First report of putative Leishmania RNA virus 2 (LRV2) in Leishmania infantum strains from canine and human visceral leishmaniasis cases in the southeast of Brazil.

BACKGROUND: Leishmania RNA virus 1 (LRV1) is commonly found in South American Leishmania parasites belonging to the subgenus Viannia, whereas Leishmania RNA virus 2 (LRV2) was previously thought to be restricted to the Old-World pathogens of the subgenus Leishmania. OBJECTIVES: In this study, we investigated the presence of LRV2 in strains of Leishmania (L.) infantum, the causative agent of visceral leishmaniasis (VL), originating from different hosts, clinical forms, and geographical regions. METHODS: A total of seventy-one isolates were screened for LRV2 using semi-nested reverse transcription-polymerase chain reaction (RT-PCR) targeting the RNA-dependent RNA polymerase (RdRp) gene. FINDINGS: We detected LRV2 in two L. infantum isolates (CUR268 and HP-EMO) from canine and human cases, respectively. MAIN CONCLUSIONS: To the best of our knowledge, this is the first detection of LRV2 in the New World.

Gene Signatures of Symptomatic and Asymptomatic Clinical-Immunological Profiles of Human Infection by Leishmania (L.) chagasi in Amazonian Brazil.

Individuals infected with Leishmania (L.) chagasi may present different asymptomatic and symptomatic stages of infection, which vary in the clinical-immunological profiles that can be classified as asymptomatic infection (AI), subclinical resistant infection (SRI), indeterminate initial infection (III), subclinical oligosymptomatic infection (SOI), and symptomatic infection (SI) (=American visceral leishmaniasis, AVL). However, little is known about the molecular differences between individuals having each profile. Here, we performed whole-blood transcriptomic analyses of 56 infected individuals from Pará State (Brazilian Amazon), covering all five profiles. We then identified the gene signatures of each profile by comparing their transcriptome with those of 11 healthy individuals from the same area. Symptomatic individuals with SI (=AVL) and SOI profiles showed higher transcriptome perturbation when compared to those asymptomatic III, AI and SRI profiles, suggesting that disease severity may be associated with greater transcriptomic changes. Although the expression of many genes was altered on each profile, very few genes were shared among the profiles. This indicated that each profile has a unique gene signature. The innate immune system pathway was strongly activated only in asymptomatic AI and SRI profiles, suggesting the control of infection. In turn, pathways such as MHC Class II antigen presentation and NF-kB activation in B cells seemed to be specifically induced in symptomatic SI (=AVL) and SOI profiles. Moreover, cellular response to starvation was down-regulated in those symptomatic profiles. Overall, this study revealed five distinct transcriptional patterns associated to the clinical-immunological (symptomatic and asymptomatic) profiles of human L. (L.) chagasi-infection in the Brazilian Amazon.

First report of putative Leishmania RNA virus 2 (LRV2) in Leishmania infantum strains from canine and human visceral leishmaniasis cases in the southeast of Brazil

BACKGROUND Leishmania RNA virus 1 (LRV1) is commonly found in South American Leishmania parasites belonging to the subgenus Viannia, whereas Leishmania RNA virus 2 (LRV2) was previously thought to be restricted to the Old-World pathogens of the subgenus Leishmania. OBJECTIVES In this study, we investigated the presence of LRV2 in strains of Leishmania (L.) infantum, the causative agent of visceral leishmaniasis (VL), originating from different hosts, clinical forms, and geographical regions. METHODS A total of seventy-one isolates were screened for LRV2 using semi-nested reverse transcription-polymerase chain reaction (RT-PCR) targeting the RNA-dependent RNA polymerase (RdRp) gene. FINDINGS We detected LRV2 in two L. infantum isolates (CUR268 and HP-EMO) from canine and human cases, respectively. MAIN CONCLUSIONS To the best of our knowledge, this is the first detection of LRV2 in the New World.

Visceral Leishmaniasis Urbanization in the Brazilian Amazon Is Supported by Significantly Higher Infection Transmission Rates Than in Rural Area.

This was an open cohort prospective study (2016−2018) that analyzed the prevalence and incidence rates of human Leishmania (L.) infantum chagasi-infection and the evolution of their clinical-immunological profiles in distinct urban and rural scenarios of American visceral leishmaniasis (AVL) in Pará State, in the Brazilian Amazon. These infection profiles were based on species-specific DTH/IFAT-IgG assays and clinical evaluation of infected individuals, comprising five profiles: three asymptomatic, Asymptomatic Infection [AI], Subclinical Resistant Infection [SRI], and Indeterminate Initial Infection [III]; and two symptomatic, Subclinical Oligosymptomatic Infection [SOI] and Symptomatic Infection [SI = AVL]. The two distinct scenarios (900 km away) were the urban area of Conceição do Araguaia municipality and the rural area of Bujaru municipality in the southeast and northeast of Pará State. Human populations were chosen based on a simple convenience sampling design (5−10% in each setting), with 1723 individuals (5.3%) of the population (32,464) in the urban area and 1568 individuals (8.9%) of the population (17,596) in the rural one. A serological survey (IFAT-IgG) of canine infection was also performed in both scenarios: 195 dogs in the urban area and 381 in the rural one. Prevalence and incidence rates of human infection were higher in the urban area (20.3% and 13.6/100 person-years [py]) than in the rural setting (14.1% and 6.8/100-py). The AI profile was the most prevalent and incident in both urban (13.4% and 8.1/100-py) and rural (8.3% and 4.2/100-py) scenarios, but with higher rates in the former. An III profile case evolved to SOI profile after four weeks of incubation and another to SI (=AVL) after six. The prevalence of canine infection in an urban setting (39.2%) was also higher (p < 0.05) than that (32%) in the rural zone. AVL urbanization in Pará State, in the Brazilian Amazon, has led to infection rates significantly higher than those in rural sites, requiring more intense control measures.

Comparative Genomic Analyses of New and Old World Viscerotropic Leishmanine Parasites: Further Insights into the Origins of Visceral Leishmaniasis Agents.

Visceral leishmaniasis (VL), also known as kala-azar, is an anthropozoonotic disease affecting human populations on five continents. Aetiologic agents belong to the Leishmania (L.) donovani complex. Until the 1990s, three leishmanine parasites comprised this complex: L. (L.) donovani Laveran & Mesnil 1903, L. (L.) infantum Nicolle 1908, and L. (L.) chagasi Lainson & Shaw 1987 (=L. chagasi Cunha & Chagas 1937). The VL causal agent in the New World (NW) was previously identified as L. (L.) chagasi. After the development of molecular characterization, however, comparisons between L. (L.) chagasi and L. (L.) infantum showed high similarity, and L. (L.) chagasi was then regarded as synonymous with L. (L.) infantum. It was, therefore, suggested that L. (L.) chagasi was not native to the NW but had been introduced from the Old World by Iberian colonizers. However, in light of ecological evidence from the NW parasite's enzootic cycle involving a wild phlebotomine vector (Lutzomyia longipalpis) and a wild mammal reservoir (the fox, Cerdocyon thous), we have recently analyzed by molecular clock comparisons of the DNA polymerase alpha subunit gene the whole-genome sequence of L. (L.) infantum chagasi of the most prevalent clinical form, atypical dermal leishmaniasis (ADL), from Honduras (Central America) with that of the same parasite from Brazil (South America), as well as those of L. (L.) donovani (India) and L. (L.) infantum (Europe), which revealed that the Honduran parasite is older ancestry (382,800 ya) than the parasite from Brazil (143,300 ya), L. (L.) donovani (33,776 ya), or L. (L.) infantum (13,000 ya). In the present work, we have now amplified the genomic comparisons among these leishmanine parasites, exploring mainly the variations in the genome for each chromosome, and the number of genomic SNPs for each chromosome. Although the results of this new analysis have confirmed a high genomic similarity (~99%) among these parasites [except L. (L.) donovani], the Honduran parasite revealed a single structural variation on chromosome 17, and the highest frequency of genomic SNPs (more than twice the number seen in the Brazilian one), which together to its extraordinary ancestry (382,800 ya) represent strong evidence that L. (L.) chagasi/L. (L.) infantum chagasi is, in fact, native to the NW, and therefore with valid taxonomic status. Furthermore, the Honduran parasite, the most ancestral viscerotropic leishmanine parasite, showed genomic and clinical taxonomic characteristics compatible with a new Leishmania species causing ADL in Central America.

Whole-Genome Sequencing of Leishmania infantum chagasi Isolates from Honduras and Brazil.

This work reports on the whole-genome sequencing of Leishmania infantum chagasi from Honduras (Central America) and Brazil (South America).

First report of cutaneous mycetoma by Paecilomyces variotii and the successful treatment with combined itraconazole and terbinafine along with resection surgeries.

Mycetoma is a progressively mutilating infectious disease of the subcutaneous tissue that affects the skin and deep structures, which is poorly responsive to chemotherapy. Here, we report a skin mycetoma caused by Paecilomyces variotii, an uncommon fungus of human infections, and the therapeutic approach that resulted in a complete cure of the patient.

Detection of Pintomyia fischeri (Diptera: Psychodidae) With Leishmania infantum (Trypanosomatida: Trypanosomatidae) Promastigotes in a Focus of Visceral Leishmaniasis in Brazil.

Visceral leishmaniasis is spreading in Brazil where the main vector of its agent, Leishmania infantum Nicolle, 1908, is the Lutzomyia longipalpis (Lutz & Neiva, 1912) species complex (Diptera: Psychodidae: Phlebotominae), on which many of the activities of the visceral leishmaniasis surveillance program are based. However, there are areas where canine, and/or human cases have been occurring without the presence of this species complex as in the western part of the Greater São Paulo Metropolitan region, where Embu das Artes municipality is situated. In this area, Pintomyia fischeri (Pinto, 1926) has been implicated as potential vector of Le. infantum but so far its natural infection with this parasite has not yet been ascertained. Therefore, the present study sought to investigate the natural infection in sand flies of a CVL focus in Embu das Artes. The sand fly collections were undertaken with Shannon and CDC traps, monthly, between 1800 and 2100 hours from November 2018 to October 2019, inclusive. A total of 951 sand flies (457 males and 494 females), belonging to 10 species, were captured. Pintomyia fischeri was the predominant species (89.5%); of which 426 females were dissected and one of them (0.23%) was found to be harboring flagellates in its midgut. A sample of these flagellates was isolated in culture and characterized by a 234 base pair fragment of Leishmania heat-shock protein 70 gene (hsp70) and restriction fragment length polymorphism with Hae III restriction enzyme as Le. infantum. This finding reinforces previous evidence of Pi. fischeri as a vector of Le. infantum in foci of visceral leishmaniasis and highlights the importance of vector surveillance in areas where this species occurs.

Reactivity of purified and axenic amastigotes as a source of antigens to be used in serodiagnosis of canine visceral leishmaniasis.

Although there is a great diversity of techniques and antigens used in the serodiagnosis of canine visceral leishmaniasis (CVL), total sensitivity and specificity have not yet been found. Since the use of amastigote forms in the indirect immunofluorescence assay has shown an improvement in the specificity of the test for the diagnosis of CVL, the performance of amastigotes forms of L. (L.) infantum chagasi as antigen source were evaluated in automatized ELISA test using crude antigen of axenic amastigote and purified amastigote from spleen of hamster chronically infected comparing with ELISA using total antigen produced with promastigote forms of L. (L.) infantum chagasi. One hundred and fifteen sera from dogs with positive parasitological diagnosis by PCR were used. The animals were classified into 2 groups: symptomatic (n = 67) and asymptomatic (n = 48) animals, in accordance with the clinical signs and laboratory tests were. As control, ninety-four sera from dogs with negative parasitological diagnosis were included. No significant difference was found in sensitivity, specificity, predictive values and accuracy between ELISA using whole antigens produced with both axenic and purified amastigotes in comparison with promastigotes forms. Correlation and concordance between the three total antigens tested in ELISA was observed. According to the similar performance among antigens, data pointed out to use antigen from promastigote forms for diagnosing canine leishmaniasis, especially due the easily in the production, lower cost and the abundance of correlative literature.

Clinical and Immunological Features of Human Leishmania (L.) infantum-Infection, Novel Insights Honduras, Central America.

Leishmania (Leishmania) infantum is the etiological agent of both American visceral leishmaniasis (AVL) and non-ulcerated cutaneous leishmaniasis (NUCL) in Honduras. Although AVL is the most severe clinical form of infection, recent studies have shown that human immune response to parasite infection can result in a clinical-immunological spectrum. The overall prevalence rate of infection and clinical-immunological profiles of the L. (L.) infantum infection in Amapala municipality, South Honduras was determined. We examined 576 individuals with diagnosis based on combined ELISA (IgG/IgM) and DTH assays. We also used genus-specific kDNA PCR and Hsp70 PCR-RFLP for NUCL cases. Clinical evaluation found 82% asymptomatic and 18% symptomatic individuals. All symptomatic cases (n = 104) showing NUCL were positive for parasites. We identified L. (L.) infantum species in 100% of the skin lesion scrapings and in 90% of the blood samples from NUCL cases studied. A total of 320 asymptomatic individuals were exposed (ELISA+ and/or DTH+), providing an overall L. (L.) infantum prevalence of 73.6%. Clinical, parasitological, and immunological evaluations suggest seven infection profiles, three asymptomatic and four symptomatic. This represents the first report on clinical and immunological features of human L. (L.) infantum-infection in Amapala municipality, Honduras.

Histopathological features of skin lesions in patients affected by non-ulcerated or atypical cutaneous leishmaniasis in Honduras, Central America.

In Honduras visceral leishmaniasis and non-ulcerated or atypical cutaneous leishmaniasis (NUCL) are caused by the species Leishmania (L.) infantum chagasi. NUCL is the most common clinical form in the southern regions of the country, mainly affecting the young. In view of the lack of knowledge about the pathogenesis of the disease pattern caused by L. (L) infantum chagasi in individuals affected by NUCL, the aim of the present study was to describe in detail the histopathological features of the skin lesion caused by the parasite. Biopsies from human NUCL lesions with a positive parasitological diagnosis were collected and processed using standard histological techniques. Paraffin sections stained by haematoxylin and eosin were used to examine the histopathological alterations seen in the skin. The lesions varied between 3 and 5 mm, and the majority of the patients (60%) had a single lesion. Lesions were more frequently seen in females (65%), with an average age of 33.4 years. Microscopically, the skin lesions were characterized by mononuclear inflammatory infiltrate in the dermis composed of lymphocytes, macrophages and a few plasma cells. The intensity of the infiltration varied from discrete to intense. In both cases, the parasitic infection was discrete. Granulomas were present in 60% of cases and were associated with intense inflammation. The data revealed by the histopathological alterations in the skin of individuals affected by NUCL suggest activation of a cellular immune response that potentially controls parasite spreading.

Value of the oral swab for the molecular diagnosis of dogs in different stages of infection with Leishmania infantum.

This study was based on the need to employ a sensitive and specific method with samples that could be easily collected for diagnosing dogs infected with Leishmania infantum. To this end, we used real time-PCR (qPCR) to assess the value of the oral swab (OS) in detecting infected sick dogs (SD; n=62), including, for the first time, the analysis of apparently healthy infected dogs (AD; n=30), both from endemic areas for visceral leishmaniasis (VL). For comparison, we also evaluated the performance of the conjunctival swab (CS), blood (BL), lymph node (LN) and serology. We detected the presence of Leishmania DNA in the oral cavity in 62 out of the 92 dogs studied. The OS positivity (67.4%) was equivalent to the CS (68.5%) (p>0.05), higher than BL (52.2%) (p≤0.05), and lower than LN (84.8%) (p≤0.05). OS and CS performed well in SD dogs (82.3% and 83.9%, respectively) but not in AD dogs (36.7% for both samples). BL showed the lowest positivity (52.2%) and provided equivalent results between AD (60.0%) and SD (48.4%) dogs (p>0.05). LN yielded the highest positivity (84.8%), and it was also higher in the SD population (93.5%) compared to the AD population (66.7%) (p≤0.05). Parasite load was high in LN, moderate in OS and CS, and low in BL, showing the relationship between the levels of parasitism and the positivity rates found in these samples. Serology was positive in 82.2% of the SD group and in 70% of the AD dogs (p>0.05). Among the 20 seronegative dogs, seven (35%) were positive in either OS or CS, and 12 (60%) were positive when both noninvasive samples were jointly considered. The OS/CS combination resulted in a significant increase of positivity (p≤0.05) for the AD dogs (from 36.7% to 63.4%), as well as OS/serology (80%) and OS/CS/serology (83.4%). For the SD population, positivity reached up to 95.2% with the same combinations, showing that combination of samples and/or tests is required for the identification of dogs infected with L. infantum and that the OS and CS combination based on qPCR notably improves the detection of both AD and SD dogs. In conclusion, OS proved to be a suitable sample for the molecular diagnosis of infected dogs with clinical signs of VL, but not for dogs with inapparent infection. For these, we recommend the combination of OS results with CS and/or serology in order to reach relevant positivity for L. infantum. Finally, another advantage of using OS or both noninvasive samples is the increased likelihood of diagnosing seronegative dogs.

Asymptomatic dogs are highly competent to transmit Leishmania (Leishmania) infantum chagasi to the natural vector.

We evaluated the ability of dogs naturally infected with Leishmania (Leishmania) infantum chagasi to transfer the parasite to the vector and the factors associated with transmission. Thirty-eight infected dogs were confirmed to be infected by direct observation of Leishmania in lymph node smears. Dogs were grouped according to external clinical signs and laboratory data into symptomatic (n=24) and asymptomatic (n=14) animals. All dogs were sedated and submitted to xenodiagnosis with F1-laboratory-reared Lutzomyia longipalpis. After blood digestion, sand flies were dissected and examined for the presence of promastigotes. Following canine euthanasia, fragments of skin, lymph nodes, and spleen were collected and processed using immunohistochemistry to evaluate tissue parasitism. Specific antibodies were detected using an enzyme-linked immunosorbent assay. Antibody levels were found to be higher in symptomatic dogs compared to asymptomatic dogs (p=0.0396). Both groups presented amastigotes in lymph nodes, while skin parasitism was observed in only 58.3% of symptomatic and in 35.7% of asymptomatic dogs. Parasites were visualized in the spleens of 66.7% and 71.4% of symptomatic and asymptomatic dogs, respectively. Parasite load varied from mild to intense, and was not significantly different between groups. All asymptomatic dogs except for one (93%) were competent to transmit Leishmania to the vector, including eight (61.5%) without skin parasitism. Sixteen symptomatic animals (67%) infected sand flies; six (37.5%) showed no amastigotes in the skin. Skin parasitism was not crucial for the ability to infect Lutzomyia longipalpis but the presence of Leishmania in lymph nodes was significantly related to a positive xenodiagnosis. Additionally, a higher proportion of infected vectors that fed on asymptomatic dogs was observed (p=0.0494). Clinical severity was inversely correlated with the infection rate of sand flies (p=0.027) and was directly correlated with antibody levels (p=0.0379). Age and gender did not influence the transmissibility. Our data show that asymptomatic dogs are highly infective and competent for establishing sand fly infection, indicating their role in maintaining L. (L.) infantum chagasi cycle as well as their involvement in VL spreading in endemic areas.

Antifungal drug susceptibility profile of Pichia anomala isolates from patients presenting with nosocomial fungemia.

In vitro susceptibility of 58 isolates of Pichia anomala to five antifungal drugs using two broth microdilution methods (CLSI and EUCAST) was analyzed. Low susceptibility to itraconazole was observed. Fluconazole, voriconazole, amphotericin B, and caspofungin showed good antifungal activity, although relatively high drug concentrations were necessary to inhibit the isolates.