American tegumentary leishmaniasis is an endemic anthropozoonosis undergoing expansion on the American continent. The disease is caused by several Leishmania species and it is manifested as cutaneous and mucocutaneous leishmaniasis. In this study, we evaluate the viability of high-resolution melt polymerase chain reaction (HRM-PCR) analysis to differentiate four closely related Leishmania species as a routine tool for the diagnosis of leishmaniasis. For this purpose, biopsy specimens from cutaneous and mucocutaneous lesions were taken from 132 individuals from endemic and non-endemic areas for leishmaniasis. Each sample was processed for parasitological, histopathological, and molecular analysis. Positive biopsy samples were analyzed by HRM-PCR of a 144-bp heat-shock protein (hsp70) gene fragment, and new cases were confirmed by sequencing. Of the 132 samples analyzed, 36 (27%) were positive for Leishmania spp., of which 86% were from cutaneous lesions and 14% from mucocutaneous lesions. We identified Leishmania (Viannia) braziliensis (84%), Leishmania (Leishmania) infantum (13%), and Leishmania (Leishmania) amazonensis (3%) in cutaneous lesions, and L. (V.) braziliensis (40%), L. (L.) infantum (20%), L. (L.) amazonensis (20%), and Leishmania (Viannia) guyanensis (20%) in mucocutaneous lesions. The main purpose of this research was to report for the first time in Paraguay the presence of L. (L.) amazonensis and L. (V.) guyanensis in patients with cutaneous and mucocutaneous lesions, using the HRM-PCR technique. In addition, we report the presence of additional new cases of L. (L.) infantum in cutaneous lesions.

Leishmaniasis is a vector-borne disease caused by a parasite protozoon from the genus Leishmania. Among the molecular techniques applied for detecting these parasites, real-time PCR with High Resolution Melting (PCR-HRM) proved advantageous since it simultaneously determines both the presence and species of the pathogen in one step, through amplification and later analysis of curves generated by melting temperature. Based on this molecular technique, the goal of this study was to estimate the PCR-HRM sensitivity for Leishmania spp. detection in different canine tissues by evaluating biological samples obtained from popliteal, submandibular, and pre-scapular lymph nodes, from bone marrow and ear pinnae of 28 stray dogs captured in the metropolitan area of Asunción (Paraguay). The rk39 immunochromatographic test showed that 25/28 tested dogs (89%) presented antibodies against L. infantum. In 20/25 dogs that tested positive for rk39 (80%), it was possible to detect Leishmania spp. by PCR-HRM and determine that the species corresponded entirely to L. infantum. Regarding the analysis of different tissues, the parasite was detected in all popliteal lymph node samples, followed by high detection in submandibular (at 95%) and pre-scapular lymph nodes (at 90%), bone marrow (at 85%), and ear pinnae (at 85%). This study demonstrated that the use of real-time PCR-HRM using the molecular marker hsp70 was a highly sensitive method for simultaneously detecting and identifying Leishmania species in different tissues taken from infected dogs. In addition, the usefulness of ear pinnae as easily accessible tissue for molecular diagnosis was emphasized.

Anaplasmataceae bacteria are emerging infectious agents transmitted by ticks. The aim of this study was to identify the molecular diversity of this bacterial family in ticks and hosts, both domestic and wild, as well as blood meal sources of free-living ticks in northeastern Paraguay. The bacteria were identified using PCR-HRM, a method optimized for this purpose, while the identification of ticks and their blood meal was performed using conventional PCR. All amplified products were subsequently sequenced. The bacteria detected in the blood hosts included Ehrlichia canis, Anaplasma platys, and Anaplasma phagocytophilum, Candidatus Anaplasma boleense, and Wolbachia spp., which had not been previously reported in the country. Free-living and parasitic ticks on dogs (Canis lupus familiaris) and wild armadillos (Dasypus novemcinctus) were collected and identified as Rhipicephalus sanguineus and Amblyomma spp. The species E. canis, A. platys, A. phagocytophilum, and Ca. A. boleense were detected in domestic dog ticks, and E. canis and A. platys were found for the first time in armadillos and free-living ticks. Blood feeding sources detected in free-living ticks were rodents, humans, armadillos and dogs. Results show a high diversity of tick-borne pathogens circulating among domestic and wild animals in the northeastern region of Paraguay.