Bartonella spp. and Typhus Group Rickettsiae among Persons Experiencing Homelessness, São Paulo, Brazil.

Persons experiencing homelessness in São Paulo, Brazil, were seropositive for Bartonella spp. (79/109, 72.5%) and typhus group rickettsiae (40/109, 36.7%). Bartonella quintana DNA was detected in 17.1% (14/82) body louse pools and 0.9% (1/114) blood samples. Clinicians should consider vectorborne agents as potential causes of febrile syndromes in this population.

Validation of the Dual-path Platform chromatographic immunoassay (DPP® CVL rapid test) for the serodiagnosis of canine visceral leishmaniasis.

BACKGROUND Visceral leishmaniasis is a major public health challenge in South America, and dogs are its main urban reservoir. OBJECTIVE Validation of the canine Dual-path Platform immunoassay for canine visceral leishmaniasis (DPP® CVL) for a sample set composed of 1446 dogs from different Brazilian endemic areas. METHODS A well-defined reference standard by means of parasitological culture, immunohistochemistry, and histopathology was used. Animals were classified as asymptomatic, oligosymptomatic, or symptomatic. Sensitivity and specificity were assessed as a single set and in clinical groups. A reproducibility assessment of the tests was conducted using the Kappa (κ) index at three different laboratories (A, B, and C). FINDINGS Overall, 89% sensitivity and 70% specificity were obtained for the entire sample set. Analysis of the clinical groups showed a gradual decrease in the sensitivity and an increase in the specificity with the reduction of clinical signs in the dogs that were assessed, reaching a sensitivity of 75% (42.8-94.5%) among asymptomatic dogs and lower specificity of 56% (46.2-66.3%) among symptomatic dogs. Inter-laboratory agreement was substantial (κAB= 0.778; κAC= 0.645; κCB= 0.711). MAIN CONCLUSIONS The test performance is somewhat dependent on canine symptomatology, but such influence was less evident than in previous studies. Favourable results for sensitivity and specificity can be obtained even in asymptomatic animals; however, caution is needed in these evaluations, and the results suggest that the immunochromatographic test may be further improved for better investigation in asymptomatic dogs. The results obtained confirm the usefulness of DPP® CVL for application in serological surveys.

Sensitivity and specificity of parallel or serial serological testing for detection of canine Leishmania infection.

In Brazil, human and canine visceral leishmaniasis (CVL) caused by Leishmania infantum has undergone urbanisation since 1980, constituting a public health problem, and serological tests are tools of choice for identifying infected dogs. Until recently, the Brazilian zoonoses control program recommended enzyme-linked immunosorbent assays (ELISA) and indirect immunofluorescence assays (IFA) as the screening and confirmatory methods, respectively, for the detection of canine infection. The purpose of this study was to estimate the accuracy of ELISA and IFA in parallel or serial combinations. The reference standard comprised the results of direct visualisation of parasites in histological sections, immunohistochemical test, or isolation of the parasite in culture. Samples from 98 cases and 1,327 noncases were included. Individually, both tests presented sensitivity of 91.8% and 90.8%, and specificity of 83.4 and 53.4%, for the ELISA and IFA, respectively. When tests were used in parallel combination, sensitivity attained 99.2%, while specificity dropped to 44.8%. When used in serial combination (ELISA followed by IFA), decreased sensitivity (83.3%) and increased specificity (92.5%) were observed. Serial testing approach improved specificity with moderate loss in sensitivity. This strategy could partially fulfill the needs of public health and dog owners for a more accurate diagnosis of CVL.

Occurrence of Leishmania infantum and associated histological alterations in the genital tract and mammary glands of naturally infected dogs.

The objectives of this study were to evaluate the occurrence of Leishmania infantum in the male and female genital tract and female mammary glands of dogs and the parasite burden and to identify histological alterations associated with this protozoan. Twenty male and 20 female Leishmania-seropositive dogs with isolation of L. infantum were examined. Tissue samples of the prepuce, glans, epididymis, testes, prostate, vulva, vagina, uterus, uterine tubes, and mammary glands were analyzed by immunohistochemistry and histopathology. For parasitological culture and in situ hybridization, samples were collected from the testis, epididymis, and uterus. Additionally, seminal fluid was aspirated from the epididymis for parasitological culture. In the genital tract, 34 (85 %) dogs, including 18 males and 16 females, were positive for Leishmania. Of these, 27 (79 %) animals were symptomatic. Leishmania was detected in the mammary glands of 13 (65 %) females. L. infantum was isolated for the first time from the seminal fluid and uterus of naturally infected dogs. The parasite burden and intensity of the inflammatory reaction were greater in the prepuce and glans of males and in the vulva and mammary glands of females. In addition to inflammation, testicular degeneration, atrophy, absence of spermatogenesis, and necrosis were observed. Detection of amastigote forms in the mammary gland lumen indicates possible elimination of this parasite in milk. The frequent parasitism observed in the genital tract of infected males and females and the viability of L. infantum in seminal fluid and uterus suggest the possibility of bidirectional venereal and vertical transmission.

Impact of 4% Deltamethrin-Impregnated Dog Collars on the Incidence of Human Visceral Leishmaniasis: A Community Intervention Trial in Brazil.

BACKGROUND: In Brazil, human visceral leishmaniasis (HVL) is caused by the protozoan parasite Leishmania infantum, primarily transmitted by the sand fly Lutzomyia longipalpis, with dogs acting as the main urban reservoir. This study aims to evaluate the effectiveness of 4% deltamethrin-impregnated dog collars (DMC) on HVL incidence. METHODS: This is a community intervention study carried out from 2012 to 2015 in the municipalities of Araguaína, State of Tocantins, and Montes Claros, State of Minas Gerais, Brazil. Two areas in each were randomly allocated to either (1) culling seropositive dogs + residual insecticide spraying (control area-CA) or (2) culling seropositive dogs + residual insecticide spraying + DMC fitted to dogs every six months for two years (intervention area-IA). Cases of HVL (n = 1202) occurring from 2008 to 2020 were identified from the Brazilian Reportable Diseases Information System and georeferenced to the control and intervention areas. The HVL cases from 2008 to 2012 were considered as occurring in the "pre-intervention" period. Those cases from 2013 to 2016 and from 2017 to 2020 were regarded as occurring in the "intervention" and "post-intervention" periods, respectively. We used a mixed-effects Poisson regression model to estimate the effectiveness of the intervention, comparing the changes from the pre-intervention period to the intervention and post-intervention periods in the control and intervention areas. RESULTS: In Araguaína, there was a statistically significant reduction in the incidence of HVL in both the control and intervention areas, comparing both the intervention and post-intervention periods with the pre-intervention period. The intervention with DMC was significantly associated with a reduction in HVL when comparing the intervention and pre-intervention periods, yielding an effectiveness estimate of the DMC of 27% (IC95% 1-46%, p = 0.045). No differences were observed when comparing the pre- and post-intervention periods (p = 0.827). In Montes Claros, cases reduced in both the control and intervention areas from the pre-intervention period to the intervention period (p = 0.913). In the post-intervention period, the incidence increased in the control area, while cases continued to decrease in the DMC area (p = 0.188). CONCLUSIONS: The use of DMC was associated with a reduction of 27% in the incidence of HVL during the period of DMC delivery, indicating that DMC is effective as an additional strategy for controlling visceral leishmaniasis in Brazil. However, no significant reduction associated with DMC was detected after the intervention period, suggesting that a control program based on the large-scale deployment of DMC might have to be maintained for more extended periods without interruption.

Therapeutic success and failure in using miltefosine to treat dogs naturally infected with Leishmania infantum.

In urban environments, domestic dogs (Canis familiaris) are a major reservoir for the parasite Leishmania infantum. Miltefosine has been used as the standard treatment for canine visceral leishmaniasis in Brazil. However, therapeutic failures have been reported. In the present study, two dogs (CG03 and CG06) with a diagnosis of infection by L. infantum underwent two cycles of treatment with miltefosine (Milteforan™ - Virbac®). Analyses showed increases in the parasite load of both CG03 and CG06, even after treatment. The clinical score of CG03 dropped from 1 to 0 (after one round of treatment), such that this dog became asymptomatic. CG06 showed clinical worsening, such that its score increased from 1 to 2. After the second therapeutic round, the parasite load in CG03 was found to have decreased, but it was still higher than before drug treatment even though this dog was physically asymptomatic. There was no decrease in the parasite load in CG06 and there was clinical worsening. The clinical response of these dogs to the treatment differed, but the parasite load remained high in both cases, which poses a risk to public health, making it essential take measures to prevent the sandfly vector from accessing the dog.

Diagnosis of fasciolosis antibodies in Brazilian cattle through ELISA employing both native and recombinant antigens.

Bovine fasciolosis is a parasitic disease with a global reach. Coprological based on egg detection in fecal samples and liver inspection to evaluate the presence of the parasite is currently the gold standard for diagnosing chronic fasciolosis in cattle. However, these techniques are labor-intensive and ineffective during the acute phase of the disease. Serodiagnosis using native and recombinant antigens has become an interesting alternative in efforts to identify cattle fasciolosis. We evaluated cattle from abattoir (n = 139) and farms (n = 500) through liver inspection and coprological examination, respectively. Our laboratory team optimized and validated enzyme-linked immunosorbent assay tests based on somatic antigen, excretory/secretory proteins, and the recombinant antigen cathepsin L-1 to detect serum antibodies against fasciolosis in cattle. For animals from abattoir, 10 were positive for fasciolosis according to liver inspection. Both FhES and FhrCL-1 presented an area under the receiver operating characteristic (AUROC) curve of 0.80, with a sensitivity of 0.80 (95% CI: 0.46-0.95) and 0.70 (95% CI: 0.38-0.90) and specificity of 0.81 (95% CI: 0.73-0.87) and 0.87 (95% CI: 0.80-0.92), respectively. For those cattle from farms, 28 were positive only for fasciolosis according to coprological examination. In this scenario, FhES gave the best performance, with an AUROC of 0.84, sensitivity of 0.79 (95% CI: 0.60-0.90), and specificity of 0.86 (95% CI: 0.82-0.89). In conclusion, our study highlights the potential of serodiagnosis for accurately screening cattle fasciolosis. The promising sensitivity and specificity values of FhES when compared to liver inspection or coprological examination enhance its importance for cattle fasciolosis diagnosis. IMPORTANCE: The aim of this article was to identify antibodies against fasciolosis in cattle in Brazil. The methodology was reproduced in our laboratory and applied for the first time to the Brazilian cattle herd. The antigens tested can be used as a screening test and thus speed up the diagnosis of bovine fascioliasis.

A peptide dataset for target analysis of human complement system proteins.

The targeted LC-MS/MS method has been widely applied for peptide quantification, offering sensibility, specificity, and reproducibility to the analysis. However, it requires the prior selection of targets, including the construction of a spectral library. Here, we present a dataset comprising peptide mass spectra for targeted LC-MS/MS method setup, applied to a set of human complement system proteins. Additionally, we selected a group of peptides and demonstrated their stability and reproducibility in quantification. This dataset is invaluable for studies aiming at the quantification of the complement system proteins by targeted LC-MS/MS, as it provides data for spectral library construction and a list of selected peptides.

In Vitro Evaluation of the Combinatorial Effect of Naringenin and Miltefosine against Leishmania amazonensis.

Leishmania amazonensis causes a clinical form called diffuse cutaneous leishmaniasis (DCL) with challenges to treatment, like low efficiency and drug toxicity. Therefore, it is necessary to investigate new therapies using less toxic leishmanicidal compounds, such as flavonoids like naringenin, and their combination with conventional drugs, such as miltefosine. Antileishmanial dose/response activity, isobologram, calculation of dose reduction index (DRI), and fractional inhibitory concentration index (FICI) tests were performed on in vitro assays using reference promastigote forms of L. amazonensis (IFLA/BR/67/PH8) to assess the combinatorial effect between naringenin and miltefosine. The in vitro results of isobologram, DRI, and FICI calculations showed that the combination of the compounds had an additive effect and was able to reduce the half maximal inhibitory concentration (IC50) of miltefosine in the promastigote forms of the parasite compared to the treatment of the drug alone. This study demonstrated in vitro the viability of a combination action of the flavonoid with the treatment with miltefosine, opening space for further investigations on the association of natural compounds with the drugs used for the treatment of L. amazonensis.

Canine visceral leishmaniasis: Seroprevalence and georeferencing in the state of Santa Catarina, Brazil.

Canine visceral leishmaniasis (CVL) has long been considered an endemic disease in the northern and northeastern regions of Brazil, while the southern region remains non-endemic. However, in recent years, several cases of CVL have been reported in southern states. The objective of this work was to determine the seroprevalence of CVL in dogs in the state of Santa Catarina, Brazil, through immunochromatographic tests (DPP®) and ELISA (Enzyme-Linked Immunosorbent Assay) and its correlation with environmental characteristics through georeferencing. Blood samples from dogs (n = 1227) were collected in six mesoregions of the state and evaluated by the rapid test (DPP®). Positive samples were sent to Lacen (Central Public Health Laboratory) in Santa Catarina to be tested using ELISA. Information obtained from the epidemiological questionnaire was subjected to statistical analysis (Chi-square and Student's t-test; P < 0.05) to verify the correlation between serology and the analyzed variables. The locations (GPS) of the samples were used for georeferencing and creating heatmaps (Kernel Method). Four animals that died from CVL were necropsied and organ samples were collected for molecular analysis (PCR), immunohistochemistry, and histopathology (HE). Of the 1227 dogs analyzed, 22 (1.8%) were reactive in the DPP® and of these, 7 (0.6%) were also positive in the ELISA. A correlation (P < 0.01) was observed between positive serology and region, environment, access to the street, and clinical signs. The positive cases were concentrated in the eastern region of the state, in low-altitude areas with average rainfall and higher average temperatures, and in more populated areas close to forest fragments. PCR, HE, and immunohistochemistry, along with serology, have proven to be efficient for characterizing positive cases.

Factors associated with adherence to the principles of responsible companion animal guardianship in a municipality in southeastern Brazil.

Responsible companion animal guardianship (RCAG) covers aspects that are relevant to both animal and human health. Understanding the factors associated with adherence to RCAG principles can guide accountability, animal welfare and disease control. The present observational study describes the level of adherence to RCAG by guardians living in a medium-sized Brazilian municipality and identifies the factors associated with inadequate guardianship. Questionnaires were administered to randomly selected guardians of 704 dogs. The binary outcome of the study, namely more and less adequate compliers to RCAG principles, was analyzed using a score list comprising eight variables relating to the care provided to the dogs, namely provision of veterinary services, vaccination against rabies, deworming, no access to the streets without supervision, walking with guardian, dog freedom at home, registration (microchipping) and satisfactory food supply. Factors possibly associated with less adequate adherence to RCAG by the guardian, such as socioeconomic and cultural features of the guardians, characteristics of the dogs and attributes of the guardian-animal interactions, were analyzed using multiple logistic regression models. The RCAG actions least adopted by guardians were animal registration, provision of veterinary care, walking with the dog and preventing access of the dogs to the streets without supervision. Individuals who cared for a single dog, a mixed breed dog or had previously lost a dog were less likely to show adequate adherence to RCAG. Conversely, guardians who owned cars, acquired dogs as puppies, lived in households with a maximum of four residents, cared for a sterilized dog or thought that caring for a dog was easier than (or as) expected, had a higher chance of showing adequate adherence to RCAG. The results verify that the socioeconomic and behavioral characteristic of guardians must be taken into consideration for understanding the adherence to RCAG. In addition, it is important to facilitate access to veterinary services and to raise awareness about the significance of a safe and healthy environment for companion animals.

Comparative Study of Colorimetric In Situ Hybridization and Quantitative Real-Time Polymerase Chain Reaction for Diagnosis of Infection by Leishmania infantum in Dogs in Formalin-Fixed and Paraffin-Embedded Skin.

The zoonotic visceral leishmaniasis is caused by the protozoan Leishmania infantum and dogs are reservoirs for this parasite. For the diagnosis of Leishmania at the species level in dogs in formalin-fixed, paraffin-embedded skin (FFPES) samples, colorimetric in situ hybridization (CISH) and quantitative real-time polymerase chain reaction (qPCR) are options, but their sensitivities are not well established. Therefore, the aim of this study was to determine the sensitivity of these two techniques in FFPES for the diagnosis of the L. infantum infection in dogs using culture as the reference standard. The FFPES of 48 dogs with cutaneous infection by L. infantum confirmed by culture and by multilocus enzyme electrophoresis were examined by CISH and qPCR using specific probes for L. infantum. The sensitivities of qPCR, CISH and their combination were, respectively, 77.0%, 58.0% and 83.3%. The sensitivities of qPCR in dogs with and without clinical signs were, respectively, 74.2% and 82.4%. The sensitivities of CISH in dogs with and without clinical signs were, respectively, 61.3% and 52.9%. The CISH and qPCR showed satisfactory sensitivities for the diagnosis of L. infantum in the FFPES of dogs, even in dogs without clinical signs, and their combination increases the sensitivity for this diagnosis.

Serosurvey of Trypanosoma cruzi in persons experiencing homelessness and shelter workers of Brazil.

Although Chagas disease, caused by Trypanosoma cruzi, has been associated with social vulnerability worldwide, producing disability and mortality, no study to date has assessed this protozoal infection in persons experiencing homelessness. Accordingly, the present study aimed to assess anti-T. cruzi antibodies by Wiener Chagatest ELISA recombinant v.3.0 in serum samples of persons experiencing homelessness and related shelter workers in São Paulo, a city with reported vectors but no recent autochthonous case report. Overall, seropositivity to T. cruzi resulted in three of 203 (1.5%) persons experiencing homelessness and two of 87 (2.3%) shelter workers, with similar seroprevalence likely associated with their past social vulnerability. Although the seropositivity in persons experiencing homelessness and shelter workers was within 0 to 25.1% seroprevalence for chronic Chagas disease in the general Brazilian population, the disease has almost decreased 2-fold from the 1980s to 2000s, and such a wide range may not reflect the local disease status. In addition, the authors hypothesized that the similar seroprevalence and exposure between homeless persons and shelter workers herein may be more associated with shared past and present low-income social vulnerability than migratory movements, which may also include infection by sharing injecting drugs, vertical transmission, or blood transfusion. Thus, future studies are needed to confirm the active transmission of Chagas disease in São Paulo city. Moreover, Chagas disease should be considered as differential diagnosis in homeless persons and shelter workers, even in major disease-free Brazilian or other worldwide cities, mostly due to early exposure and vulnerable living conditions.

Serological diagnosis of fasciolosis (Fasciola hepatica) in humans, cattle, and sheep: a meta-analysis.

Fasciola hepatica can cause problems in both animals and humans. Fasciolosis can be diagnosed through the indirect ELISA immunodiagnostic test. Serological diagnosis of Fasciola is based on recombinant antigens secreted by this worm. We used PubMed and Google Scholar databases to review the published literature on 'antigens with immunogenic potential' used in serological tests to identify antibodies against F. hepatica in humans, cattle, and sheep. Studies that investigated diagnostic tests with common reference standards were included in the sensitivity and/or specificity bivariate meta-analysis. In the quality and susceptibility to bias analysis of the 33 included studies, 26 fulfilled at least six (75%) of the eight QUADAS criteria and were considered good-quality papers. We found that most of the studies used native excretory-secretory antigens and recombinant cathepsin in ELISA tests for serological diagnosis of fascioliasis in humans, cattle, and sheep. The meta-analysis revealed that all antigens demonstrated good accuracy. The best results in terms of sensitivity [0.931-2.5% confidence interval (CI) and 0.985-97.5% CI] and specificity (0.959-2.5% CI and 0.997-97.5% CI) were found in human FhES. FhrCL-1, FhES, and FhrSAP-2 antigens gave the best results for the serum diagnosis of human and animal fasciolosis.

A case of canine visceral leishmaniasis of unknown origin in Curitiba (state of Paraná, Brazil) treated successfully with miltefosine.

There are no records of autochthonous cases of canine visceral leishmaniasis in the city of Curitiba, Paraná state, Brazil. In 2020, a male French bulldog (CW01), approximately 2 years old was taken by its owners to a private veterinarian clinic. The suspicion of CVL was confirmed by means of a serology test (ELISA/IFAT reagent), rapid chromatographic immunoassay (DPP®) (ELISA - Biomanguinhos®), parasitological culture and quantitative polymerase chain reaction (qPCR). The animal routinely frequented parks in Curitiba and was taken on several trips to the municipalities of Bombinhas and Balneário Camboriú (Santa Catarina) and to Matinhos (Paraná) where CVL had not previously been reported. Treatment was initiated orally with Milteforan™ which resulted in a significant reduction in the parasitic load. The suspicion of autochthony was investigated through entomological research. A total of 10 traps were installed, one at the animal's home, seven in adjacent city blocks and two in a forest edge. No sandflies were trapped in the dog's home and adjacent houses. The traps in the forest edge caught one Migonemyia migonei female and five Brumptomyia spp. females. This case serves as a warning of the possible introduction of CVL in the city of Curitiba.

Correction: Frequency of co-seropositivities for certain pathogens and their relationship with clinical and histopathological changes and parasite load in dogs infected with Leishmania infantum.

[This corrects the article DOI: 10.1371/journal.pone.0247560.].

Leishmania spp. in indigenous populations: A mini-review.

Leishmaniasis, considered a neglected vector-borne disease complex of global concern, has a significant impact on indigenous communities due to daily human and animal exposure in periurban, rural, and naturally preserved areas. This mini-review aims to assess and discuss studies of leishmaniasis in these communities of the New World and Old World, particularly those in the Americas and Asia. Such indigenous communities have been mostly built in poor traditional households with no mosquito-net protection, mostly located in environmentally protected areas, favoring vectors and reservoirs. The presence of leishmaniasis cases surrounding such indigenous areas indicated a high risk of infection, which may have been historically underestimated due to a lack of surveillance, even at present. The absence of studies of indigenous populations in recognized endemic areas may reflect insufficient health services. In conclusion, the persistence of this neglectful scenario may impact tragic outcomes and potential outbreaks in indigenous peoples and surroundings populations worldwide.

Factors associated with Leishmania infantum infection in dogs from urban areas endemic for visceral leishmaniasis in Brazil.

There are gaps in the knowledge of the factors associated with canine visceral leishmaniasis (CVL). Moreover, there is a need for studies conducted with appropriate sample selection and with standardized diagnostic methods, to assess the current infection status of animals. This study analyzed factors associated with Leishmania infantum infection in dogs from urban areas endemic for CVL in Brazil. Data from four cities in different regions of the country (Fortaleza, Brasília, Palmas, and Bauru) were included, encompassing a large and heterogeneous sample group. Dogs were considered infected if they had positive results in parasitological tests (skin lesion culture, histological or immunohistochemical analysis of skin). Data analysis consisted of logistic regression with adjustment for the effect of cities as cluster variables, using generalized estimating equations. The canine positivity for L. infantum was 9.8%. Bauru had the highest percentage (18.7%), followed by Brasília (8.4%), Fortaleza (7.9%), and Palmas (4%). Male animals were twice as likely to be infected as females (Confidence Interval (CI): 1.5-2.8). Dogs living outdoors were 1.5 times more likely to be positive (CI: 1.4-1.6). Dogs living in households rearing chickens were 40% less likely to be infected (CI: 0.5-0.8). A significant multiplicative interaction indicated that a protective association between purebred and CVL was stronger among dogs older than seven years. These findings provide evidence for the role of individual and environmental factors on L. infantum infection in dogs from endemic urban areas. They may be used for identifying groups of animals at higher risk for delivering control interventions.

In vitro anti-Leishmania activity of triclabendazole and its synergic effect with amphotericin B.

Introduction: Leishmaniasis is a neglected tropical disease, with approximately 1 million new cases and 30,000 deaths reported every year worldwide. Given the lack of adequate medication for treating leishmaniasis, drug repositioning is essential to save time and money when searching for new therapeutic approaches. This is particularly important given leishmaniasis's status as a neglected disease. Available treatments are still far from being fully effective for treating the different clinical forms of the disease. They are also administered parenterally, making it challenging to ensure complete treatment, and they are extremely toxic, in some cases, causing death. Triclabendazole (TCBZ) is a benzimidazole used to treat fasciolosis in adults and children. It presents a lower toxicity profile than amphotericin B (AmpB) and is administered orally, making it an attractive candidate for treating other parasitoses. The mechanism of action for TCBZ is not yet well understood, although microtubules or polyamines could potentially act as a pharmacological target. TCBZ has already shown antiproliferative activity against T. cruzi, T. brucei, and L. infantum. However, further investigations are still necessary to elucidate the mechanisms of action of TCBZ. Methods: Cytotoxicity assay was performed by MTT assay. Cell inhibition (CI) values were obtained according to the equation CI = (O.D treatment x 100/O.D. negative control). For Infection evaluation, fixated cells were stained with Hoechst and read at Operetta High Content Imaging System (Perkin Elmer). For growth curves, cell culture absorbance was measured daily at 600 nm. For the synergism effect, Fractional Inhibitory Concentrations (FICs) were calculated for the IC50 of the drugs alone or combined. Mitochondrial membrane potential (DYm), cell cycle, and cell death analysis were evaluated by flow cytometry. Reactive oxygen species (ROS) and lipid quantification were also determined by fluorimetry. Treated parasites morphology and ultrastructure were analyzed by electron microscopy. Results: The selectivity index (SI = CC50/IC50) of TCBZ was comparable with AmpB in promastigotes and amastigotes of Leishmania amazonensis. Evaluation of the cell cycle showed an increase of up to 13% of cells concentrated in S and G2, and morphological analysis with scanning electron microscopy showed a high frequency of dividing cells. The ultrastructural analysis demonstrated large cytoplasmic lipid accumulation, which could suggest alterations in lipid metabolism. Combined administration of TCBZ and AmpB demonstrated a synergistic effect in vitro against intracellular amastigote forms with cSFICs of 0.25. Conclusions: Considering that TCBZ has the advantage of being inexpensive and administrated orally, our results suggest that TCBZ, combined with AmpB, is a promising candidate for treating leishmaniasis with reduced toxicity.

Epidemiology of Rickettsia spp. in Atlantic rainforest areas of island and seashore mainland, southern Brazil.

Non-fatal cases of rickettsial infection with different clinical features than the classic BSF (Brazilian Spotted Fever) have been reported in seashore areas of Paraná state, southern Brazil. In addition, Amblyomma ovale tick infected by Rickettsia parkeri strain Atlantic rainforest has been also described in this area. Accordingly, the aim of this study was to investigate the occurrence of anti-Rickettsia spp. antibodies in human and dog populations, and Rickettsia spp. infection in ticks from oceanic islands and seashore mainland cities of southern Brazil. Serum samples were collected from 328 persons and their 282 dogs from three islands and two seashore mainland cities. A total of 211 ticks were collected from dogs, identified as A. ovale and R. sanguineus. In overall, 40 of 328 (12.2%) human samples were seropositive for Rickettsia spp., including 21 of 190 (11.1%) on islands and 19 of 138 (13.7%) on seashore mainland, and 62 of 282 (22.0%) dog samples, including 31 of 153 (20.3%) on islands and 31 of 129 (24.0%) in seashore mainland areas. In overall, nine of 82 (11.0%) ticks were positive to real-time PCR assay targeting a fragment of the rickettsial gltA gene, including two of 64 (3.1%) Rickettsia sanguineus and seven of 18 (38.9%) A. ovale, of which four were infected with the R. parkeri strain Atlantic rainforest. Despite no association between risk factors and Rickettsia spp. seropositivity was found in human beings, access to natural areas (p = .011) and tick infestation (p = .004) was significantly associated to dog seropositivity. The serological and molecular findings herein have confirmed previous tick and clinical case reports and enlarged the geographical occurrence of A. ovale infected by R. parkeri strain Atlantic rainforest in oceanic islands and seashore mainland cities of Paraná State, indicating a new likely transmission area of this new rickettsial infection in human beings and dogs of southern Brazil.