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Leishmaniases are neglected tropical diseases caused by Leishmania spp. parasites transmitted by the bite of phlebotomine sand flies. In Argentina, the most endemic area of American tegumentary leishmaniasis (ATL) has been Orán department, Province of Salta, where Leishmania (Viannia) braziliensis prevails and Nyssomyia neivai is considered as its vector, although there is no accurate and sufficient information in this regard. The aim of this work was to search for natural infection by Leishmania spp. in sand flies from peri-urban and rural sites with ATL background in Orán department. For this, sand flies were caught at five sites; female sand flies captured with Shannon trap were dissected to microscopically examine their gut contents, while females captured with CDC traps were molecularly analyzed by duplex PCR with two primer pairs to simultaneously amplify kinetoplast DNA (kDNA) and mammalian actin. A total of 1921 females were captured, with Ny. neivai being the most abundant species (89%), followed by Migonemyia migonei (6%) and cortelezzii complex (3%). No natural infection was found in any of them neither by dissection nor by PCR, although the detection limit of kDNA PCR was up to 25 promastigotes. The absence of infected females in peri-urban sites suggest that the transmission did not take place in those environments during the study period. Future searches for natural infection should focus on rural settings to deepen knowledge and elucidate the role of the circulating sand fly species as all have been linked to ATL transmission at other sites., (Copyright © 2021. Published by Elsevier B.V.)

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., Competing Interests: The authors have declared that no competing interests exist.

Cutaneous leishmaniasis is endemic in Salta province, which belongs to the northwest of Argentina. Leishmania spp. DNA from Giemsa-stained slides of up to 12 years in storage of patients from Salta was characterized through polymerase chain reaction (PCR) restriction fragment length polymorphism (RFLP). One hundred smears positive for microscopy, classified in a semiquantitative scale for amastigote density, were analyzed. Also, Leishmanin skin test (LST) results were included. DNA extraction was carried out applying lysis buffer with proteinase K, and then DNA was amplified with ribosomal internal transcribed spacer 1 primers. PCR products were digested with HaeIII enzyme. All PCR-positive smears (74/100) belonged to Viannia subgenus. A statistically significant, directly proportional relationship between semiquantitative microscopy and PCR results was detected. All patients had LST-positive results (induration ≥ 5 mm), and the smears of those with smaller induration (LST < 19 mm) gave a higher proportion of positive PCR results. This study determined that smear age did not affect PCR positivity, which allows retrospective analyzes and suggests smears might be useful for molecular complementary diagnosis. Because Leishmania ( Viannia ) braziliensis is the main circulating species in the study area, determining Viannia subgenus in all analyzed samples confirms previous findings. PCR positivity showed statistically significant differences according to semiquantitative microscopy, highlighting the importance of parasite burden in the diagnostic sensitivity of the method. Considering that smears of patients with smaller LST induration were more positive in PCR, a negative smear from patients with positive LST response, but < 19 mm, could actually represent a false-negative result.

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.

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.