Assessment of pathogenic potential of Acanthamoeba isolates by in vitro and in vivo tests.

Acanthamoeba are free-living protozoa present ubiquitously in numerous environmental reservoirs that exist as an actively feeding trophozoite or a dormant cyst stage. The pathogenic Acanthamoeba are known to cause Acanthamoeba keratitis (AK) and granulomatous amoebic encephalitis (GAE). Despite their omnipresence, the number of infections is quite low. The reason behind this low frequency of Acanthamoeba infections could be the existence of many non-pathogenic strains or a successful host immune response to these infections. Studies in the past have proposed a few physiological parameters for the differentiation of pathogenic and non-pathogenic strains. Additionally, in vivo experiments are known to play an essential role in understanding the virulence of parasites, immunological aspects, and disease pathogenesis. The thermotolerance (30 °C, 37 °C, and 40 °C) and osmotolerance (0.5 M, 1 M, and 1.5 M) tests were performed on 43 Acanthamoeba isolates from patients with keratitis (n = 22), encephalitis (n = 5), and water samples (n = 16). In addition, the genotype of 10 Acanthamoeba isolates (keratitis (n = 2), encephalitis (n = 2), water (n = 6)) was determined and were then evaluated for pathogenicity on mouse model by inducing Acanthamoeba keratitis and amoebic encephalitis. The results of the thermotolerance and osmotolerance assays categorized 29/43 (67.4%) isolates as pathogenic, 8 as low pathogenic (18.6%), and the remaining 6 (13.9%) as non-pathogenic. The 10 Acanthamoeba isolates were categorized as T11 (5 isolates), T5 (2 isolates), T4 (2 isolates), and T10 (1 isolate) genotypes. Out of 10 Acanthamoeba isolates, 9 were successful in establishing AK, amoebic encephalitis, or both in the mice model, and a single isolate was found non-pathogenic. Two isolates from water samples were non-pathogenic in the physiological tests but successfully established Acanthamoeba infection in the mice model. The results of the physiological assays and in vivo experiments were analogous for 7 isolates while 1 isolate from the water was low pathogenic in the physiological assays but failed to produce pathogenicity during in vivo experiments. The physiological parameters are not very dependable to test the pathogenic potential of Acanthamoeba isolates, and thus results must always be validated by in vivo experiments. There is no infallible approach for determining the potential pathogenicity of environmental isolates of Acanthamoeba because several parameters regulate the pathogenic potential.

Next-generation sequencing for hypothesis-free genomic detection of invasive tropical infections in poly-microbially contaminated, formalin-fixed, paraffin-embedded tissue samples - a proof-of-principle assessment.

BACKGROUND: The potential of next-generation sequencing (NGS) for hypothesis-free pathogen diagnosis from (poly-)microbially contaminated, formalin-fixed, paraffin embedded tissue samples from patients with invasive fungal infections and amebiasis was investigated. Samples from patients with chromoblastomycosis (n = 3), coccidioidomycosis (n = 2), histoplasmosis (n = 4), histoplasmosis or cryptococcosis with poor histological discriminability (n = 1), mucormycosis (n = 2), mycetoma (n = 3), rhinosporidiosis (n = 2), and invasive Entamoeba histolytica infections (n = 6) were analyzed by NGS (each one Illumina v3 run per sample). To discriminate contamination from putative infections in NGS analysis, mean and standard deviation of the number of specific sequence fragments (paired reads) were determined and compared in all samples examined for the pathogens in question. RESULTS: For matches between NGS results and histological diagnoses, a percentage of species-specific reads greater than the 4th standard deviation above the mean value of all 23 assessed sample materials was required. Potentially etiologically relevant pathogens could be identified by NGS in 5 out of 17 samples of patients with invasive mycoses and in 1 out of 6 samples of patients with amebiasis. CONCLUSIONS: The use of NGS for hypothesis-free pathogen diagnosis from contamination-prone formalin-fixed, paraffin-embedded tissue requires further standardization.

Functional Assessment of 2,177 U.S. and International Drugs Identifies the Quinoline Nitroxoline as a Potent Amoebicidal Agent against the Pathogen Balamuthia mandrillaris.

Balamuthia mandrillaris is a pathogenic free-living amoeba that causes a rare but almost always fatal infection of the central nervous system called granulomatous amoebic encephalitis (GAE). Two distinct forms of B. mandrillaris-a proliferative trophozoite form and a nonproliferative cyst form, which is highly resistant to harsh physical and chemical conditions-have been isolated from environmental samples worldwide and are both observed in infected tissue. Patients suffering from GAE are typically treated with aggressive and prolonged multidrug regimens that often include the antimicrobial agents miltefosine and pentamidine isethionate. However, survival rates remain low, and studies evaluating the susceptibility of B. mandrillaris to these compounds and other potential therapeutics are limited. To address the need for more-effective treatments, we screened 2,177 clinically approved compounds for in vitro activity against B. mandrillaris The quinoline antibiotic nitroxoline (8-hydroxy-5-nitroquinoline), which has safely been used in humans to treat urinary tract infections, was identified as a lead compound. We show that nitroxoline inhibits both trophozoites and cysts at low micromolar concentrations, which are within a pharmacologically relevant range. We compared the in vitro efficacy of nitroxoline to that of drugs currently used in the standard of care for GAE and found that nitroxoline is the most potent and selective inhibitor of B. mandrillaris tested. Furthermore, we demonstrate that nitroxoline prevents B. mandrillaris-mediated destruction of host cells in cultured fibroblast and primary brain explant models also at pharmacologically relevant concentrations. Taken together, our findings indicate that nitroxoline is a promising candidate for repurposing as a novel treatment of B. mandrillaris infections.IMPORTANCEBalamuthia mandrillaris is responsible for hundreds of reported cases of amoebic encephalitis, the majority of which have been fatal. Despite being an exceptionally deadly pathogen, B. mandrillaris is understudied, leaving many open questions regarding epidemiology, diagnosis, and treatment. Due to the lack of effective drugs to fight B. mandrillaris infections, mortality rates remain high even for patients receiving intensive care. This report addresses the need for new treatment options through a drug repurposing screen to identify novel B. mandrillaris inhibitors. The most promising candidate identified was the quinoline antibiotic nitroxoline, which has a long history of safe use in humans. We show that nitroxoline kills B. mandrillaris at pharmacologically relevant concentrations and exhibits greater potency and selectivity than drugs commonly used in the current standard of care. The findings that we present demonstrate the potential of nitroxoline to be an important new tool in the treatment of life-threatening B. mandrillaris infections.

Assessment of blood-brain barrier penetration of miltefosine used to treat a fatal case of granulomatous amebic encephalitis possibly caused by an unusual Balamuthia mandrillaris strain.

Balamuthia mandrillaris, a free-living ameba, causes rare but frequently fatal granulomatous amebic encephalitis (GAE). Few patients have survived after receiving experimental drug combinations, with or without brain lesion excisions. Some GAE survivors have been treated with a multi-drug regimen including miltefosine, an investigational anti-leishmanial agent with in vitro amebacidal activity. Miltefosine dosing for GAE has been based on leishmaniasis dosing because no data exist in humans concerning its pharmacologic distribution in the central nervous system. We describe results of limited cerebrospinal fluid (CSF) and serum drug level testing performed during clinical management of a child with fatal GAE who was treated with a multiple drug regimen including miltefosine. Brain biopsy specimens, CSF, and sera were tested for B. mandrillaris using multiple techniques, including culture, real-time polymerase chain reaction, immunohistochemical techniques, and serology. CSF and serum miltefosine levels were determined using a liquid chromatography method coupled to tandem mass spectrometry. The CSF miltefosine concentration on hospital admission day 12 was 0.4 µg/mL. The serum miltefosine concentration on day 37, about 80 h post-miltefosine treatment, was 15.3 µg/mL. These are the first results confirming some blood-brain barrier penetration by miltefosine in a human, although with low-level CSF accumulation. Further evaluation of brain parenchyma penetration is required to determine optimal miltefosine dosing for Balamuthia GAE, balanced with the drug's toxicity profile. Additionally, the Balamuthia isolate was evaluated by real-time polymerase chain reaction (PCR), demonstrating genetic variability in 18S ribosomal RNA (18S rRNA) sequences and possibly signaling the first identification of multiple Balamuthia strains with varying pathogenicities.

A amebíase deveria detectar-se com metodologias diagnósticas mais económicas e específicas / Cheaper, more specific diagnostic procedures recommended to detect amoebiasis

A (La) amebíase é uma (es una) infecção parasitária causada pela (por) Entamoeba histolytica, protozoário que representa um risco à saúde nos (un riesgo para la salud en los) países onde as (donde las) barreiras sanitárias são inadequadas. Estima-se que 500 milhões de indivíduos em todo o mundo estejam (se encuentran) infectados por este patógeno, havendo até (hay hasta) 100 000 óbitos anuais (muertes anuales). O (El) diagnóstico laboratorial é realizado rotineiramente através da (rutinariamente por medio de la) demonstração microscópica de cistos e/ou (o) trofozoítos no (en el) sedimento fecal, não permitindo a (no permitiendo la) detecção de variações interespecíficas entre a E. histolytica e espécies não patogênicas como a Entamoeba dispar e a (y) Entamoeba moshkovskii. Metodologias imunológicas para a pesquisa de anticorpos contra a (la búsqueda de anticuerpos contra) E. histolytica já foram usadas esão (ya fueron utilizadas y son) capazes de diferenciar as espécies do (entre las especies del) complexo E.histolytica/dispar/moshkovskii, apesar de apresentarem resultados falso positivos e negativos. De modo semelhante a pesquisa de antígenos específicos por técnicas imunológicas são de utilidade limitada. Areação em cadeia da (La reacción en cadena de la) polimerase ainda constitui a metodologia de escolha para o (todavía es la metodología de elección para el) diagnóstico específico da amebíase, entretanto metodologias mais recentes foram empregadas no (fueron empleadas en el) diagnóstico da amebías elevando a resultados promissores (promisorios). A PCR em tempo real e as tecnologias baseadas na(basadas en la) detecção de microesferas magnéticas acopladas foram utilizadas para o diagnóstico da amebíase e apresentaram bom desempenho. Apesar do avanço no (del avance en el) diagnóstico laboratorial da amebíase ao longo dos anos, ainda há necessidade do desenvolvimento de (a lo largo de los años, todavía hay necesidad de desarrollar)...

The association between campylobacteriosis, agriculture and drinking water: a case-case study in a region of British Columbia, Canada, 2005-2009.

We studied the association between drinking water, agriculture and sporadic human campylobacteriosis in one region of British Columbia (BC), Canada. We compared 2992 cases of campylobacteriosis to 4816 cases of other reportable enteric diseases in 2005-2009 using multivariate regression. Cases were geocoded and assigned drinking water source, rural/urban environment and socioeconomic status (SES) according to the location of their residence using geographical information systems analysis methods. The odds of campylobacteriosis compared to enteric disease controls were higher for individuals serviced by private wells than municipal surface water systems (odds ratio 1·4, 95% confidence interval 1·1-1·8). In rural settings, the odds of campylobacteriosis were higher in November (P = 0·014). The odds of campylobacteriosis were higher in individuals aged ⩾15 years, especially in those with higher SES. In this region of BC, campylobacteriosis risk, compared to other enteric diseases, seems to be mediated by vulnerable drinking water sources and rural factors. Consideration should be given to further support well-water users and to further study the microbiological impact of agriculture on water.

War on terror cells: killing the host that harbours 'superbugs' is an infection control strategy in our fight against infectious diseases.

It is proposed that targeting the environmental host that harbour 'superbugs' is an effective strategy in our fight against infectious diseases.

Rapid detection and simultaneous molecular profile characterization of Acanthamoeba infections.

Diagnosis of Acanthamoeba by microscopic examination, culture, and polymerase chain reactions (PCRs) has several limitations (sensitivity, specificity, lack of detection of several strains, cost of testing for discrimination among strains). We developed a new high-resolution melting real-time PCR (HRM) to detect and characterize Acanthamoeba infections. HRM performances were evaluated with strains from the American Type Culture Collection (ATCC) and with 20 corneal scrapings. The DNA extracted from specimens were amplified, detected, and characterized in 1 run using 2 original primers diluted in a solution containing an intercalating dye. Detection and molecular characterization of Acanthamoeba infections could be achieved in less than 2.5 h with a dramatic reduction in cost of reactants (postamplification procedures and radioactive or fluorescent-labeled molecular probes were unnecessary). HRM detection limits were 0.1 cyst/µL or less (including genotypes T5 and T11), and its sensitivity and specificity were higher than other molecular tests. For the tested strains from the ATCC, the HRM drafted 4 different profiles: Type I (genotypes T2 and T4), Type II (T5 and T7), Type III (T8), and Type IV (T1, T3, T6, T9, T11, T12, and T13).

Antiprotozoal compounds: state of the art and new developments.

Protozoa can cause severe diseases, including malaria, leishmaniasis, Chagas disease, sleeping sickness and amoebiasis, all being responsible for morbidity and mortality particularly in tropical countries. To date there are no protective vaccines against any of these diseases, and many of the available drugs are old or elicit serious adverse reactions. Moreover, parasite resistance to existing drugs has become a serious problem. Owing to lack of financial returns, research in this field is of limited interest to pharmaceutical companies and largely depends on funding by public authorities. This article aims to provide a concise overview of the state-of-the-art treatment for the most important tropical protozoal infections as well as new approaches.

Assessment of Balamuthia mandrillaris-specific serum antibody concentrations by flow cytometry.

A flow cytometry (fluorescence-activated cell sorter)-based assay was adapted to detect and quantify antibodies to Balamuthia mandrillaris, a causative agent of fatal amoebic encephalitis (BAE), and to Acanthamoeba species. With sera from BAE patients for positive and a group of inconspicuous volunteers for negative reference, most of the 237 sera from random blood donors, patients with atypical encephalitis, atypical pneumonitis, visceral amoebiasis and toxoplasmosis and from subjects working with primates and other mammals were rated negative, 19% elevated and of these 2% high. In comparison, 23 of 25 West Africans living in rural areas revealed elevated, of these 15 high, and one very high B. mandrillaris-binding antibody titers, the latter well in the range of BAE patients. To date, none of the tested individuals have developed symptoms indicative of BAE. Criss-cross analysis with rabbit hyper immune sera against B. mandrillaris, Acanthamoeba comandoni (group 1), Acanthamoeba castellanii (group 2) and Acanthamoeba lenticulata (group 3) confirmed that cross-reactivity between B. mandrillaris and Acanthamoeba sp. is negligible while accentuating antigenic differences between the three morphological groups of Acanthamoeba.

Investigation of the prevalence of amoebiasis in Izmir province and determination of Entamoeba spp. using PCR and enzyme immunoassay.

Amoebiasis is a common and life-threatening disease. The discrimination of the pathogenic Entamoeba histolytica from the non-pathogenic Entamoeba dispar could be done by advanced methods such as enzyme immunoassay (EIA) and PCR. The aim of this study was to investigate the prevalence of amoebiasis in Izmir province, and differentiate the Entamoeba species by PCR and EIA. Stool samples of 2,047 individuals were examined by direct microscopy, formalin ethyl acetate concentration, trichrome staining and culture, and those found to be positive for E. histolytica/dispar by any of these methods were further analyzed by PCR and EIA for species identification. Fifty-nine of 2,047 (2.9%) stool samples were found to be positive for E. histolytica/dispar with microscopy and/or culture. Among these positive samples, E. histolytica was detected in 14 (23.7%) and 5 (8.5%) samples with PCR and antigen-specific ELISA (EIA), respectively. E. dispar was diagnosed in 31 (52.5%) and 52 (88.1%) of 59 samples with species-specific PCR and EIA, respectively. Risk factors related to infection with Entamoeba spp. and other intestinal parasites included living in shanty houses (p < 0.01), a history of recent immigration to Izmir (p < 0.01), having no social security (p < 0.05) and living with a crowded family (p < 0.01). The results demonstrated the significance of amoebiasis as a public health problem among people with low socio-economic status in Izmir province.

Studies on the development of colon targeted oral drug delivery systems for ornidazole in the treatment of amoebiasis.

The aim of the present study is to develop colon-targeted drug delivery systems for ornidazole using guar gum as a carrier. The core formulation containing ornidazole was directly compressed. Compression-coated tablets of ornidazole containing various proportions of guar gum in the coat were prepared. All the formulations were evaluated for hardness and drug content uniformity and were subjected to in vitro drug release studies. The amount of ornidazole released from tablets at different time intervals was estimated by the HPLC method. The compression-coated formulations released less than 8% of ornidazole in the physiological environment of stomach and small intestine. The compression-coated tablets with 85%, 75%, and 65% of guar gum coat released about 21%, 38%, and 73% of ornidazole, respectively, in simulated colonic fluids indicating the susceptibility of the guar gum formulations to the rat caecal contents. The results of the study show that compression-coated ornidazole tablets with either 65% (OLV-65) or 75% (OLV-75) of guar gum coat are most likely to provide targeting of ornidazole for local action in the colon owing to its minimal release of the drug in the first 5 hr. The ornidazole compression-coated tablets showed no change in physical appearance, drug content, or in dissolution pattern after storage at 40 degrees C/75% relative humidity for 6 months.

Epidemiological Bulletin, v. 18, n. 1, Mar. 1997

IN THIS ISSUE: Implementation of the International Statistical Classification of Diseases and Related Health Problems, Tenth Revision (ICD-10) | Cholera situation in the Americas, 1996 | Health situation in the Americas, selection of basic indicators | Situation of blood banks in the region, 1994-1995 | Consultation meeting report with amoebiasis experts | Scientific Advisory Committee (SAC) Meeting Report | AIDS Surveillance | New publications | Congresses and new publications

Boletín Epidemiológico, v. 18, n. 1 Mar. 1997

EN ESTE NÚMERO: Implementación de la clasificación estadística internacional de enfermedades y problemas relacionados con la salud, décima revisión (CIE-10) | Situación del cólera en las Américas, 1996 | Situación de salud en las Américas, selección de indicadores básicos | Situación de los bancos de sangre en la región, 1994-1995 | Informe de reunión de consulta con expertos en Amibiasis | Informe de reunión del comité asesor científico (CAC) | Vigilancia del SIDA | Nuevas publicaciones | Congresos y nuevas publicaciones

La amibiasis y el absceso hepático amibiano en México, un problema de salud pública de actualidad / The amebiasis and the amebic liver abscess in Mexico, a present time public health problem

Antecedentes: La amibiasis continúa siendo un problema de salud pública a nivel mundial. En México es un motivo de consulta frecuente y tan sólo en el Instituto Mexicano del Seguro Social se atienden más de medio millón de casos al año. La información epidemiológica en México es aún incompleta. Objetivo: Describir el comportamiento secular de la amibiasis en todas sus formas de presentación clínica y del abseco hepático amibiano, en la población mexicana y en la amparada por IMSS-Solidaridad. Métodos: Se realizó un estudio ecológico de tendencia. Se graficaron las tasas de incidencia de la amibiasis en todas sus formas, así como del absceso hepático amibiano por cada uno de los años de período analizado. Resultados: La incidencia de amibiasis en todas sus formas mostró una tendencia estable en el período de estudio, situación similar a lo observado con el absceso hepático amibiano. La amibiasis es más frecuente durante el primer año de vida. Por el contrario, el absceso hepático amibiano muestra un patrón de "J" invertida, es decir, la ocurrencia es mayor en los extremos de la vida. La letalidad ha mostrado una tendencia al descenso constante: Conclusiones: La amibiasis en México es un reflejo de las condiciones socioeconómicas y de la cultura inacabada mexicana. Es necesario propiciar la educación para la salud, al igual que el mejor diagnóstico y detección de portadores asintomáticos. Con las madres portadoras debieran redefinirse las políticas de salud, dada la elevada frecuencia de amibiasis y absceso hepático amibiano en los menores de un año.

[Amebiasis and amebic liver abscess in Mexico: a present-day public health problem]. / La amibiasis y el absceso hepático amibiano en México, un problema de salud pública de actualidad.

BACKGROUND: Amebiasis still remains as a major public health problem in the world. It is one of the most common reasons for medical consult. There are more than half a million cases of amebiasis just at the Mexican Institute of the Social Security. There is still a lack of epidemiologic information on amebiasis in Mexico. AIM: To describe the secular trend fro amebiasis and for amebic liver abscess in the Mexican population, as well as in those covered by IMSS Solidaridad. METHOD: An ecologic trend study was carried on. Incidence rate of amebiasis in all of its forms of presentation, and of amebic liver abscess, were plotted against each year for the 1986-1994 period. RESULTS: Amebiasis incidence in all of its forms of presentation showed a stable trend in this period, as it was seen with amebic liver abscess. Amebiasis is more common in the first years of life. On the contrary, amebic liver abscess showed an inverted 'J' pattern; its occurrence is higher in the extreme years of life. Fatality rates have shown a descendent trend. CONCLUSIONS: Amebiasis reflects socioeconomic conditions in Mexico and the fact that Mexican is still an endless culture. There is a need to promote health education, better diagnostic procedures and detection of asymptomatic carriers. Health policies for mothers that are asymptomatic carriers should be reviewed, due to the high rates of amebiasis and amebic liver abscess in children under one year of age.

The future of surgery in Papua New Guinea and the South Pacific.

Surgery in the South Pacific is different in many respects from surgery in Australia and New Zealand. It is primarily the surgery of trauma, infection, advanced malignancy, hollow-tube obstruction and congenital abnormalities. Specific tropical infections such as tuberculosis, typhoid, pigbel and amoebiasis occur regularly but constitute only a small proportion of all cases. The patients tend to be young, rural and poor, and often present late because access to surgical services is limited. The treatment patients receive is also compromised by a lack of resources--the result of underfunding and inefficient administration. Standards for appropriate surgical practice should be determined in-country and based at least on surgical audit and clinical studies. Even though Western diseases are emerging in the tropics, the best management may sometimes be different. Training of national surgeons is a priority if a sustainable surgical service is to be established. Such training is more effectively carried out in the home country, or at least in one with similar pathology and problems, rather than overseas. Project aid should support these schemes and encourage regional co-operation through the Fiji and Papua New Guinea medical schools. There remains an important role for visiting surgical specialists, but they need to ensure that they transfer skills and encourage the professional development of promising local doctors rather than simply focusing on treating patients.

Salmonellas

Trata de pesquisas realizadas pelo Instituto Evandro Chagas sobre a qualidade da água da orla de Belém. Os resultados mostram que 79% das amostras retiradas estavam contaminadas pela bactéria que causa a diarréia, a Salmonella, que é uma bactéria que representa risco à saúde da população. Também trata da amebíase transmitida pelo protozoário Entamoeba histolytica