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Background Cutaneous leishmaniasis (CL) is an infectious vector-borne disease caused by protozoa of the Leishmania genus that affects humans and animals. The distribution of parasites in the lesion is not uniform, and there are divergences in the literature about the choice of the better sampling site for diagnosis–inner or outer edge of the ulcerated skin lesion. In this context, determining the region of the lesion with the highest parasite density and, consequently, the appropriate site for collecting samples can define the success of the laboratory diagnosis. Hence, this study aims to comparatively evaluate the parasite load by qPCR, quantification of amastigotes forms in the direct exam, and the histopathological profile on the inner and outer edges of ulcerated CL lesions. Methods Samples from ulcerated skin lesions from 39 patients with confirmed CL were examined. We performed scraping of the ulcer inner edge (base) and outer edge (raised border) and lesion biopsy for imprint and histopathological examination. Slides smears were stained by Giemsa and observed in optical microscopy, the material contained on the smears was used to determine parasite load by quantitative real-time PCR (qPCR) with primers directed to the Leishmania (Viannia) minicircle kinetoplast DNA. The histopathological exam was performed to evaluate cell profile, tissue alterations and semi-quantitative assessment of amastigote forms in inner and outer edges. Principal findings Parasite loads were higher on the inner edge compared to the outer edge of the lesions, either by qPCR technique (PP< 0.003). There was no significant difference in the parasite load between the imprint and scraping on the outer edge (P = 1.0000). Conclusion/Significance The results suggest that clinical specimens from the inner edge of the ulcerated CL lesions are the most suitable for both molecular diagnosis and direct parasitological examination.

Background Treatment of cutaneous leishmaniasis (CL) remains challenging since the drugs currently used are quite toxic, thus contributing to lethality unrelated to the disease itself but to adverse events (AE). The main objective was to evaluate different treatment regimens with meglumine antimoniate (MA), in a reference center in Rio de Janeiro, Brazil. Methodology A historical cohort of 592 patients that underwent physical and laboratory examination were enrolled between 2000 and 2017. The outcome measures of effectiveness were epithelialization and complete healing of cutaneous lesions. AE were graded using a standardized scale. Three groups were evaluated: Standard regimen (SR): intramuscular (IM) MA 10–20 mg Sb5+/kg/day during 20 days (n = 46); Alternative regimen (AR): IM MA 5 mg Sb5+/kg/day during 30 days (n = 456); Intralesional route (IL): MA infiltration in the lesion(s) through subcutaneous injections (n = 90). Statistical analysis was performed through Fisher exact and Pearson Chi-square tests, Kruskal-Wallis, Kaplan-Meier and log-rank tests. Results SR, AR and IL showed efficacy of 95.3%, 84.3% and 75.9%, with abandonment rate of 6.5%, 2.4% and 3.4%, respectively. IL patients had more comorbidities (58.9%; p = 0.001), were mostly over 50 years of age (55.6%), and had an evolution time longer than 2 months (65.6%; p = 0.02). Time for epithelialization and complete healing were similar in IL and IM MA groups (p = 0.9 and p = 0.5; respectively). Total AE and moderate to severe AE that frequently led to treatment interruption were more common in SR group, while AR and IL showed less toxicity. Conclusions/Significance AR and IL showed less toxicity and may be good options especially in CL cases with comorbidities, although SR treatment was more effective. IL treatment was an effective and safe strategy, and it may be used as first therapy option as well as a rescue scheme in patients initially treated with other drugs., Author summary Treatment of cutaneous leishmaniasis remains a challenge since the drugs used are quite toxic. Currently, there is a global effort to reduce the morbidity associated with the treatment of this disease and life-threatening complications due to drugs or treatment approaches. Meglumine antimoniate (MA) in different regimens was evaluated in cutaneous leishmaniasis patients in the state of Rio de Janeiro, Brazil. Effectiveness and toxicity were compared among the groups: standard regimen (SR) [intramuscular (IM) MA in the dosage of 10 to 20 mg of pentavalent antimony (Sb5+)/kg/day]; alternative regimen (AR) [IM MA in the dosage of 5 mg Sb5+/kg/day]; and intralesional route (IL) [patients treated with MA through the infiltration of the lesion]. AR and IL regimens demonstrated good effectiveness, with reduced abandonment rate and toxicity. Total adverse events were higher in the SR group, which frequently led to treatment interruptions. AR and IL showed less toxicity especially in CL cases with comorbidities, although SR treatment was more effective than AR and IL regimens. IL was an effective and safe treatment and may be used as a first therapy option as well as a rescue scheme.

Background We identified the species of Leishmania isolated from traveling and migrant patients attended in a reference center from 2000 to 2015, we performed the georeferencing of these species in Rio de Janeiro (RJ) state and we had knowledge about the human flows between the likely location of infection (LLI) and place of residence (PR) in RJ state, Brazil. Methodology/Principal findings This is a retrospective cross-sectional study including 171 patients diagnosed with ATL. Google Maps, OpenStreetMap, and Bing Maps were tools used to georeference LLI and PR. For etiological identification, we used isoenzyme electrophoresis, polymerase chain reaction-restriction fragment length polymorphism (molecular target hsp70C with restriction enzymes HaeIII and BstUI), and sequencing of the internal transcribed spacer of ribosomal DNA. ARCGIS software was used to create maps of the geographic distribution of Leishmania species in the state and municipality of RJ, together with flows between the LLI and PR. Isolates from 104 patients were identified as: L. (Viannia) braziliensis (80.8%), L. (V.) naiffi (7.7%), L. (V.) guyanensis (6.7%), L. (Leishmania) amazonensis (1%), and genetic variants of L. (V.) braziliensis (3.8%). The flow maps showed that the LLI included 4 countries, 19 Brazilian states, and 18 municipalities of RJ state. The Brazilian states with the highest density of cases were Amazonas (n = 32), Bahia (n = 18), and Ceará (n = 15). Conclusions/Significance This work is the first contribution to the knowledge of the routes of Leishmania species introduced in RJ state by migrants and travelers patients. L. (V.) braziliensis, L. (V.) guyanensis, L. (V.) naiffi, L. (L.) amazonensis, and genetic variants of L. (V.) braziliensis were identified in RJ state. To determine whether the autochthonous transmission of these imported species is possible it is necessary the adaptation of these species to environmental conditions as well as the presence of reservoirs and phlebotomine vectors in this region., Author summary Leishmaniasis is one of the most important neglected parasitic diseases in humans worldwide. American Tegumentary Leishmaniasis (ATL) is a disease caused by protozoa of the genus Leishmania, which are injected into the skin by small insects known by sandflies. Many different Leishmania species may cause ATL, which can affect the skin and/or oral and nasal mucosa. In this study, we analyzed flows between the likely location of infection (LLI) and the place of residence (PR) in Rio de Janeiro (RJ) state and identified Leishmania species isolated from travelers and migrants attended between 2000 and 2015 in a reference hospital for the care of patients with ATL. This work is the first contribution to knowledge about the routes of Leishmania species introduced to RJ state by migrant and traveling patients. In addition to Leishmania (Viannia) braziliensis, which was the most common species, other species have also been introduced to RJ state, including L. (V.) guyanensis, L. (V.) naiffi, L. (Leishmania) amazonensis, and genetic variants of L. (V.) braziliensis. Knowledge of the migratory flow and geographic location of these imported Leishmania species may contribute to the surveillance and control of ATL in RJ state, Brazil.

Although direct examination methods are important for diagnosing leishmaniasis, such methods are often neglected because of their low sensitivity relative to other techniques. Our study aimed to evaluate the performance of bone marrow (BM) thick smears and cytocentrifugation tests as alternatives to direct examination for diagnosing canine visceral leishmaniasis (CVL). Ninety-two dogs exhibiting leishmaniasis seroreactivity were evaluated. The animals were euthanized; and healthy skin, spleen, popliteal lymph node, and BM puncture samples were cultured. BM cultures were used as the reference standard. Of the 92 dogs studied, 85.9% exhibited positive cultures, and Leishmania infantum (synonym Leishmania chagasi) was confirmed in all positive culture cases. The sensitivity rates for cytocentrifugation as well as thin and thick smears were 47.1%, 52.8%, and 77%, respectively. However, no association between the dogs' clinical status and culture or direct examination results was found. To our knowledge, this was the first study to use thick smears and cytocentrifugation for diagnosing CVL. Our results indicate that BM thick smears have a good sensitivity and their use reduces the time required to read slides. Therefore, thick smears can provide a rapid and safe alternative to parasitological confirmation of seroreactive dogs.

The most severe clinical form of American tegumentary leishmaniasis (ATL) due to Leishmania braziliensis is mucosal leishmaniasis (ML), characterized by destructive lesions in the facial mucosa. We performed a retrospective cohort study of 109 ATL patients from Rio de Janeiro State, Brazil, where ATL is caused by L. braziliensis, to evaluate the influence of intestinal parasite coinfections in the clinical course of ATL. Parasitological stool examination (PSE) was performed with samples from all patients by the sedimentation, Kato-Katz and Baermann-Moraes methods. The diagnosis of ATL was made from lesion biopsies by direct observation of amastigotes in Giemsa-stained imprints, isolation of Leishmania promastigotes or histopathological examination.All patients were treated with meglumine antimoniate. Patients with positive PSE had a frequency of mucosal lesions significantly higher than those with negative PSE (p < 0.005). The same was observed for infections with helminths in general (p < 0.05), with nematodes (p < 0.05) and with Ascaris lumbricoides (p < 0.05), but not for protozoan infections. Patients with intestinal parasites had poor response to therapy (therapeutic failure or relapse) significantly more frequently than the patients with negative stool examination (p < 0.005). A similar difference (p < 0.005) was observed between patients with positive and negative results for intestinal helminths, but not for intestinal protozoa. Patients with positive PSE took significantly longer to heal than those with negative PSE (p < 0.005). A similar difference was observed for intestinal helminth infections (p < 0.005), but not for protozoan infections. Our results indicate a deleterious influence of intestinal helminth infections in the clinical course of ATL and evidence for the first time an association between ML and these coinfections, particularly with nematodes and A. lumbricoides.

Background Skin ulcers in American cutaneous leishmaniasis (ACL) may heal spontaneously after months/years. However, few cases may present quick heal even during diagnosis procedure (early spontaneous healing- ESH). The main objective of this study was to compare ESH patients with cases requiring specific treatment [non-ESH (NESH)]. Methods A historical cohort study of ACL patients (n = 445) were divided into 2 groups: ESH – spontaneously healed patients (n = 13; 2.90%), and NESH- treated patients (n = 432; 97.10%). We compared clinical and laboratorial findings at diagnosis, including the lesion healing process. Results ESH patients had a higher percentage of single lesions (p = 0.027), epithelialized lesion on initial examination (p = 0.001), lesions located in the dorsal trunk (p = 0.017), besides earlier healing (p

Objective: To elaborate figures that highlight the microscopic appearance of amastigote forms of the genus Leishmania and other structures subject to confusion during the routine of the direct examination of tegumentary leishmaniasis (TL). Study Design: We reviewed imprint and scraping slides previously prepared from patients with a definite diagnosis. Smear examinations were performed with an immersion objective (×1,000) selecting structures of interest for photodocumentation and elaboration of the illustrations. Results: We elaborated two 13 × 17 cm figures containing photomicrographs of amastigote forms with typical and atypical morphology of Leishmania parasites and other microorganisms, mainly fungi in the yeast-like phase and bacteria which could be confused with amastigote forms during the direct examination. Conclusions: The production of material like we show here is important and should be encouraged because of the need for constant training of professionals working in the area of TL diagnosis.

A leishmaniose visceral vem apresentando mudanças epidemiológicas marcantes nas últimas décadas, com acentuada expansão e surgimento de casos em áreas urbanas do Norte, Sudeste e Centro-Oeste do Brasil. O caso de Calazar aqui relatado, apesar de ser muito característico, apresentou grande dificuldade de diagnóstico, por se tratar de doença não-endêmica em Volta Redonda. A criança passou por duas internações em hospitais diferentes, porém, só obteve o diagnóstico correto após 11 meses do início dos sintomas. Neste relato são discutidos os principais diagnósticos diferenciais e chama-se a atenção de sempre considerar a hipótese de leishmaniose visceral em todo paciente com febre prolongada, hepatoesplenomegalia e pancitopenia, mesmo em áreas tradicionalmente não endêmicas. Visceral Leishmaniasis has been showing remarkable epidemiological changes in recent decades, with marked expansion and an emergence of cases in urban areas of the North, Southeast and Midwest regions of Brazil. The Kala-azar cases reported here, despite being very characteristic, presented a great difficulty of diagnosis, because the disease is not endemic in Volta Redonda. The child underwent two hospitalizations in different hospitals, but got the correct diagnosis only after 11 months of symptom onset. In this report we discuss the main differential diagnoses and call attention to the suspected symptoms of visceral leishmaniasis in patients with prolonged fever, hepatosplenomegaly and pancytopenia, even in areas not traditionally endemic for the disease.

Introduction In Brazil, visceral leishmaniasis (VL) has spread to various regions. This study reports canine cases of VL in Barra Mansa, where human VL cases were recently reported. Methods Using the human index case, a canine survey was performed by dual-path platform immunochromatography and enzyme-linked immunosorbent assay. Seropositive animals were euthanized. Cultures were collected to detect Leishmania parasites. Results Serological tests detected 141 canine VL cases, and Leishmania chagasi were isolated from 82.2% animals. Conclusions Leishmania chagasi is in circulation in Barra Mansa. This study broadens information on the parasite's distribution in the State of Rio de Janeiro.

Introduction Nine cases of visceral leishmaniasis occurred recently in Barra Mansa, State of Rio de Janeiro, with a high mortality rate. Methods We reviewed the medical records of the patients. Results Eight were male; 7 were adults. Patients who died progressed to death quickly and presented with aggravating factors: systemic steroid therapy before diagnosis, bleeding, severe liver involvement, infection, and/or refusal to receive transfusion. Conclusions We warn clinicians to be aware of the emergence of visceral leishmaniasis in new areas and to keep in mind the possibility of atypical clinical pictures and aggravating factors, so timely diagnosis can be made and prompt and adequate treatment can be initiated.

American tegumentary leishmaniasis (ATL) is an infectious disease that presents a wide spectrum of clinical manifestations making parasitological tests important for its diagnosis. Direct examination, although considered of low sensitivity is still employed mainly in areas with poor laboratory infrastructure. The aim of this study was to standardize the method of collecting and reading the scraping procedure and to then compare sensitivity of this procedure on two sites of the lesion (outer edge-OE and inner edge-IE) and of the imprint against the reference method (isolation in culture) in a group of 110 patients treated at a Referral Center in Rio de Janeiro, Brazil. ATL diagnosis was confirmed in 40 patients (36.4%), 39 cases were caused by L. braziliensis and 1 by L. amazonensis. Imprint was positive in 28 patients and scraping in OE in 17 and in IE in 25 patients, resulting in sensitivity of 70%, 42.5%, and 62.5% respectively. When the three direct examinations were combined, sensitivity value attained 77.5%. Aspects related to ease and quality of the collected material, pain intensity and frequency of bleeding in the scraping procedure were also broached and discussed in this study. The parameters of accuracy presented indicate that the direct methods can be safely used in ATL diagnosis, principally in IE scraping, as it is easy to produce and the examination is not costly, which allows the procedure to be repeated at different moments which, in turn, increases the possibility of finding the parasite. Despite that the direct methods are technically widespread, they are not standardized and the parameters of accuracy are unknown. If we consider the high incidence of leishmaniasis in low-income areas, the implantation of standardized and selective methods would provide advances in the diagnosis of leishmaniasis.

Dear Sir: We read with interest the article in the November 2014 issue of the Journal by Sousa and others entitled: “Press Imprint Smear: A Rapid, Simple, and Cheap Method for the Diagnosis of Cutaneous Leishmaniasis Caused by Leishmania (Viannia) braziliensis.” We agree with those authors that direct diagnosis by microscopic visualization of amastigotes is the first choice for the diagnosis of leishmaniasis. Advantages include speed, simple execution, and low operational cost, allowing use in poor endemic areas.1 The results obtained using a press imprint smear were very satisfactory, reaching a sensitivity of approximately 85%, while other direct diagnostic tests, such as traditional imprint, skin scraping, and slit skin smear, for leishmaniasis have had sensitivity values around 60–70%.2 The authors state that their method is cheaper than skin scraping. However, they did not consider the necessity of using anesthetic drugs, suturing material, a minimally structured outpatient setting, and sterile equipment in biopsy procedures. In addition, the described procedure must be performed by medical professionals, and this is the biggest drawback to the method in endemic settings. All of these aspects increase the cost of the imprint smear method, making it undoubtedly more expensive than skin scraping. In addition, skin scraping may be performed by a well-trained technician, facilitating the use of this procedure in areas with poor infrastructure. In relation to slide interpretation, the authors state that “A technician can learn to identify amastigotes with a short training course.” In fact, the finding of amastigotes of Leishmania sp. is not always an easy task. The possibility of the presence of other microorganisms in the lesion, such as Sporothrix schenckii and Histoplasma capsulatum, besides artifacts, highlights the need for skilled professionals for this procedure.3,4 We have conducted training courses in some Brazilian regions for professionals who conduct direct examination for leishmaniasis, and so appreciate the challenges in this activity. In addition to training, it is important to produce and disseminate informational materials, such as photomicrographs with amastigotes and other microorganisms, to enforce training, as exists for other health problems, such as malaria.5 We are pleased to note that other groups share our enthusiasm, and have worked to improve direct examination, which is the tool of choice, to safely diagnose leishmaniasis in endemic areas.