Comparative Assessment of DNA Targets and Amplification Methods for Leishmania (Viannia) Detection in Human Samples.

Multiple polymerase chain reaction (PCR)-based approaches have been developed for Leishmania detection in clinical and laboratory samples, and this diversity limits inter-study comparisons, meta-analyses, and generalization of findings. Towards harmonization of a molecular tool for detection of Leishmania (Viannia) for research purposes, we evaluated the concordance of 18SrDNA quantitative polymerase chain reaction (qPCR) and minicircle kinetoplastid DNA (mkDNA) PCR followed by Southern blot (PCR-SB) in in vitro infection systems and in lesion and mucosal swab samples from Colombian patients with cutaneous leishmaniasis caused by L. (Viannia). The lower limit of parasite detection of 18SrDNA qPCR and mkDNA PCR-SB was 10-1 promastigotes and one intracellular amastigote per reaction. From cutaneous lesions (n = 63), an almost perfect concordance was found between the methods (κ = 0.92, 95% CI: 0.82-1.00). Despite equal limits of detection, mkDNA PCR-SB was more efficient for parasite detection in mucosal samples than 18SrDNA qPCR or 18SrDNA digital droplet PCR. The high concordance, sensitivity, scaling potential, and feasibility of implementation of the 18SrDNA qPCR, support its selection as the L. (Viannia) in research laboratories, as a first step towards harmonization of research protocols in the region.

Case Report: Mucosal Leishmaniasis Presenting with Nasal Septum Perforation after Almost Thirty Years.

Mucosal leishmaniasis (ML) is associated with progressive tissue destruction and granuloma formation, often after a considerable period of latency from an initial cutaneous infection. We report a case of recurrent epistaxis of 3 years duration and nasopharyngeal obstruction in a woman with treated cutaneous leishmaniasis nearly 30 years before and with no further exposure to Leishmania. Computed tomography revealed nasal septal perforation and histopathology demonstrated chronic inflammation. Microscopy was negative for amastigotes, but molecular testing of nasal mucosa biopsy detected Leishmania (Viannia) braziliensis. The patient underwent 28 days of treatment with IV sodium stibogluconate and her symptoms improved significantly. Sixteen months after treatment, she continues to have episodic epistaxis and detectable parasite load in her nasal lesion. Although ML is known to take years to decades to develop, there are few reported cases in the literature of such a long latency period. This report highlights the importance of considering ML in the differential diagnosis of chronic epistaxis in countries where leishmaniasis is endemic or in immigrants from these countries, even when presentation occurs decades after leaving an endemic region.

In vivo CNS infection model of Acanthamoeba genotype T4: the early stages of infection lack presence of host inflammatory response and are a slow and contact-dependent process.

This study was developed in order to describe the early morphological events observed during the invasion of two pathogenic strains of Acanthamoeba (genotype T4); A. castellanii and A. culbertsoni, at the olfactory meatus and cerebral, pulmonary, renal, hepatic and splenic tissues levels, an in vivo invasion study. Histological and immunohistochemical description of the events at 24, 48, 72, and 96 h postintranasal inoculations of BALB/c mice was performed. A. castellanii showed a higher invasion rate than A. culbertsoni, which was only able to reach lung and brain tissue in the in vivo model. The current study supports previous evidence of lack of inflammatory response during the early stages of infection. Acanthamoeba invasion of the CNS and other organs is a slow and contact-dependent process. The early morphological events during the invasion of amoebae include the penetration of trophozoites into different epithelia: olfactory, respiratory, alveolar space, and renal tubule, which resemble the process of amoebae invasion described in corneal tissue. The data suggest that after reaching the nasal epithelium, trophozoites continued invasion, separating and lifting the most superficial cells, then migrating and penetrating between the cell junctions without causing a cytolytic effect on adjacent cells. These results reaffirm the idea that contact-dependent mechanisms are relevant for amoebae of Acanthamoeba genus regardless of the invasion site.

Miasis nasal: informe de un caso y revisión del tema / Nasal myiasis: report of a case and literature review / Miíase nasal: informe de um caso e revisão do assunto

La miasis es la infección de los tejidos u órganos de animales y seres humanos por larvas de dípteros; puede afectar a individuos de cualquier grupo etario, pero es más común en pacientes de edad media o avanzada. La miasis nasal, una infección de las cavidades nasales y paranasales por dichas larvas, es común en los países tropicales y en vías de desarrollo. Los casos informados de miasis nasal han sido causados por varias especies diferentes, entre ellas: Lucilia sericata en Corea e Irán, Estro ovis en Argelia y Francia, Lucilia cuprina y Phaenicia sericata en Malasia, Cochliomyia hominivorax en Guayana Francesa, Drosophila melanogaster en Turquía, Eristalis tenax en Irán y Oestrus ovis en Israel. Los principales signos y síntomas están relacionados con la presencia y el movimiento de las larvas, e incluyen sensación de cuerpo extraño, sangrado o secreción nasal mucopurulenta. La prevención se puede hacer con repelentes de insectos. El tratamiento de la miasis nasal se basa en el uso de antiparasitarios y técnicas para la eliminación de las larvas, pero puede incluir el uso de antibióticos profilácticos, sean tópicos o sistémicos, para las posibles infecciones secundarias. Se presenta un caso de miasis nasal y del seno maxilar izquierdo en una mujer de edad avanzada, que evolucionó favorablemente con el tratamiento.

Myiasis is the infection of animal or human tissues or organs by larvae of Diptera. It may affect individuals of any age, but is more common in middle-aged and elderly patients. Nasal myiasis, an infection of the nasal and paranasal cavities by such larvae, is a common disease in tropical and developing countries. Reported cases of nasal myiasis have been caused by several different species, such as Lucilia sericata in Korea and Iran, Estro ovis in Algeria and France, Lucilia cuprina and Phaenicia sericata in Malaysia, Cochliomyia hominivorax in French Guiana, Drosophila melanogaster in Turkey, Eristalis tenax in Iran and Oestrus ovis in Israel. Signs and symptoms are related to the presence and movement of the larvae, and include foreign body sensation, bloody or muco-purulent nasal discharge. Prevention may be done with insect repellent. Treatment is based on antiparasitic drugs and techniques for removal of larvae, but may include the use of prophylactic topical or systemic antibiotics for possible secondary infections. We report a case of nasal and left maxillary sinus myiasis in an elderly woman, whoresponded favorably to treatment.

A miíase é a infecção dos tecidos ou órgãos de animais e seres humanos por larvas de dípteros; pode afetar a indivíduos de qualquer faixa etária, mas é mais comum em pacientes de meia idade ou avançada. A miíase nasal, uma infecção das cavidades nasais e paranasais por ditas larvas, é comum nos países tropicais e em via de desenvolvimento. Os casos informados de miíase nasal tem sido causados porvarias espécies diferentes, entre elas: Lucilia sericata na Coreia e no Iran, Estro ovis na Argélia e na França, Lucilia cuprina e Phaenicia sericata na Malásia, Cochliomyia hominivorax na Guayana Francesa, Drosophila melanogaster na Turquía, Eristalis tenax na Iran e Oestrus ovis em Israel. Os principais signos e sintomas estão relacionados com a presencia e o movimento das larvas, e incluem sensação de corpo estranho, sangrado ou secreção nasal mucopurulenta. A prevenção se pode fazer com repelentes de insetos. O tratamento da miíase nasal se baseia no uso de antiparasitários e técnicas para a eliminação das larvas, mas pode incluir o uso de antibióticos profilácticos, sejam tópicos ou sistémicos, para as possíveis infecções secundárias. Se apresenta um caso de miíase nasal e do seno maxilar esquerdo numa mulher de idade avançada, que evolucionou favoravelmente com o tratamento.

Neutrophils extracellular traps damage Naegleria fowleri trophozoites opsonized with human IgG.

Naegleria fowleri infects humans through the nasal mucosa causing a disease in the central nervous system known as primary amoebic meningoencephalitis (PAM). Polymorphonuclear cells (PMNs) play a critical role in the early phase of N. fowleri infection. Recently, a new biological defence mechanism called neutrophil extracellular traps (NETs) has been attracting attention. NETs are composed of nuclear DNA combined with histones and antibacterial proteins, and these structures are released from the cell to direct its antimicrobial attack. In this work, we evaluate the capacity of N. fowleri to induce the liberation of NETs by human PMN cells. Neutrophils were cocultured with unopsonized or IgG-opsonized N. fowleri trophozoites. DNA, histone, myeloperoxidase (MPO) and neutrophil elastase (NE) were stained, and the formation of NETs was evaluated by confocal microscopy and by quantifying the levels of extracellular DNA. Our results showed N. fowleri induce the liberation of NETs including release of MPO and NE by human PMN cells as exposure interaction time is increased, but N. fowleri trophozoites evaded killing. However, when trophozoites were opsonized, they were susceptible to the neutrophils activity. Therefore, our study suggests that antibody-mediated PMNs activation through NET formation may be crucial for antimicrobial responses against N. fowleri.

Correlation between presence of Leishmania RNA virus 1 and clinical characteristics of nasal mucosal leishmaniosis / Correlação entre a presença de Leishmania RNA Vírus 1 e as características clínicas da leishmaniose de mucosa nasal

ABSTRACT INTRODUCTION: Mucosal leishmaniosis (ML) is a severe clinical form of leishmaniosis. Complex factors related to the parasite and the host are attributed to the development of mucosal lesions. Leishmania RNA virus 1 (LRV1) can disrupt immune response, and may be the main determinant of severity of the disease; it should be investigated. OBJECTIVE: To study the existence of clinical differences between patients with ML with endosymbiosis by LRV1 and. those without it. METHODS: A cross-sectional cohort study with clinical evaluation, polymerase chain reaction (PCR) detection of Leishmania, species classification, and search of LRV1 was performed. Only patients with confirmed diagnosis of ML by positive PCR and with nasal mucosa injuries were included in this analysis. RESULTS: Out of 37 patients, 30 (81.1%) were diagnosed with Leishmania braziliensis, five (13.5%) with Leishmania guyanensis, and two (5.4%) with mixed infection of L. braziliensis and L. guyanensis. LVR1 virus was present in 26 (70.3%) of the cases. CONCLUSION: Correlation between clinical phenotype and presence of LRV1 was not observed, although the frequency of the virus is two-fold higher in mucosal lesions than that found in the literature on skin lesions in the same geographical area.

RESUMO Introdução: A leishmaniose de mucosa (LM) é uma forma clínica grave da leishmaniose. Fatores complexos ligados ao parasita e ao hospedeiro são atribuídos ao desenvolvimento das lesões de mucosa. Leishmania RNA Vírus 1 (LRV1) pode subverter a resposta imune, podendo ser o principal determinante da gravidade da doença e deve ser pesquisado. Objetivo: Estudar a existência de diferenças clínicas entre pacientes portadores de LM com endosimbiose por LRV1 e as que não possuem. Métodos: Foi realizado um estudo de coorte histórica com corte transversal com avaliação clínica, detecção da Leishmania por técnica de PCR, classificação da espécie e pesquisa de LRV1. Foram incluídos na análise da pesquisa somente os pacientes com diagnóstico confirmado de LM com PCR positivo, com lesão de mucosa nasal. Resultados: Dos 37 pacientes, 30 (81,1%) foram diagnosticados com L. braziliensis, 5 (13,5%) com L. guyanensis e 2 (5,4%) com infecção mista de L. braziliensis e L. guyanensis. O vírus LVR1 estava presente em 26 casos (70,3%). Conclusão: A correlação entre o fenótipo clínico e a presença do LRV1 não foi constatada, porém a frequência do vírus é duas vezes maior em lesão de mucosa do que encontrado em trabalho, da mesma região, sobre lesão cutânea.

Correlation between presence of Leishmania RNA virus 1 and clinical characteristics of nasal mucosal leishmaniosis.

INTRODUCTION: Mucosal leishmaniosis (ML) is a severe clinical form of leishmaniosis. Complex factors related to the parasite and the host are attributed to the development of mucosal lesions. Leishmania RNA virus 1 (LRV1) can disrupt immune response, and may be the main determinant of severity of the disease; it should be investigated. OBJECTIVE: To study the existence of clinical differences between patients with ML with endosymbiosis by LRV1 and. those without it. METHODS: A cross-sectional cohort study with clinical evaluation, polymerase chain reaction (PCR) detection of Leishmania, species classification, and search of LRV1 was performed. Only patients with confirmed diagnosis of ML by positive PCR and with nasal mucosa injuries were included in this analysis. RESULTS: Out of 37 patients, 30 (81.1%) were diagnosed with Leishmania braziliensis, five (13.5%) with Leishmania guyanensis, and two (5.4%) with mixed infection of L. braziliensis and L. guyanensis. LVR1 virus was present in 26 (70.3%) of the cases. CONCLUSION: Correlation between clinical phenotype and presence of LRV1 was not observed, although the frequency of the virus is two-fold higher in mucosal lesions than that found in the literature on skin lesions in the same geographical area.

Intranasal coadministration of Cholera toxin with amoeba lysates modulates the secretion of IgA and IgG antibodies, production of cytokines and expression of pIgR in the nasal cavity of mice in the model of Naegleria fowleri meningoencephalitis.

The nasal mucosa is the first contact with antigens to induce IgA response. The role of this site has rarely been studied. We have shown than intranasal administration with Naegleria fowleri lysates plus Cholera toxin (CT) increased the protection (survival up to 100%) against N. fowleri infection in mice and apparently antibodies IgA and IgG together with polymorphonuclear (PMN) cells avoid the attachment of N. fowleri to apical side of the nasal epithelium. We also observed that nasal immunization resulted in the induction of antigen-specific IgG subclasses (IgG1 and IgG2a) in nasal washes at days 3 and 9 after the challenge and IgA and IgG in the nasal cavity, compared to healthy and infected mice. We found that immunization with both treatments, N. fowleri lysates plus CT or CT alone, increased the expression of the genes for alpha chain, its receptor (pIgR), and it also increased the expression of the corresponding proteins evidenced by the ∼65 and ∼74kDa bands, respectively. Since the production of pIgR, IgA and IgG antibodies, is up-regulated by some factors, we analyzed the expression of genes for IL-10, IL-6, IFN-γ, TNF-α and IL-1ß by using RT-PCR of nasal passages. Immunization resulted in an increased expression of IL-10, IL-6, and IFN-γ cytokines. We also aimed to examine the possible influences of immunization and challenge on the production of inflammatory cytokines (TNF-α and IL-1ß). We observed that the stimulus of immunization inhibits the production of TNF-α compared to the infected group where the infection without immunization causes an increase in it. Thus, it is possible that the coexistence of selected cytokines produced by our immunization model may provide a highly effective immunological environment for the production of IgA, IgG and pIgR as well as a strong activation of the PMN in mucosal effector tissue such as nasal passages.

Endosymbiotic Mycobacterium chelonae in a Vermamoeba vermiformis strain isolated from the nasal mucosa of an HIV patient in Lima, Peru.

In March 2010, a 35 year-old HIV/AIDS female patient was admitted to hospital to start treatment with Highly Active Antiretroviral Therapy (HAART) since during a routine control a dramatic decrease in the CD4(+) levels was detected. At this stage, a nasal swab from each nostril was collected from the patient to include it in the samples for the case study mentioned above. Moreover, it is important to mention that the patient was diagnosed in 2009 with invasive pneumococcal disease, acute cholecystitis, pancreatitis and pulmonary tuberculosis. The collected nasal swabs from both nostrils were positive for Vermamoeba vermiformis species which was identified using morphological and PCR/DNA sequencing approaches. Basic Local Alignment Search Tool (BLAST) homology and phylogenetic analysis confirmed the amoebic strain to belong to V.vermiformis species. Molecular identification of the Mycobacterium strain was carried out using a bacterial universal primer pair for the 16S rDNA gene at the genus level and the rpoB gene was amplified and sequenced as previously described to identify the Mycobacterium species (Shin et al., 2008; Sheen et al., 2013). Homology and phylogenetic analyses of the rpoB gene confirmed the species as Mycobacterium chelonae. In parallel, collected swabs were tested by PCR and were positive for the presence of V.vermiformis and M.chelonae. This work describes the identification of an emerging bacterial pathogen,M.chelonae from a Free-Living Amoebae (FLA) strain belonging to the species V.vermiformis that colonized the nasal cavities of an HIV/AIDS patient, previously diagnosed with TB. Awareness within clinicians and public health professionals should be raised, as pathogenic agents such as M.chelonae may be using FLA to propagate and survive in the environment.

Genotyping of potentially pathogenic Acanthamoeba strains isolated from nasal swabs of healthy individuals in Peru.

Free Living Amoebae (FLA) of Acanthamoeba genus are widely distributed in the environment and can be found in the air, soil and water; and have also been isolated from air-conditioning units. In humans, they are causative agents of a sight-threating infection of the cornea, Acanthamoeba keratitis (AK) and a fatal infection of the central nervous system known as Granulomatous Amoebic Encephalitis (GAE). In this study, a survey was conducted in order to determine the presence and pathogenic potential of free-living amoebae of Acanthamoeba genus in nasal swabs from individuals in two regions of Peru. Identification of isolates was based on cyst morphology and PCR-sequencing of the Diagnostic Fragment 3 to identify strains at the genotype level. The pathogenic potential of the isolates was also assayed using temperature and osmotolerance assays and extracellular proteases zymograms. The obtained results revealed that all isolated strains exhibited pathogenic potential. After sequencing the highly variable DF3 (Diagnostic Fragment 3) region in the 18S rRNA gene as previously described, genotype T4 was found to be the most common one in the samples included in this study but also genotype T15 was identified. To the best of our knowledge, this is the first study on the characterization of Acanthamoeba strains at the genotype level and the first report of genotype T4 and T15 in Peru.

Genetic polymorphism in Leishmania (Viannia) braziliensis detected in mucosal leishmaniasis of HIV-infected and non-HIV-infected patients.

The genetic polymorphism of Leishmania (Viannia) braziliensis detected in cases of mucosal leishmaniasis (ML) from HIV-infected and non HIV-infected patients was evaluated. Nine samples from three HIV-infected patients and five samples from five non HIV-infected patients were analysed by polymerase chain reaction (PCR), low-stringency single-specific primer PCR (LSSP-PCR) and phenetic analysis. The presence of L. (V.) braziliensis DNA was detected in all samples by specific PCR assay. The intraspecific polymorphism of the variable region of L. (V.) braziliensis kDNA minicircles was investigated by LSSP-PCR. Phenetic analysis grouped the genetic profiles into two distinct clusters, which discriminated between samples obtained from HIV-infected and non HIV-infected patients. In two HIV-infected patients, identical genetic profiles were detected in lesions biopsied at different times after the treatment of the initial lesion. Interestingly, genetically divergent profiles were detected in the cutaneous and mucosal lesions of the same HIV-infected patient collected at the same time. This is the first work comparing genetic polymorphism of L. (V.) braziliensis in cases of mucosal leishmaniasis from HIV-infected and non HIV-infected patients.

Immune responses in sheep naturally infected with Oestrus ovis (Diptera: Oestridae) and gastrointestinal nematodes.

This study was carried out to evaluate the immune response in young Ile de France (IF) and Santa Ines (SI) sheep naturally infected by Oestrus ovis and gastrointestinal nematodes (GIN). Mast cells, eosinophils and globule leucocytes were enumerated in the upper respiratory tract (septum, middle meatus and ventral nasal conchae) and in the mucosa of abomasum and small intestine. Immunoglobulin G (IgG) levels in serum samples and immunoglobulin A (IgA) levels in mucus from the nasal, abomasum and small intestinal mucosae were determined against O. ovis, Haemonchus contortus and Trichostrongylus colubriformis antigens. Significant positive correlation coefficients were observed in both breeds between the number of O. ovis larvae×IgG against Oestrus crude extract (IF: r=0.58; SI: r=0.66; P<0.05), and between O. ovis larvae x IgG against Oestrus excretory and secretory products (IF: r=0.59; SI: r=0.63; P<0.05). Apparently, the presence of antibodies in the serum or nasal mucus, as well as inflammatory cells, was not able to efficiently protect against O. ovis infestation. With regard to GIN, the levels of immunoglobulins and the inflammatory cell numbers in the gastrointestinal mucosa presented a significant inverse relationship with H. contortus worm burden in SI animals and this may have contributed to the fact that these animals presented the lowest FEC and worm burden compared to IF. In conclusion, the immune responses against O. ovis and GIN are very similar and involve the recruitment of inflammatory cells and production of immunoglobulins against the parasites.

A proposed new clinical staging system for patients with mucosal leishmaniasis.

Mucosal leishmaniasis (ML) occurs mainly in areas where Leishmania braziliensis is transmitted. It affects predominantly the nasal mucosa and, in more severe forms, can lead to significant tissue destruction. There is no standard method for grading the severity of disease. We categorised 50 patients with ML according to a proposed new clinical staging system. Their age ranged from 10 to 86 y (mean ± SD: 36 ± 16 y) and 43 (86%) patients were male. The different degrees of evolution of mucosal disease, from the initial stage to the more severe long-term cases, enabled ML to be graded into five stages. Stage I is characterised by nodular lesions of the mucosa without ulceration. Stage II is represented by superficial mucosal ulcerations with concomitant fine granular lesions. Stage III is characterised by deep mucosal ulcerations with granular tissue formation. In stage IV there are irreversible lesions leading to perforation of the cartilaginous nasal septum with necrosis. In stage V the nasal pyramid is compromised with alterations of facial features as a consequence of severe tissue destruction. These stages may be useful in characterising the severity of the lesion and optimising therapeutic outcome.

kDNA minicircle signatures of Leishmania (Viannia) braziliensis in oral and nasal mucosa from mucosal leishmaniasis patients.

Polymerase chain reaction (PCR) and low-stringency single-specific primer PCR (LSSP-PCR) analyses were used to detect Leishmania (Viannia) braziliensis DNA and investigate kDNA signatures of parasite populations present in oral and nasal mucosa lesions from mucosal leishmaniasis patients. A total of 25 samples from 22 patients were processed by specific PCR/hybridization assays. Parasite DNA was detected in all samples analyzed. The intraspecific polymorphism of the variable region of L. (V.) braziliensis kDNA minicircles was also investigated by LSSP-PCR. Similar kDNA signatures were observed in parasites recovered from nasal and oral mucosa lesions of the same patient. In contrast, genetically divergent profiles were detected in lesions from patients biopsied at different times within a period of 1 year. This is the first work to report genetic typing of L. (V.) braziliensis directly from human oral and nasal mucosal lesions.

Intranasal immunization with Naegleria fowleri lysates and Cry1Ac induces metaplasia in the olfactory epithelium and increases IgA secretion.

According to previous reports, intranasal administration of the Cry1Ac protein alone or with amoebic lysates increases protection against Naegleria fowleri meningoencephalitis in mice, apparently by eliciting IgA responses in the nasal mucosa. In the current study, we performed an immunohistochemical analysis of IgA in the nasal mucosa of mice immunized intranasally with Cry1Ac, and amoebic lysates or a combination of both. The animals were sacrificed 24 h after the last immunization or after an intranasal lethal challenge with N. fowleri. Our results indicate that all of the intranasal immunizations provoked an increase in areas with metaplasia in the olfactory epithelium, allowing for secretion of IgA. As a result, IgA antibodies were found interacting with trophozoites in the nasal lumen, and there was a marked increase of IgA in the metaplasic epithelium. On the other hand in nonimmunized mice trophozoites were observed invading the nasal mucosa, which was not the case for immunized mice. Our results suggest that intranasal immunization provokes cellular changes in the olfactory epithelium, leading to greater protection against N. fowleri that is probably caused by an increased secretion of IgA. The increased IgA response induced in the nasal mucosa by immunization probably impedes both amoebic adhesion and subsequent invasion of the parasite to the nasal epithelium.

Parenteral aminosidine is not effective for Peruvian mucocutaneous leishmaniasis.

Few therapeutic options are available for mucocutaneous leishmaniasis (MCL). We conducted a randomized open trial to evaluate the efficacy, safety, and tolerance of parenteral aminosidine sulphate (AS) 14 mg/kg/d for 21 days compared with intravenous meglumine antimonate (MA) 20 mg/kg/d for 28 days in patients with moderate MCL in Cuzco, Peru. Cure was defined as complete healing with re-epithelialization within 1 year of follow-up. The trial was stopped after 38 patients were enrolled (17 in the MA group and 21 in the AS group) because of marked differences in response. Study groups were comparable in baseline characteristics. Cure rates were 0/21 in the AS group compared with 8/17 (47%, 95% confidence interval: 23-71%) in the MA group (P < 0.001). Side effects and laboratory abnormalities were mild in both groups. We conclude that parenteral AS given on its own is not effective for MCL in Peru.

Concomitant early mucosal and cutaneous leishmaniasis in Brazil.

Mucosal leishmaniasis (ML) is often clinically silent until reaching a highly advanced state. In this prospective study, 6 of 220 patients with early cutaneous leishmaniasis were diagnosed with mucosal involvement by otorhinolaryngological examination (a rate similar to the reported rate of late ML). Detection of early ML may represent an important strategy in preventing severe mucosal destruction in human leishmaniasis.

Leishmania (Viannia) braziliensis-induced chronic granulomatous cutaneous lesions affecting the nasal mucosa in the rhesus monkey (Macaca mulatta) model.

The present studies on infections with Leishmania (Viannia) braziliensis in rhesus macaques were made to characterize the evolution of different parasite strains and the immune responses they elicited in this experimental host. A standardized inoculum of promastigotes was injected intradermally either above the eyelid or on the forearm of each monkey. Sixteen infected monkeys developed longstanding infections which lasted until the end of the observation period (33 months). The time required for lesion development was very variable, not only for the isolates showing molecular differences but also for individual animals in groups infected with the same parasite strain. The inocula produced lesions of variable severity, ranging from localized cutaneous leishmaniasis (CL) with a tendency to spontaneous healing to non-healing disease. One infected animal developed persistent metastatic skin and mucosal lesions. Anti-Leishmania antibodies and parasite-specific T-cell responses were induced by the experimental infections. As the granulomatous inflammatory response found at the lesions in L. (V.) braziliensis-infected M. mulatta was similar to that in patients with CL, this primate model could be useful for studying the pathophysiology and immunoregulatory events associated with disease evolution, as well as for the evaluation of new drugs or candidate vaccines.

Biological behavior of Leishmania (L.) amazonensis isolated from a human diffuse cutaneous leishmaniasis in inbred strains of mice.

After a subcutaneous injection of 100000 purified amastigotes of an isolate from a diffuse case of cutaneous leishmaniasis caused by the MHOM/BR/76/Ma-5 strain of Leishmania amazonensis, three inbred mouse strains developed a progressive nodular lesion, which evolved to an ulcerated lesion. Based on these data, mice of BALB/c, C57BL/6 or C57BL/10 could be classified as susceptible. The majority of mice developed metastases in the footpads, ear, tail, nose and oral mucosa. Amputation of the members related to the primary lesion was frequent. Experiments using the limiting dilution analysis showed that there was no correlation between lesion and parasite load. It has been demonstrated that these mouse strains could be considered excellent models for mucocutaneous leishmaniasis when infected with L. amazonensis. Metastatic lesions caused destruction of the nasal region with many parasitized macrophages under the epithelial surface of the nasal mucosa. Bone destruction occurred with an extensive inflammatory reaction presenting macrophages heavily parasitized by amastigotes. The parasites also spread to the periodontal ligament and other structures of the oral cavity, which could induce a severe inflammatory process. This study indicates that both nasal and oral lesions in mice infected by L. amazonensis were characterized by an inflammatory reaction with the presence of a high parasite load within macrophages.

Mucosal leishmaniasis: quantitative nasal cytology as a marker of disease activity and indicator of healing.

Mucosal leishmaniasis (ML) follow-up is based on subjective parameters. Using simplified quantitative cytology of nasal lavages (QNCs), we studied 20 ML patients, 10 patients with cutaneous leishmaniasis (CL), and 10 healthy subjects. Patients with ML were treated with antimony and followed up with otolaryngological examination plus QNCs for 6 months. At the first evaluation, the median total number of cells in ML patients (1,540,000) was greater than that in CL patients (215,000) or that in healthy subjects (250,000). Neutrophils were predominant in ML patients, in contrast to both sets of controls, in whom epithelial cells were more frequent. During treatment, we found a significant reduction in total nasal cell counts in ML patients who were cured, and encountered a switch in predominant cell type. The cytology of 2 patients who did not respond to antimony remained the same. It is therefore possible to detect nasal inflammation in ML patients through QNCs, which may indicate extension of mucosal involvement, providing an objective parameter to monitor therapy.