RESUMEN
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.
Asunto(s)
Leishmania , Leishmaniasis Cutánea , Psychodidae , Animales , Argentina , Femenino , Insectos Vectores/parasitología , Leishmania/genética , Leishmaniasis Cutánea/transmisión , Psychodidae/parasitologíaRESUMEN
Leishmaniases are vector-borne diseases that in the Americas are distributed from southern United States to northern Argentina. The vectors for this disease are small dipterans known as sand flies that are usually identified morphologically by observing structures with taxonomic value; but it is time-consuming, laborious, and requires entomological expertise. Then, this work was aimed at identifying sand flies with molecular techniques, using the morphological identification as a reference technique, in an endemic area of American Tegumentary Leishmaniasis (ATL) located in northern Argentina. For this, sand flies were caught at two patches of vegetation adjacent to rural areas in Orán department, Salta Province. Females were dissected with sterile needles; the head and last abdominal segments were analyzed for morphological identification. The remaining thorax and abdominal segments were used to extract DNA, which was amplified by PCR of the small subunit (SSU), 18S rRNA gene. PCR products were digested with CviQI and DdeI enzymes to identify sand fly species by Restriction Fragment Length Polymorphism (RFLP) analysis. Thus, the restriction pattern of each caught species was defined according to morphological identification. A total of 1501 females, belonging to four sand fly species, were captured. Nyssomyia neivai (1347/1501) was the most abundant species, followed by Migonemyia migonei (90/1501). From the total, 801 females were morphologically and molecularly identified, while 700 females were characterized only molecularly. For those females analyzed by both methods, there was total coincidence in the achieved result. Besides, the 5% (38/801) of females that could not be determined morphologically due to inadequate mounting were molecularly identified. All the females characterized just by PCR-RFLP, were successfully identified. Our results indicate that the explored method is capable of identifying the sand fly species that circulate in an ATL endemic area. Since this method is based on the analysis of markedly different patterns, the identification process might be more easily reproduced, as the bias introduced by the technician's lack of experience is removed.
Asunto(s)
Insectos Vectores/clasificación , Insectos Vectores/genética , Polimorfismo de Longitud del Fragmento de Restricción , Psychodidae/clasificación , Psychodidae/genética , ARN Ribosómico 18S/genética , Animales , Argentina/epidemiología , Femenino , Leishmaniasis Cutánea/epidemiologíaRESUMEN
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.