[First case report of the Balamuthia mandrillaris in the Camaná district of Arequipa, Peru]. / Primer informe clínico de Balamuthia mandrillaris en el distrito de Camaná, Arequipa, Perú.

Infections by free-living amoebas cause cutaneous and neurological compromise. These conditions have a low frequency, but a high lethality more than 98%. Generally, the clinical picture is nonspecific; the laboratory tests dont help, so it represents a diagnostic and therapeutic challenge. In this report, we present the case of a 21-year-old patient, who was hospitalized for a convulsive syndrome with brain tumors, in addition to a cutaneous lesion on the right thigh. Histopathological analysis, PCR and the clinical picture allowed the diagnosis of granulomatous amebic encephalitis. Despite the treatment, the patient had a fatal outcome.

Fatal granulomatous amoebic encephalitis due to free-living amoebae in two boys in two different hospitals in Lima, Perú.

Granulomatous amoebic encephalitis caused by free-living amoebae is a rare condition that is difficult to diagnose and hard to treat, generally being fatal. Anti-amoebic treatment is often delayed because clinical signs and symptoms may hide the probable causing agent misleading the appropriate diagnostic test. There are four genera of free-living amoeba associated with human infection, Naegleria, Acanthamoeba sp., Balamuthia and Sappinia. Two boys were admitted with diagnosis of acute encephalitis. The history of having been in contact with swimming pools and rivers, supports the suspicion of an infection due to free-living amoebae. In both cases a brain biopsy was done, the histology confirmed granulomatous amoebic encephalitis with the presence of amoebic trophozoites.

Aislamiento y caracterización molecular de micobacterias no tuberculosas en el sistema de distribución de agua en un hospital de lima (perú) / Isolation and molecular characterization of nontuberculous mycobacteria in the water distribution system in a hospital of lima (perú)

RESUMEN Introducción: Las infecciones por micobacterias pueden causar lesiones cutáneas y pulmonares; las micobacterias pueden hallarse en fuentes de agua intrahospitalarias, pudiendo llegar a los pacientes inmunosuprimidos y provocar enfermedades severas. Objetivo: Aislar y caracterizar micobacterias no tuberculosas en el sistema de distribución de agua en un hospital de Lima. Materiales y métodos: Se aislaron las micobacterias en medios convencionales a base de huevo; y se emplearon pruebas bioquímicas y herramientas moleculares como la reacción en cadena de la polimerasa ―PCR― y secuenciación para su identificación. Para el análisis de secuenciación se empleó el GenBank y para el árbol filogenético se utilizó MEGA 6. Resultados: Fue posible el aislamiento de bacilos ácido alcohol resistentes en muestras de agua potable fría, aunque no en agua caliente del hospital. Se aislaron e identificaron varias especies de micobacterias no tuberculosas en las muestras de agua, siendo el 100 % de crecimiento rápido, estas fueron: Mycobacterium canariasense; M. frederiksbergense; M. mucogenicum; Mycobacterium fortuitum y Mycobacterium chelonae. Conclusiones: Esta es la primera vez que se aíslan micobacterias en agua potable de un hospital en Perú. Se concluye que la existencia de micobacterias en el agua del hospital podría ser fuente potencial de brotes nosocomiales, por lo que debería tenerse en cuenta su estudio periódicamente.

ABSTRACT Introduction: Mycobacterial infections can cause skin and lung lesions. Mycobacteria can be found in intra-hospital water sources which can reach immuno-suppressed patients and cause severe disease. Objective: To isolate and characterize non-tuberculous mycobacteria in the water distribution system in a hospital in Lima. Materials and Methods: Mycobacteria were isolated in conventional culture egg-based media and biochemical tests and molecular tools such as polymerase chain reaction (PCR) and sequencing were used for their identification. The GenBank was used for sequencing analysis and MEGA 6 was used for the phylogenetic tree. Results: Isolation of acid-fast bacilli was possible in samples of cold drinking water, although not in the hospital hot water. Several species of non-tuberculous mycobacteria were isolated and identified in the water samples being a 100% of rapid growth such as Mycobacterium canariasense, M. frederiksbergense, Mycobacterium fortuitum, Mycobacterium chelonae, and M. mucogenicum. Conclusions: This is the first time that mycobacteria are isolated in drinking water at a hospital in Perú. It is concluded that the existence of mycobacteria in hospital water could be a potential source of nosocomial outbreaks and should be analyzed periodically.

Balamuthia mandrillaris en el Perú, lesiones cutáneas, meningoencefalitis y métodos de cultivo / Balamuthia mandrillaris in Perú, skin lesions, meningoencephalitis and cultivation methods

Las amebas de vida libre son microorganismos abundantes en el suelo y agua en todo el planeta. Algunas especies de estas amebas son capaces de causar muerte en el ser humano y animales, asi como Naegleria fowleri (N. fowleri), Acanthamoeba sp. T4 principalmente y Balamuthia mandrillaris (B. mandrillaris). El 98% de los casos reportados en el mundo han fallecido. En Peru alrededor de 8 casos de meningoencefalitis por Acanthamoeba sp. se han reportado y mas de 55 casos por B. mandrillaris . Ningun caso por N. fowleri ha sido documentado oficialmente en el Peru. B. mandrillaris es de dificil diagnostico y aislamiento en medios de cultivo. En esta revision pretendo documentar la historia de casos reportados de los ultimos 40 años y describir los medios de cultivo utiles para su crecimiento. La identificacion morfologica y molecular de Balamuthia es critica en el diagnostico de meningoencefalitis amebiana.

Free-living amoebae are abundant microorganisms in soil and water worldwide. Some species of these amoebae are capable of causing death in humans and animals, such as Naegleria fowleri (N. fowleri) , Acanthamoeba sp . T4 , and Balamuthia mandrillaris (B. mandrillaris) . Some 98% of cases reported in the world have resulted in death. In Peru, 8 cases of meningoencephalitis due to Acanthamoeba sp. have been reported and more than 55 cases per B. mandrillaris . No case of N. fowleri has been officially documented in Peru. B. mandrillaris is difficult to diagnose and isolate in culture media. In this review we document the history of reported cases of the last 40 years and describe useful methods for their growth. Morphological and molecular identification of Balamuthia is critical to the diagnosis of amoebic meningoencephalitis.

Isolation and molecular characterization of Acanthamoeba and Balamuthia mandrillaris from combination shower units in Costa Rica.

Free living amoebae (FLA) are ubiquitous protozoa, which may behave as parasites under certain conditions. Four genera are recognized as causal agents of infections in humans and animals: Naegleria, Sappinia, Acanthamoeba and Balamuthia. This work determines the presence of FLA in combination shower units and employs molecular biology for the characterization of isolates. The morphological analysis and partial sequencing of the 18S rDNA gene revealed the presence of Acanthamoeba genotype T4 in 30% of the units sampled. In addition to Acanthamoeba cysts, trophozoites with morphological characteristics similar to Balamuthia were identified. PCR assay using the mitochondrial 16S rRNA gene as a target confirmed the identification of the amoeba as Balamuthia mandrillaris. Up to date, this is the first report of the isolation of B. mandrillaris in Central America and the fifth report worldwide.

The isolation of Balamuthia mandrillaris from environmental sources from Peru.

Balamuthia mandrillaris is an opportunistic free-living amoeba that has been reported to cause skin lesions and the fatal Balamuthia amoebic encephalitis (BAE) in humans and other animals. Currently, around 200 human BAE cases have been reported worldwide, although this number is considered to be underestimated. The highest number of BAE cases has been reported in the American continent, mainly in the southwest of the USA. Peru seems to be another hotspot for BAE with around 55 human cases having been identified, usually involving cutaneous infection, especially lesions in the central face area. The isolation of Balamuthia from environmental sources has been reported on only three prior occasions, twice from Californian soils and once from dust in Iran and so it seems that this amoeba is relatively rarely encountered in samples from the environment. We investigated that possibility of finding the amoebae in soil samples from different regions where clinical cases have been reported in Peru. Twenty-one samples were cultured in non-nutrient agar plates and were checked for the presence of B. mandrillaris-like trophozoites and/or cysts. Those samples that were positive for these amoebae by microscopic criteria were then confirmed by PCR amplification and DNA sequencing of the mitochondrial 16S rDNA gene of B. mandrillaris. We have detected the presence of B. mandrillaris in four samples collected in the regions of Piura (3) and Lima (1) where infection cases have been previously reported. We hypothesize that B. mandrillaris is present in Peru in soil and dust which therefore constitutes a source of the infection for the BAE cases previously reported in this country. Further studies should be carried out in the area to confirm the generality of this finding.

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.

Isolation and characterization of Acanthamoeba strains from soil samples in Gran Canaria, Canary Islands, Spain.

Free-living Amoebae of Acanthamoeba genus include non-pathogenic and pathogenic strains that are currently classified in 18 different genotypes, T1-T18. In this study, a survey was carried out to evaluate the presence of Acanthamoeba strains in soil samples collected between 2012 and 2013 in Gran Canaria Island, Canary Islands, Spain. Samples were inoculated onto non-nutrient agar (NNA) plates and were checked for the presence of Acanthamoeba. Identification of Acanthamoeba strains was based on the morphology of the cyst and trophozoite forms. Subsequently, positive samples were cloned for their molecular characterization at the genotype level by sequencing the DF3 region located in the 18S rDNA gene of Acanthamoeba as previously described. Sequencing results revealed the presence of T2, T5 and T4 genotypes within the studied samples. To the best of our knowledge, this is the first report demonstrating the presence of Acanthamoeba in Gran Canaria Island and the first study at the genotype level in the Canary Islands.

Voriconazole as a first-line treatment against potentially pathogenic Acanthamoeba strains from Peru.

Pathogenic strains of Acanthamoeba genus are the causative agents of fatal granulomatous amoebic encephalitis and a serious sight-threatening infection of the eye known as Acanthamoeba keratitis. In a previous study, Acanthamoeba strains were isolated from nasal swabs collected from healthy individuals in Peru. In the present study, the pathogenic potential of the isolated strains was established based on temperature and osmotolerance assays as well as the secretion rate of extracellular proteases. Based on these experiments, four strains that showed the highest pathogenic potential were selected for sensitivity assays against two molecules (voriconazole and chlorhexidine) which are currently used for the treatment of Acanthamoeba infections. After performing sensitivity and activity assays, it was found that both drugs were active against the tested strains. However, voriconazole showed higher activity against the studied strains compared to chlorhexidine. Therefore, voriconazole should be established as a first-line treatment against Acanthamoeba infections at least in the studied region of Peru.

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.

Is Balamuthia mandrillaris a public health concern worldwide?

Balamuthia mandrillaris is an opportunistic, free-living amoeba that can cause skin lesions and the typically fatal Balamuthia amoebic encephalitis (BAE) both in immunocompromised and immunocompetent individuals. Available data for BAE cases indicate that this disease is difficult to detect because knowledge of predisposing factors is lacking, causing a challenge for diagnosing BAE. The number of reported BAE cases is increasing worldwide, and this is a major concern because little is known about the pathogen, no standardized detection tools are available, and most of the treatments are almost empirical. The recently reported cases, novel diagnostics tools, and successful therapeutic approaches against BAE infections are reviewed here.