RESUMO
OBJECTIVE: To provide primary evidence of Trypanosoma cruzi landscape genetics in the Mexican Neotropics. MATERIALS AND METHODS: Trypanosoma cruzi and discrete typing units (DTU) prevalence were analyzed in landscape communities of vectors, wildlife, livestock, pets, and sympatric human populations using endpoint PCR and sequencing of all relevant amplicons from mitochondrial (kDNA) and nuclear (ME, 18S, 24Sα) gene markers. RESULTS: Although 98% of the infected sample-set (N=2 963) contained single or mixed infections of DTUI (TcI, 96.2%) and TcVI (22.6%), TcIV and TcII were also identified. Sensitivity of individual markers varied and was dependent on host taxon; kDNA, ME and 18S combined identified 95% of infections. ME genotyped 90% of vector infections, but 60% of mammals (36% wildlife), while neither 18S nor 24Sα typed more than 20% of mammal infections. CONCLUSION: Available gene fragments to identify or genotype T. cruzi are not universally sensitive for all landscape parasite populations, highlighting important T. cruzi heteroge- neity among mammal reservoir taxa and triatomine species.
Assuntos
Doença de Chagas , Trypanosoma cruzi , Animais , Humanos , Trypanosoma cruzi/genética , Animais Selvagens/genética , Doença de Chagas/epidemiologia , Doença de Chagas/veterinária , Doença de Chagas/parasitologia , Gado/genética , DNA de Cinetoplasto/genética , Mamíferos/genética , Mamíferos/parasitologia , GenótipoRESUMO
Leishmania infantum is a protozoan that causes visceral leishmaniasis (VL) in the Americas and some regions of Europe. The disease is mainly characterized by hepatosplenomegaly and fever, and can be fatal. Factors related to the host and parasite can contribute to the transmission of Leishmania and the clinical outcome. The intraspecific genetic variability of L. infantum strains may be one of these factors. In this study, we evaluated the genetic variability of L. infantum obtained from bone marrow smear slides from patients in the Sao Paulo State, Brazil. For this, the minicircle of the kDNA hypervariable region was used as target by Sanger sequencing. By analyzing the similarity of the nucleotides and the maximum likelihood tree (Fasttree), we observed a high similarity (98%) among samples. Moreover, we identified four different profiles of L. infantum. In conclusion, L. infantum strains from Sao Paulo State, Brazil, showed low diversity measured by minicircle of the kDNA hypervariable region.
Assuntos
Doenças do Cão , Leishmania infantum , Leishmaniose Visceral , Animais , Cães , Humanos , Leishmania infantum/genética , Leishmaniose Visceral/parasitologia , DNA de Cinetoplasto/genética , Brasil , Doenças do Cão/parasitologiaRESUMO
BACKGROUND: The Gran Chaco region is a major hotspot of Chagas disease. We implemented a 9-year program aimed at suppressing house infestation with Triatoma infestans and stopping vector-borne transmission to creole and indigenous (Qom) residents across Pampa del Indio municipality (Argentine Chaco). The aim of the present study was to assess the intervention effects on parasite-based transmission indices and the spatial distribution of the parasite, and test whether house-level variations in triatomine infection with Trypanosoma cruzi declined postintervention and were influenced by household ethnicity, persistent infestation linked to pyrethroid resistance and other determinants of bug infection. METHODS: This longitudinal study assessed house infestation and bug infection with T. cruzi before and after spraying houses with pyrethroids and implemented systematic surveillance-and-response measures across four operational areas over the period 2007-2016. Live triatomines were individually examined for infection by optical microscopy or kinetoplast DNA (kDNA)-PCR and declared to be infected with T. cruzi when assessed positive by either method. RESULTS: The prevalence of infection with T. cruzi was 19.4% among 6397 T. infestans examined. Infection ranged widely among the study areas (12.5-26.0%), household ethnicity (15.3-26.9%), bug ecotopes (1.8-27.2%) and developmental stages (5.9-27.6%), and decreased from 24.1% (baseline) to 0.9% (endpoint). Using random-intercept multiple logistic regression, the relative odds of bug infection strongly decreased as the intervention period progressed, and increased with baseline domestic infestation and bug stage and in Qom households. The abundance of infected bugs and the proportion of houses with ≥ 1 infected bug remained depressed postintervention and were more informative of area-wide risk status than the prevalence of bug infection. Global spatial analysis revealed sharp changes in the aggregation of bug infection after the attack phase. Baseline domestic infestation and baseline bug infection strongly predicted the future occurrence of bug infection, as did persistent domestic infestation in the area with multiple pyrethroid-resistant foci. Only 19% of houses had a baseline domestic infestation and 56% had ever had ≥ 1 infected bug. CONCLUSIONS: Persistent bug infection postintervention was closely associated with persistent foci generated by pyrethroid resistance. Postintervention parasite-based indices closely agreed with human serosurveys at the study endpoint, suggesting transmission blockage. The program identified households and population subgroups for targeted interventions and opened new opportunities for risk prioritization and sustainable vector control and disease prevention.
Assuntos
Doença de Chagas , Piretrinas , Triatoma , Trypanosoma cruzi , Animais , Humanos , Triatoma/parasitologia , Prevalência , Estudos Longitudinais , Insetos Vetores/parasitologia , Doença de Chagas/epidemiologia , Doença de Chagas/prevenção & controle , Piretrinas/farmacologia , DNA de Cinetoplasto , Argentina/epidemiologiaRESUMO
BACKGROUND: Cutaneous leishmaniasis (CL) is a neglected disease and a public health problem in Latin America. The diagnosis of CL in poor hyperendemic regions relies to large extent on the identification of amastigotes in Giemsa-stained smears. There is an urgent need for a rapid, sensitive and low cost diagnostic method for use in field conditions for CL as current modalities are not readily available. The primary objective of this study was to determine the sensitivity and specificity of the FDA-cleared CL Detect Rapid Test in Peru, using modified test procedures rather than the instructions-for-use, by 1) increasing the extraction time and 2) increasing the volume of the sample added to the test strip. CL Detect Rapid Test results were compared against microscopy and kDNA-PCR, for the diagnosis of CL in ulcerated lesions. In addition, we compared two collection methods the dental broach used and mentioned in the CL Detect insert and the standard less invasive and easier to conduct scrapping method. METHODOLOGY: Participants were patients who presented for medical consultation due to a suspected CL lesion. Four samples from the index lesion were collected using a dental broach, per package insert, and lancet scraping and tested by the modified CL Detect Rapid Test, microscopy, and PCR. PRINCIPAL FINDINGS: A total of 156 subjects were eligible and evaluated. The modified CL Detect sensitivity was higher in specimens obtained by scraping (83.3%) than those from dental broach (64.2%). The specificity was lower in scrapings (77.8%) with a false positive rate of 22.2% compared with dental broach samples (91.7%) with a false positive rate of 8.3%. However, molecular analysis showed that all 8 false negative microscopy scrapings (those positive by modified CL Detect and negative by microscopy) were positive by kDNA-PCR, meaning that the modified CL Detect was more sensitive than microscopy. CONCLUSIONS: These modifications to the package insert that resulted in a diagnostic sensitivity (83.3%) comparable to microscopy for species found in Peru may enable earlier anti-leishmanial drug treatment decisions based on a positive result from the CL Detect Rapid Test alone until further diagnostic tests like microscopy and PCR can be performed. TRIAL REGISTRATION: NCT03762070; Clinicaltrials.gov.
Assuntos
Leishmania , Leishmaniose Cutânea , Humanos , DNA de Cinetoplasto , Peru , Leishmaniose Cutânea/diagnóstico , Leishmania/genética , Reação em Cadeia da Polimerase/métodos , Sensibilidade e EspecificidadeRESUMO
BACKGROUND: This study aimed to describe the kinetics of Leishmania parasite load determined using kinetoplast DNA (kDNA)-based quantitative polymerase chain reaction (qPCR) in visceral leishmaniasis (VL) patients. METHODS: Parasite load in blood was assessed by qPCR at five time points, up to 12 months post-diagnosis. Sixteen patients were followed up. RESULTS: A significant reduction in the parasite load was observed after treatment (P < 0.0001). One patient had an increased parasite load 3 months post-treatment and relapsed clinically at month six. CONCLUSIONS: We have described the use of kDNA-based qPCR in the post-treatment follow-up of VL cases.
Assuntos
Leishmania , Leishmaniose Visceral , Humanos , Leishmaniose Visceral/diagnóstico , Leishmaniose Visceral/tratamento farmacológico , Leishmaniose Visceral/parasitologia , DNA de Cinetoplasto/genética , Brasil , Leishmania/genética , Carga ParasitáriaRESUMO
The aim of this study was to characterize Leishmania spp. from canine and feline samples using Polymerase Chain Reaction (PCR)- Restriction Fragment Length Polymorphism (RFLP). It was conducted in the southern region of Brazil, located at border crossings to Argentina and Uruguay. Samples were collected from 116 dogs (Canis lupus familiaris) and 89 cats (Felis catus). The PCR was performed to screen for an LT1 fragment from kinetoplast DNA (kDNA) target gene, and positive samples were subjected to a second PCR for an internal transcribed spacers (ITS1) region from ribosomal DNA (rDNA) target. RFLP was performed using the Haemophilus aegyptius (HAE III) restriction endonuclease (Fermentas ®). Positive samples by PCR ITS1 were sequenced and deposited in NCBI GenBank, and a phylogenetic analysis was developed. We found that 12.9% (15/116) of the samples from dogs were positive. All the 89 cat samples were negative. Positive samples were tested against Leishmania reference strains presenting different patterns in PCR-RFLP, and these samples showed bands denoting similarity to the standard species of Leishmania infantum, proven through sequencing and phylogenetic analysis. The RFLP technique, alone, was shown to be feasible for practical application and confirmation of the involved Leishmania spp.
Assuntos
Doenças do Gato , Doenças do Cão , Leishmania infantum , Leishmaniose Visceral , Animais , Animais Domésticos , Brasil , Doenças do Gato/diagnóstico , Doenças do Gato/epidemiologia , Doenças do Gato/parasitologia , Gatos , DNA de Cinetoplasto/genética , Doenças do Cão/diagnóstico , Doenças do Cão/parasitologia , Cães , Leishmania infantum/genética , Leishmaniose Visceral/veterinária , Filogenia , Reação em Cadeia da Polimerase/veterináriaRESUMO
One strategy to identify transmission foci is based on vector monitoring, and efficient methods are important for vector control. Our study evaluated different light sources (red, green, ultraviolet, blue, and incandescent light) to capture sand fly in Porto Velho, Brazil. We also evaluated Leishmania and blood meal sources in females. A total of 1,943 individuals were identified in 45 species level-taxa, with Trichophoromyia ubiquitalis (Mangabeira) (n = 364), Nyssomyia antunesi (n = 241), Bichromomyia flaviscutellata (Mangabeira) (n = 222), and Psychodopygus davisi (Root) (n = 148) being the most abundant. Incandescent light captured most individuals (n = 589), followed by blue (n = 471), green (n = 452), ultraviolet (n = 281) and red (n = 150). No significant difference was observed between the species composition and lights (PERMANOVA: Pseudo F = 1.29, p = 0.14, NMDS: Stress 0.18). The Shannon and Simpson indices demonstrated a high diversity captured using all lights. Our data demonstrated that LEDs are alternative devices for sand fly capture, with blue and green LEDs presenting similar results to incandescent light. 53 pools were analyzed, only one pool was positive for kDNA and hsp70 targets [Nyssomyia fraihai (Martins, Falão & Silva)] and identified as Endotrypanum spp., suggesting that other trypanosomatids may circulate in the locality. Choloepus hoffmanni (two-toed sloth) Peters, Homo sapiens Linnaeus, Proechimys gardneri (Gardner's spiny rat) Da Silva, and Tamandua tetradactyla (lesser anteater) (Linnaeus) were blood meal sources identified in females, increasing the knowledge about sources used by these insects.
Assuntos
Leishmania , Phlebotomus , Psychodidae , Animais , Brasil , DNA de Cinetoplasto , Feminino , Insetos Vetores , Leishmania/genética , Phlebotomus/genética , Psychodidae/genéticaRESUMO
Leishmaniases comprise a spectrum of diseases caused by protozoan parasites of the genus Leishmania, with some species of rodents being incriminated as reservoirs. The capybara is the largest extant rodent species in the world and is widely distributed in South America. The occurrence of infection by Leishmania spp. was investigated in capybaras captured in Brazil during 20152019 from established populations in five highly anthropic areas of the state of São Paulo and two natural areas of the states of Mato Grosso and Mato Grosso do Sul. A total of 186 individuals were captured and subjected to abdominal skin biopsy. All skin samples were Leishmania kDNA-negative, suggesting that capybaras have no role in the transmission cycles of Leishmania species in the studied areas despite the well-known role of other rodents in the life cycle of Leishmania spp.(AU)
As leishmanioses compreendem um espectro de doenças causadas por protozoários do gênero Leishmania e algumas espécies de roedores são incriminadas como reservatórios de Leishmania spp. As capivaras compreendem a maior espécie de roedores existentes e são amplamente distribuídas na América do Sul. Para investigar a ocorrência de infecção por Leishmania spp. em capivaras, durante os anos de 2015-2019 capivaras foram capturadas em cinco áreas antrópicas do estado de São Paulo e em duas áreas naturais dos estados do Mato Grosso e do Mato Grosso do Sul, todos esses ambientes com populações de capivaras estabelecidas. Um total de 186 indivíduos foram capturados e submetidos à biópsia de pele abdominal. Todas as amostras de pele foram negativas para o alvo kDNA, assim, os dados sugerem que nas áreas estudadas as capivaras não têm papel no ciclo de transmissão de espécies de Leishmania spp., apesar do papel bem conhecido de outros roedores no ciclo de vida de Leishmania spp.(AU)
Assuntos
Animais , Infecções Protozoárias em Animais/diagnóstico , Roedores/microbiologia , Leishmaniose/diagnóstico , Pele/microbiologia , Biópsia/instrumentação , Brasil , DNA de Cinetoplasto/análise , Leishmania/genéticaRESUMO
The study aimed to develop a multiplex qPCR to detect Leishmania infantum load in different sandfly sample settings using Leishmania kDNA and sandfly vacuolar ATPase (VATP) subunit C as internal control gene. The amplification of Lutzomyia longipalpis VATP gene was evaluated together with Leishmania infantum kDNA in a multiplex reaction. The concentration of VATP gene oligonucleotides was adjusted until no statistically significant difference was observed between all multiplex standard curves and singleplex curves, that is, only kDNA amplification. Limit of detection (LoD) was measured using a probit model and a cut-off defined by receiver operating characteristic analysis. Limit of quantification (LoQ) was assessed by a linear model using the coefficient of variation threshold of 25%. After assuring VATP gene amplification, its primer-probe concentrations were best at 100 nM/10 nM, respectively. The cut-off Cq value for L. infantum kDNA was defined as 35.46 with 100% of sensitivity and specificity. A total of 95% LoD was determined to be of 0.162 parasites while LoQ was 5.858. Our VATP/kDNA multiplex qPCR assay shows that it can be used to evaluate both DNA integrity and determine L. infantum load in L. longipalpis even for low yielded samples, that is, individual midguts.
Assuntos
Leishmania infantum , Phlebotomus , Psychodidae , Animais , DNA de Cinetoplasto/genética , Leishmania infantum/genética , Psychodidae/parasitologia , Reação em Cadeia da Polimerase em Tempo Real/veterináriaRESUMO
Studying the occurrence of Leishmania in bats would help toward clarifying the role of these animals in epidemiological cycles. Therefore, the objective of this study was to detect Leishmania spp. in bats from the Brazilian State of Espírito Santo. In total, 105 bats from 11 species which had been collected from 17 municipalities of Espírito Santo were obtained from the IDAF Rabies Diagnostic Laboratory between December 2018 and June 2019. DNA was extracted from the bone marrow, skin, and spleen samples and analysed using conventional PCR, with primers specific for the kinetoplastic DNA minicircle of the parasitic species Leishmania (Viannnia) braziliensis, Leishmania (Leishmania) infantum, and Leishmania (Leishmania) amazonensis. To identify the Leishmania species, the positive PCR products were analysed using PCR-RFLP with the HaeIII enzyme and 8% polyacrylamide gel electrophoresis. Positivity for L. (V.) braziliensis was observed in five (4.76%) bats: namely, one male Eumops glaucinus from Vitória (in a skin sample), one male Artibeus lituratus from Vila Velha and one female Eumops glaucinus from Vitória (both in bone marrow samples), and one male A. lituratus from Vitória and one male Molossus rufus from Guarapari (both in the spleen samples). To our best knowledge, this is the first report of the detection of Leishmania in bats from Espírito Santo as well as of Leishmania DNA in the bone marrow of bats.
Assuntos
Quirópteros , Leishmania braziliensis , Leishmania infantum , Animais , Brasil/epidemiologia , DNA de Cinetoplasto , Feminino , Leishmania braziliensis/genética , Leishmania infantum/genética , MasculinoRESUMO
Trypanosoma cruzi, as other kinetoplastids, has a complex mechanism of editing of mitochondrial mRNAs that requires guide RNAs (gRNAs) coded in DNA minicircles in the kinetoplast. There are many variations on this mechanism among species. mRNA editing and gRNA repertoires are almost unknown in T. cruzi. Here, gRNAs were inferred based on deep-sequenced minicircle hypervariable regions (mHVRs) and editing cascades were rebuilt in strains belonging to the six main T. cruzi lineages. Inferred gRNAs were clustered according to their sequence similarity to constitute gRNA classes. Extreme diversity of gRNA classes was observed, which implied highly divergent gRNA repertoires among different lineages, even within some lineages. In addition, a variable gRNA class redundancy (i.e., different gRNA classes editing the same mRNA region) was detected among strains. Some strains had upon four times more gRNA classes than others. Such variations in redundancy affected gRNA classes of all mRNAs in a concerted way, i.e., there are correlated variations in the number of gRNAs classes editing each mRNA. Interestingly, cascades were incomplete for components of the respiratory complex I in several strains. Finally, gRNA classes of different strains may potentially edit mitochondrial mRNAs from other lineages in the same way as they edit their own mitochondrial mRNAs, which is a prerequisite for biparental inheritance of minicircle in hybrids. We propose that genetic exchange and biparental inheritance of minicircles combined with minicircle drift due to (partial) random segregation of minicircles during kDNA replication is a suitable hypothesis to explain the divergences among strains and the high levels of gRNA redundancy in some strains. In addition, our results support that the complex I may not be required in some stages in the life cycle as previously shown and that linkage (in the same minicircle) of gRNAs that edit different mRNAs may prevent gRNA class lost in such stage.
Assuntos
RNA Guia de Cinetoplastídeos , Trypanosoma brucei brucei , Trypanosoma cruzi , Sequência de Bases , DNA de Cinetoplasto , RNA Guia de Cinetoplastídeos/genética , Trypanosoma brucei brucei/genética , Trypanosoma cruzi/genéticaRESUMO
BACKGROUND: Control of cutaneous leishmaniasis by public health systems in the Americas relies on case identification and treatment. Point-of-care diagnostics that can be performed by health workers within or near affected communities could effectively bring the health system to the resource-limited sites providing early diagnosis and treatment, reducing morbidity and the burden of disease. METHODOLOGY/PRINCIPAL FINDINGS: A cross-sectional study was undertaken to evaluate the diagnostic test performance of Isothermal Recombinase Polymerase Amplification (RPA) targeting Leishmania kinetoplast DNA, coupled with a lateral flow (LF) immunochromatographic strip, in a field setting and a laboratory reference center. Minimally invasive swab and FTA filter paper samples were obtained by community health workers and highly trained technicians from ulcerated lesions of > 2 weeks' evolution from 118 patients' ≥ 2 years of age in the municipality of Tumaco, Nariño. Extracted DNA was processed by RPA-LF at a reference center or in a primary health facility in the field. Evaluation was based on a composite "gold standard" that included microscopy, culture, biopsy and real-time polymerase chain reaction detection of Leishmania 18S rDNA. Standard of care routine diagnostic tests were explored as comparators. Sensitivity and specificity of RPA-LF in the reference lab scenario were 87% (95%CI 74-94) and 86% (95%CI 74-97), respectively. In the field scenario, the sensitivity was 75% (95%CI 65-84) and specificity 89% (95%CI 78-99). Positive likelihood ratios in both scenarios were higher than 6 while negative likelihood ratios ranged to 0.2-0.3 supporting the usefulness of RPA-LF to rule-in and potentially to rule-out infection. CONCLUSIONS/SIGNIFICANCE: The low complexity requirements of RPA-LF combined with non-invasive sampling support the feasibility of its utilization by community health workers with the goal of strengthening the diagnostic capacity for cutaneous leishmaniasis in Colombia. TRIAL REGISTRATION: ClinicalTrials.gov NCT04500873.
Assuntos
Leishmania/genética , Leishmania/isolamento & purificação , Leishmaniose Cutânea/diagnóstico , Técnicas de Diagnóstico Molecular/métodos , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Criança , Pré-Escolar , Cromatografia de Afinidade , Colômbia , Estudos Transversais , Primers do DNA/genética , DNA de Cinetoplasto/genética , DNA de Protozoário/genética , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Técnicas de Amplificação de Ácido Nucleico , Sensibilidade e Especificidade , Adulto JovemRESUMO
Angomonas deanei coevolves in a mutualistic relationship with a symbiotic bacterium that divides in synchronicity with other host cell structures. Trypanosomatid mitochondrial DNA is contained in the kinetoplast and is composed of thousands of interlocked DNA circles (kDNA). The arrangement of kDNA is related to the presence of histone-like proteins, known as KAPs (kinetoplast-associated proteins), that neutralize the negatively charged kDNA, thereby affecting the activity of mitochondrial enzymes involved in replication, transcription and repair. In this study, CRISPR-Cas9 was used to delete both alleles of the A. deanei KAP4 gene. Gene-deficient mutants exhibited high compaction of the kDNA network and displayed atypical phenotypes, such as the appearance of a filamentous symbionts, cells containing two nuclei and one kinetoplast, and division blocks. Treatment with cisplatin and UV showed that Δkap4 null mutants were not more sensitive to DNA damage and repair than wild-type cells. Notably, lesions caused by these genotoxic agents in the mitochondrial DNA could be repaired, suggesting that the kDNA in the kinetoplast of trypanosomatids has unique repair mechanisms. Taken together, our data indicate that although KAP4 is not an essential protein, it plays important roles in kDNA arrangement and replication, as well as in the maintenance of symbiosis.
Assuntos
Bactérias/metabolismo , Replicação do DNA , DNA de Cinetoplasto/genética , DNA de Protozoário/genética , Mitocôndrias/genética , Proteínas de Protozoários/genética , Trypanosomatina/genética , Divisão Celular , Núcleo Celular , DNA de Cinetoplasto/metabolismo , DNA Mitocondrial/genética , DNA Mitocondrial/metabolismo , DNA de Protozoário/metabolismo , Mitocôndrias/metabolismo , Proteínas de Protozoários/metabolismo , Simbiose , Trypanosomatina/metabolismo , Trypanosomatina/microbiologiaRESUMO
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 (P<0.001) and histopathological examination (P< 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.
Assuntos
DNA de Cinetoplasto/análise , Leishmania braziliensis , Leishmaniose Cutânea/parasitologia , Reação em Cadeia da Polimerase em Tempo Real/métodos , Úlcera/parasitologia , Adulto , Feminino , Humanos , Leishmania braziliensis/genética , Leishmania braziliensis/isolamento & purificação , Masculino , Pessoa de Meia-Idade , Carga ParasitáriaRESUMO
The main clinical, anatomopathological, and molecular aspects of the infection by Leishmania infantum are described in two cats with multicentric cutaneous, nodular, and ulcerated lesions. The animals were submitted to a clinical examination, followed by serological, molecular and parasitological exams, with culture and isolation of the parasite, and subsequent isoenzymatic characterization. The animals were euthanized and necropsied. Case 1 was an adult, female, mixed-bred stray cat. Case 2 was an adult, male, mixed-bred and domiciled cat. Both were positive for the presence of anti-L. infantum antibodies. In the cytology of the cutaneous nodules and lymph nodes, amastigote forms of Leishmania spp. could be visualized, free and in the interior of the macrophages. In the histopathology, the lesions were characterized by nodular granulomatous and/or ulcerative dermatitis, associated to amastigote forms of Leishmania spp. By means of the polymerase chain reaction, the sequence of the L. infantum kDNA minicircle was amplified. It is concluded that the infection by L. infantum occurs in cats in the State of Paraíba, Northeast region of Brazil and the need to understand the immunological profile of the visceral leishmaniasis in the feline population is highlighted with aimed at the control measures in public health.
Assuntos
Doenças do Gato , Leishmania infantum , Leishmaniose Visceral , Animais , Anticorpos Antiprotozoários/sangue , Brasil , Doenças do Gato/diagnóstico , Gatos , DNA de Cinetoplasto/genética , Eutanásia Animal , Feminino , Leishmania infantum/genética , Leishmaniose Visceral/diagnóstico , Leishmaniose Visceral/transmissão , Leishmaniose Visceral/veterinária , Macrófagos/parasitologia , Masculino , Reação em Cadeia da Polimerase/veterináriaRESUMO
BACKGROUND: There is little information about the frequency of Leishmania infection in asymptomatic people living with HIV (PLWH) and about the performance of laboratory diagnostic methods in coinfected patients in Latin America. The main objective of this study is to evaluate the frequency of Leishmania spp. infection in HIV-infected patients living in an urban area in Brazil. METHODS: To detect Leishmania infection, diagnostic tests were performed to detect anti-Leishmania antibodies (ELISA using Leptomonas seymouri antigens; ELISA using rK39 antigens; ELISA using rK28 antigens; indirect fluorescent-antibody test (IFAT); direct agglutination test (DAT)) and Leishmania DNA (polymerase chain reaction (PCR) with the target genes kDNA and ITS-1). RESULTS: The frequency of at least one positive test was 15%. For ELISA using Leptomonas antigens and IFAT, there was an association between CD4+ T lymphocyte counts and test positivity, with a higher positivity of these tests in more immunosuppressed patients (CD4+ T cell count < 200/mm3). CONCLUSIONS: According to our data, there was a high prevalence of Leishmania spp. infections in this population living with HIV. Although there is the possibility of cross-reaction, some tests that are considered highly specific for the diagnosis of Leishmania infection were positive. There was also an association between the positivity of some tests studied and lower values of CD4+ T lymphocytes.
Assuntos
Coinfecção/epidemiologia , Infecções por HIV/epidemiologia , HIV , Leishmania/genética , Leishmania/imunologia , Leishmaniose Visceral/diagnóstico , Leishmaniose Visceral/epidemiologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Testes de Aglutinação , Animais , Brasil/epidemiologia , Contagem de Linfócito CD4 , Estudos de Coortes , Coinfecção/virologia , Estudos Transversais , DNA de Cinetoplasto/genética , Testes Diagnósticos de Rotina/métodos , Ensaio de Imunoadsorção Enzimática , Feminino , Técnica Indireta de Fluorescência para Anticorpo , Infecções por HIV/virologia , Humanos , Leishmaniose Visceral/parasitologia , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , Prevalência , Adulto JovemRESUMO
Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of the mannose phosphate isomerase (mpi) gene was applied to 134 skin samples collected from patients with cutaneous leishmaniasis (CL) in Peru for identification of the infecting parasite at the species level, and the results were compared with those of cytochrome b (cyt b) gene sequencing obtained in previous studies. Although most results (121/134) including 4 hybrids of Leishmania (Viannia) braziliensis and L. (V.) peruviana corresponded to those obtained in the previous study, PCR-RFLP analyses revealed the distribution of putative hybrid strains between L. (V.) peruviana and L. (V.) lainsoni in two samples, which has never been reported. Moreover, parasite strains showing discordance between kinetoplast and nuclear genes (kDNA and nDNA), so-called mito-nuclear discordance, were identified in 11 samples. Of these, six strains had the kDNAs of L. (V.) braziliensis or L. (V.) peruviana and nDNAs of L. (V.) guyanensis, and three strains had the kDNAs of L. (V.) shawi and nDNAs of L. (V.) braziliensis. The rest were identified as mito-nuclear discordance strains having kDNAs of L. (V.) braziliensis or L. (V.) peruviana and nDNAs of L. (V.) lainsoni, and kDNAs of L. (V.) lainsoni and nDNAs of L. (V.) braziliensis. The results demonstrate that Leishmania strains in Peru are genetically more complex than previously considered.
Assuntos
Núcleo Celular/parasitologia , DNA de Cinetoplasto/genética , DNA de Protozoário/genética , Leishmania/genética , Leishmaniose Cutânea/parasitologia , Cruzamentos Genéticos , Citocromos b/genética , Humanos , Leishmania/classificação , Leishmania/isolamento & purificação , Peru , Filogenia , Proteínas de Protozoários/genéticaRESUMO
BACKGROUND: Almost 1000 cases of American cutaneous leishmaniasis have been registered yearly in Rondônia State, Brazil. Little is known about the Leishmania transmission cycle (vectors and reservoirs) in the state. This study aimed to evaluate sand fly fauna from two vertical stratification layers in order to identify potential vectors and their blood-meal sources. METHODS: The study was conducted in Jamari National Forest. Sand flies were collected in the canopy (15 m) and at ground level (1 m) using HP light traps during four months, February, April, August and October, 2018. Insects were identified to the species level, and females were subjected to DNA extraction and PCR targeting minicircle kDNA and hsp70 (for Leishmania detection and species identification), and cytb (to identify blood-meal sources). Exploratory data analysis was used to determine mean of abundance and species richness between stratifications. The hsp70 and cytb sequences were analyzed and compared with sequences from GenBank. RESULTS: Overall, 68 species were identified from 15,457 individuals. On the Potosi trail, 7531 individuals of 49 species were collected; canopy captures totaled 6463 individuals of 46 species, while ground captures totaled 1068 individuals of 38 species. On the Santa Maria trail, 7926 individuals of 61 species were collected; canopy captures totaled 6136 individuals of 51 species, while ground captures totaled 1790 individuals of 53 species. A total of 23 pools were positive for kDNA (canopy n = 21, ground n = 2). Only two samples were sequenced for hsp70 (both in canopy); one sequence exhibited similarity with Leishmania braziliensis (Lutzomyia davisi pool) and another with L. naiffi (Lu. antunesi pool). The cytb fragment was amplified in 11 of 86 samples. Sample sequencing identified cytb DNA from 5 blood-meal sources: Micrastur gilvicollis, Psophia viridis, Tamandua tetradactyla, Homo sapiens and Choloepus didactylus. CONCLUSIONS: Sand fly fauna is more diverse in the canopy than at ground level. Factors such as blood-meal sources, resting sites, and abiotic components probably contribute to high abundance in the canopy. Our results reinforce the possibility that Lu. antunesi and Lu. davisi participate in Leishmania transmission in forest environments and may play an important role in transmission from sylvatic to human hosts.
Assuntos
Leishmania , Leishmaniose Cutânea/transmissão , Psychodidae/parasitologia , Animais , Biodiversidade , Sangue/parasitologia , Brasil , Citocromos b/genética , DNA de Cinetoplasto , DNA de Protozoário/genética , Reservatórios de Doenças , Feminino , Proteínas de Choque Térmico HSP70/genética , Humanos , Leishmania/genética , Leishmania/isolamento & purificação , Mamíferos/sangue , Mamíferos/genética , Mamíferos/parasitologia , Floresta ÚmidaRESUMO
BACKGROUND: The blood-feeding behaviour of female sand flies may increase their likelihood of acquiring and transmitting Leishmania parasites. Studies on the host usage by these insects may thus improve our understanding of the Leishmania transmission risk in leishmaniasis-endemic areas. Here, we developed a fast multiplex real-time PCR assay for simultaneous detection of dog, human and Leishmania DNA in sand flies. METHODS: Primers and TaqMan probes targeting the mitochondrial cytochrome c oxidase subunit 1 and cytochrome b genes of dog and human, respectively, were combined in a multiplex assay, which also includes primers and a TaqMan probe targeting the Leishmania minicircle kinetoplast DNA. RESULTS: The multiplex assay was 100% specific, with analytical sensitivities of 103 fg/reaction for dog and human and 1 fg for Leishmania. By testing field-collected engorged female sand flies (95 Migonemyia migonei and two Nyssomyia intermedia), 50 M. migonei were positive for one or two targets (positivity rates: 45.4% for human, 4.1% for dog and 12.4% for Leishmania DNA). CONCLUSIONS: This multiplex real-time PCR assay represents a novel fast assay for detecting dog, human and Leishmania DNA in female sand flies and therefore a tool for assessing the risk of Leishmania transmission to these hosts in areas of active transmission.
Assuntos
Leishmania/genética , Leishmaniose/diagnóstico , Leishmaniose/veterinária , Reação em Cadeia da Polimerase Multiplex/métodos , Psychodidae/parasitologia , Animais , Ciclo-Oxigenase 1/genética , Primers do DNA/genética , DNA de Cinetoplasto/genética , Cães , Feminino , Humanos , Proteínas de Insetos/genética , Insetos Vetores/parasitologia , Leishmaniose/transmissão , Sensibilidade e Especificidade , Fatores de TempoRESUMO
The diagnosis of canine visceral leishmaniasis (CVL) has been a problem for public health services due to the variety of clinical signs similar to other diseases and low sensitivity and specificity of available tests. In this sense, our main objective was to develop a simple, rapid, and accurate quantitative real-time PCR (qPCR) diagnosis for CVL. Thus, low-invasive samples from bone marrow (BM), popliteal lymph nodes (PLN), and conjunctival swabs (CS) were selected from negative and VL-positive dogs, using as gold standard, immunological and parasitological tests performed with different tissues. Oligonucleotides for Leishmania infantum kDNA were designed and the limit of quantification and amplification efficiency of the qPCR were determined using tissue-specific standards produced with DNA from those different tissues, mixed with DNA from a known amount of L. infantum promastigotes. Endogenous control was used to validate a comparative Ct method, and tissue parasite concentrations were estimated by comparison with tissue-specific reference standard samples. The overall analysis of the qPCR data suggests the following ranking for tissue choice: PLN > BM > CS. Finally, we have concluded that this molecular approach simplifies and accelerates the quantitative diagnostic process because it is easy to perform, requiring no DNA dosing or standard curve application, and it shows good diagnostic parameters, especially when using popliteal lymph node samples.