tegumentaria y flebótomos en la localidad de Colonia Santa Rosa del norte de Argentina / Tegumentary leishmaniasis and sandflies in Colonia Santa Rosa locality in northern Argentina

Resumen La leishmaniasis tegumentaria (LT) es causada por parásitos del género Leishmania y transmitida por especies de flebótomos, insectos pertenecientes al orden Diptera, familia Psychodidae. El objetivo de este trabajo fue analizar la presencia de casos de LT y su variación temporal entre 1985 y 2019, las formas clínicas, la ubicación de las lesiones en los pacientes y la presencia de flebótomos en la localidad de Colonia Santa Rosa, del norte de Argentina. Los pacientes se diagnosticaron mediante frotis e intradermorreacción de Montenegro. Se colocaron trampas CDC en 14 sitios en el horario de 19 a 7. Se diagnosticaron 120 casos de LT y la prevalencia global fue del 0,75% (^16.000 habitantes). Los pacientes presentaron formas cutáneas simples y múltiples (88,79%) y mucocutáneas (10,83%). Las lesiones cutáneas fueron más frecuentes en extremidades inferiores. Del total de flebótomos, Nyssomyia neivai fue la especie predominante (95%), y, en menor proporción, estuvieron presentes Migonemyia migonei (1,9%), complejo cortelezzii (1,3%) y Evandromyia sallesi (0,09%). La persistente ocurrencia de casos y la presencia de flebótomos en la citada localidad nos sugiere la transmisión endémica en la zona. Esto senala la necesidad de planificar medidas preventivas y de control de la LT en el norte de Argentina.

Abstract Tegumentary leishmaniasis (TL) is caused by parasites of the genus Leishmania and transmitted by the sandfly species, insects belonging to the order Diptera, family Psychodidae. Historically, the most endemic area of TL in Argentina has been the northern region. The aim of this work was to analyze the presence and temporal variation of TL cases reported between 1985 and 2019 in Colonia Santa Rosa locality, northern Argentina. Furthermore, its clinical forms were characterized and sandflies were captured. Patients were diagnosed by smear and the Montenegro skin test. For sampling, CDC light traps were placed at 14 sites from 7pm to 7am. The correlation between vegetation cover and sandfly abundance was also studied. One hundred and twenty TL cases were diagnosed and the overall prevalence was 0.75% (M6 000 inhabitants). Patients presented simple and multiple cutaneous leishmaniasis (88.79%) and the mucocutaneous form (10.83%). Skin lesions were more frequent on the lower extremities (46.73%). Of the total number of sandflies, Nyssomyia neivai (95%) was the predominant species followed by Migonemyia migonei (1.9%), cortelezzii complex (1.3%) and Evandromyia sallesi (0.09%). The persistent occurrence of cases and the presence of sandflies in the locality suggest the existence of endemic transmission in the area. This highlights the need to design prevention and control measures for TL in northern Argentina.

[Tegumentary leishmaniasis and sandflies in Colonia Santa Rosa locality in northern Argentina]. / Leishmaniasis tegumentaria y flebótomos en la localidad de Colonia Santa Rosa del norte de Argentina.

Tegumentary leishmaniasis (TL) is caused by parasites of the genus Leishmania and transmitted by the sandfly species, insects belonging to the order Diptera, family Psychodidae. Historically, the most endemic area of TL in Argentina has been the northern region. The aim of this work was to analyze the presence and temporal variation of TL cases reported between 1985 and 2019 in Colonia Santa Rosa locality, northern Argentina. Furthermore, its clinical forms were characterized and sandflies were captured. Patients were diagnosed by smear and the Montenegro skin test. For sampling, CDC light traps were placed at 14 sites from 7pm to 7am. The correlation between vegetation cover and sandfly abundance was also studied. One hundred and twenty TL cases were diagnosed and the overall prevalence was 0.75% (≈16 000 inhabitants). Patients presented simple and multiple cutaneous leishmaniasis (88.79%) and the mucocutaneous form (10.83%). Skin lesions were more frequent on the lower extremities (46.73%). Of the total number of sandflies, Nyssomyia neivai (95%) was the predominant species followed by Migonemyia migonei (1.9%), cortelezzii complex (1.3%) and Evandromyia sallesi (0.09%). The persistent occurrence of cases and the presence of sandflies in the locality suggest the existence of endemic transmission in the area. This highlights the need to design prevention and control measures for TL in northern Argentina.

High performance of an enzyme linked immunosorbent assay for American tegumentary leishmaniasis diagnosis with Leishmania (Viannia) braziliensis amastigotes membrane crude antigens.

The diagnosis of American tegumentary leishmaniasis (ATL) still requires the design of more effective tools. Leishmania (Viannia) braziliensis is the causal agent of the 90% of Argentinean ATL cases. Considering the current knowledge, an ELISA based crude antigen (CA) for the diagnosis was designed. Ninety-nine subjects diagnosed as ATL, 27 as no-ATL, and 84 donors from non-ATL-endemic areas were included in this study. The current ATL diagnosis was based four techniques, dermal smear microscopic examination (parasitological test), PCR, Leishmanin skin test, and clinical records. We obtained CA extracts from promastigotes and amastigotes from macrophage cultures of different zymodemes of endemic Leishmania species circulating in the study area. Crude antigens from the 'local' main zymodeme of L. (V.) braziliensis showed the highest reactivity against anti-Leishmania antibodies compared to the other included species. The CA of amastigotes of this zymodeme was 3.4 fold more reactive than promastigotes one. Moreover, amastigote-membrane CA (MCA) were 3.6 fold more reactive than the soluble antigens. The MCA-ELISA reached a sensitivity and specificity of 98% (CI = 94.7%-100%) and 63.6% (53.9-73.1), respectively. When anti-Trypanosoma cruzi reactive sera were excluded, the specificity reached 98.4% (94.4-100), while the sensitivity was similar, with a positive predictive value (PV) of 98.6% (94.6-100) and negative PV of 96.3% (91.6-100). The performance of the MCA-ELISA results strongly contribute to the final diagnostic decision, since a non-reactive serological result almost discards the suspected ATL, because of its high negative PV. The developed MCA-ELISA showed a high diagnostic performance, which makes it a good candidate for ATL diagnosis, for seroprevalence studies, or for monitoring treatments efficacy.

Vegetation Cover and Microspatial Distribution of Sand Flies (Diptera: Psychodidae) in an Endemic Locality for Cutaneous Leishmaniasis in Northern Argentina.

The sand fly fauna in Hipólito Yrigoyen, Argentina, a locality where cutaneous leishmaniasis cases occur, was surveyed with zones of higher abundance of sand flies correlated to vegetation cover estimated through normalized difference vegetation index (NDVI). Sand flies were collected with 10 CDC traps during six nights, from December 2009 to January 2010. A map was built of expected sand flies abundance in which levels of NDVI were categorized. In total, 1,392 Phlebotominae (Diptera: Psychodidae) specimens were collected, comprised of the following species: Nyssomyia neivai (Pinto 1926), Migonemyia migonei (França 1920), species of the cortelezzii complex (Brèthes 1923), Evandromyia sallesi (Galvão & Coutinho 1940), and Psathyromyia shannoni (Dyar 1929). Positive correlations were found between the abundance of sand flies and the NDVI (P < 0.05) for buffer areas of <150 m radii from the trap location points, i.e., the sand fly abundance was greater where vegetation cover and density were greater. In this context, plant cover should be taken into account to prioritize surveillance and control areas within the program of sand flies control in northern Argentina.

Epidemiology of American Tegumentary Leishmaniasis and Trypanosoma cruzi Infection in the Northwestern Argentina.

Background. Endemic areas of tegumentary leishmaniasis (TL) in Salta, Argentina, present some overlap zones with the geographical distribution of Chagas disease, with mixed infection cases being often detected. Objectives. The purpose of this study was to determine the magnitude of Leishmania sp. infection and potential associated risk factors, the serologic prevalence of T. cruzi, and the presence of T. cruzi-Leishmania sp. mixed infection in a region of the northwest of Argentina. Methods. Cross-sectional studies were conducted to detect TL prevalence and T. cruzi seroprevalence. A case-control study was conducted to examine leishmaniasis risk factors. Results. Prevalence of TL was 0.17%, seroprevalence of T. cruzi infection was 9.73%, and mixed infection proportion-within the leishmaniasic patients group-was 16.67%. The risk factors associated with TL transmission were sex, age, exposure to bites at work, staying outdoors more than 10 hours/day, bathing in the river, and living with people who had lesions or were infected during the study. Discussion. The endemic pattern of TL seems to involve exposure of patients to vectors in wild as well as peridomestic environment. Cases of T. cruzi infection are apparently due to migration. Therefore, a careful epidemiological surveillance is necessary due to the contraindication of antimonial administration to chagasic patients.

Genetic and clinical characterization of canine leishmaniasis caused by Leishmania (Leishmania) infantum in northeastern Argentina.

Leishmaniases comprise zoonotic diseases caused by protozoan flagellates of the Leishmania genus. They are endemic to South America, and the visceral form has been recently reported in Argentina. Dogs can play different roles in the Leishmania transmission cycles, depending mainly on the species of parasite involved. Here we focused on the clinical characterization of canine leishmaniasis (CanL) in Northeast Argentina and on the molecular typing of its etiological agent. The nested polymerase chain reaction and sequence analysis of the Leishmania cytochrome b (cyt b) gene was performed on DNA templates purified from lymph nodes, bone marrow or spleen aspirates obtained from 48 dogs previously diagnosed by the observation of Leishmania amastigotes on smears from these aspirates. Their clinical and epidemiological data were also recorded. Systemic abnormalities were observed in 46 subjects (95.8%), most frequently lymphadenopathy, and emaciation (89.6 and 75%). Furthermore, 87% also presented tegumentary abnormalities, such as alopecia (54.2%) or secondary skin lesions (47.9%), among others. Twenty three dogs were positive for cyt b amplification. The sequence analysis showed the presence of two genotypes, LiA1 and LiA2, assigned to Leishmania (Leishmania) infantum, with 99.9 and 100% homology with the reference strain MHOM/TN/80/IPT1 respectively. LiA1 was identified in 18 cases (78.3%) and LiA2 in five (21.7%). Two cyt b variants of L. (L.) infantum were incriminated as the causative agents of CanL cases from three cities: Posadas, Garupá, and Ituzaingó. All three cities are located in the northeastern area of the country, where these parasites seem to be spreading in urban areas.

Visceral Leishmaniasis Caused by Leishmania infantum in Salta, Argentina: Possible Reservoirs and Vectors.

Cases of human visceral leishmaniasis (HVL) were not recorded until recently in the Chaco region of northwestern Argentina. Dogs were surveyed at the sites of infection of two HVL index cases in the Chaco region of Salta province. Canine cases (CanL) were diagnosed by two parasitological methods, two molecular methods targeting mini- and maxicircle DNA, and immunochromatographic dipstick. Among 77 dogs studied, 10 (13%) were found infected with Leishmania spp. In seven dogs and two humans, the infecting species was typed as Leishmania (Leishmania) infantum. The same genotype was detected in the human and two of the CanL. Although several diagnostic methods displayed weak or moderate agreement, the concordance values for serology versus maxicircle PCR were very good (Kappa index = 0.84). Sandflies captured in the area were identified as Lutzomyia migonei and Lu. cortelezzii/Lu. sallesi (cortelezzii complex). The focal appearance of leishmaniasis in dogs and humans in a sylvatic region and its relatively low prevalence of infection suggests that L. (L.) infantum transmission to dogs and humans may, in this region, stem from sylvatic reservoirs.

Multilocus sequence typing approach for a broader range of species of Leishmania genus: describing parasite diversity in Argentina.

Leishmaniasis is a vector-borne protozoan infection affecting over 350 million people around the world. In Argentina cutaneous leishmaniasis is endemic in nine provinces and visceral leishmaniasis is spreading from autochthonous transmission foci in seven provinces. However, there is limited information about the diversity of the parasite in this country. Implementation of molecular strategies for parasite typing, particularly multilocus sequence typing (MLST), represents an improved approach for genetic variability and population dynamics analyses. We selected six loci as candidates implemented in reference strains and Argentinean isolates. Phylogenetic analysis showed high correlation with taxonomic classification of the parasite. Autochthonous Leishmania (Viannia) braziliensis showed higher genetic diversity than L. (Leishmania) infantum but low support was obtained for intra-L. braziliensis complex variants suggesting the need of new loci that contribute to phylogenetic resolution for an improved MLST or nested-MLST scheme. This study represents the first characterization of genetic variability of Leishmania spp. in Argentina.

Restricted outbreak of American tegumentary leishmaniasis with high microfocal transmission.

Cutaneous leishmaniasis is endemic in Salta, the northwestern province of Argentina. We describe an outbreak involving five recreational hunters whose exposure was limited to several hours in a residual patch of primary forest. All patients presented with typical cutaneous lesions after a mean incubation period of 59 days (range 15-78), and one developed simultaneous mucosal involvement. Polymerase chain reaction analysis of lesions confirmed Leishmania (V.) braziliensis as the etiologic agent in three cases. All patients were cured with anti-Leishmania treatment. Entomologic surveys in the transmission area revealed a predominance of Lutzomyia neivai. This outbreak report confirms a microfocal transmission pattern of tegumentary leishmaniasis in the Americas and based on a well-determined exposure, allows the determination of incubation times for leishmaniasis caused by Leishmania braziliensis.

[Role of three ELISA tests using promastigote homogenates of Leishmania braziliensis, L. amazonensis and L. guyanensis in the diagnosis of tegumentary leishmaniasis]. / El rol de tres pruebas de ELISA con antígenos de promastigotes de Leishmania braziliensis, L. amazonensis y L. guyanensis en el diagnóstico de leishmaniasis tegumentaria.

It is important to know whether the variability of species of Leishmania parasites circulating in a region affects the performance of the ELISA test for the diagnosis of leishmaniasis. Therefore, the aim of this study was to analyze the reactivity of the ELISA using homogenates of promastigotes of Leishmania (V.) braziliensis (ELISAb), Leishmania (L) amazonensis (ELISAa) and Leishmania (V.) guyanensis (ELISAg) against different sera groups. Samples from individuals with cutaneous leishmaniasis (n = 37), mucocutaneous leishmaniasis (n = 8), healthy controls (n = 52), persons infected with Trypanosoma cruzi (n = 11) and mixed infections (n = 14) were included in the study. We calculated sensitivities, specificities, cut offs, and predictive values for the three tests and compared them using ANOVA, kappa index, ROC curves comparison, and confidence intervals calculated by the bootstrap method. Significant differences were found when comparing the OD levels of sera from patients with cutaneous leishmaniasis against healthy controls, but there were no differences when comparing the different ELISAs. The sensitivities calculated for ELISAb and ELISAa were 84.6 and of 88.5% for ELISAg, while the value of specificity for the three tests was 96.2. The kappa index (0.87) and comparison of ROC curves showed similar performance for the three ELISAs (p = 0.225). The high reactivity obtained for these ELISAs in sera of patients with mucocutaneous leishmaniasis indicates this test as an important complement in the diagnosis of the disease.

El rol de tres pruebas de ELISA con antígenos de promastigotes de Leishmania braziliensis, L. amazonensis y L. guyanensis en el diagnóstico de leishmaniasis tegumentaria / Role of three ELISA tests using promastigote homogenates of Leishmania braziliensis, L. amazonensis and L. guyanensis in the diagnosis of tegumentary leishmaniasis

Es importante conocer si la variabilidad de especies de Leishmania circulantes en una región afecta la performance de las pruebas de ELISA estandarizadas para el diagnostico de la leishmaniasis. El objetivo de este trabajo fue analizar la reactividad de la prueba de ELISA utilizando homogenados de promastigotes de Leishmania (V.) braziliensis (ELISAb), L (L) amazonensis (ELISAa) y L (V.) guyanensis (ELISAg) frente a distintos grupos de sueros. Se estudiaron muestras de personas con leishmaniasis cutánea (n = 37), leishmaniasis mucocutánea (n = 8), no infectados (n = 52), infectadas por Trypanosoma cruzi (n = 11) e infecciones mixtas (n = 14). Se calcularon las sensibilidades, especificidades, cut off, valores predictivos, y se compararon las tres pruebas usando ANOVA, índice de concordancia kappa, comparación de curvas ROC e intervalos de confianza construidos por el método de bootstrap. Se encontraron diferencias significativas al comparar los niveles de DO de los sueros de pacientes con leishmaniasis cutánea respecto a los controles negativos, pero no se encontraron diferencias entre pruebas. Las sensibilidades calculadas fueron de 84.6% para ELISAb y ELISAa y de 88.5 para ELISAg, mientras que el valor de especificidad para las tres pruebas fue de 96.2. El índice de concordancia kappa y la comparación de curvas ROC mostraron performances similares para las tres pruebas (p = 0.225). La elevada reactividad obtenida para estas ELISAs frente a sueros de pacientes con leishmaniasis mucocutánea indica un importante potencial de esta técnica como complemento en el diagnóstico de la enfermedad.

It is important to know whether the variability of species of Leishmania parasites circulating in a region affects the performance of the ELISA test for the diagnosis of leishmaniasis. Therefore, the aim of this study was to analyze the reactivity of the ELISA using homogenates of promastigotes of Leishmania (V.) braziliensis (ELISAb), Leishmania (L) amazonensis (ELISAa) and Leishmania (V.) guyanensis (ELISAg) against different sera groups. Samples from individuals with cutaneous leishmaniasis (n = 37), mucocutaneous leishmaniasis (n = 8), healthy controls (n = 52), persons infected with Trypanosoma cruzi (n = 11) and mixed infections (n = 14) were included in the study. We calculated sensitivities, specificities, cut offs, and predictive values for the three tests and compared them using ANOVA, kappa index, ROC curves comparison, and confidence intervals calculated by the bootstrap method. Significant differences were found when comparing the OD levels of sera from patients with cutaneous leishmaniasis against healthy controls, but there were no differences when comparing the different ELISAs. The sensitivities calculated for ELISAb and ELISAa were 84.6 and of 88.5% for ELISAg, while the value of specificity for the three tests was 96.2. The kappa index (0.87) and comparison of ROC curves showed similar performance for the three ELISAs (p = 0.225). The high reactivity obtained for these ELISAs in sera of patients with mucocutaneous leishmaniasis indicates this test as an important complement in the diagnosis of the disease.