Isolation and Identification of Pathogenic Acanthamoeba Species from Air Conditioning Systems, Egypt.

Acanthamoeba are free-living amoebae that cause granulomatous amoebic encephalitis and keratitis. In this study, we aimed to isolate and identify Acanthamoeba from air conditioning systems using in vitro cell culture and polymerase chain reaction assays. We also estimated the pathogenicity of the isolates by measuring their thermotolerance and studying mice models inoculated with these isolates. Of the 80 dust samples acquired, 41 (51.25%) were found to be positive for Acanthamoeba spp. using in vitro cell culture and the results were validated using PCR. Out of these 41 samples, 27 (65.9%) were thermotolerant and 16 (39%) samples could infect mice and cause histopathological effects. Highly pathogenic Acanthamoeba isolates were characterized by their thermotolerance and the ability to disseminate in all organs after infection, causing early death of infected animals. Our study thus validated the presence of pathogenic isolates of Acanthamoeba in air conditioners that may be potentially infectious to humans.

Cecal amebiasis mimicking inflammatory bowel disease.

Amebiasis is a frequently occurring parasitic infection in South East Asia. We present a case of a 54-year-old man with right lower quadrant abdominal pain that persisted for longer than 1 year. He had been diagnosed with inflammatory bowel disease in Indonesia. His abdominal pain persisted, despite therapy, and he visited Malaysia for transnational medical advice. Abdominal ultrasound showed fatty liver, gallbladder polyps, and a small left renal stone. Colonoscopy showed multiple ulcers in the cecum and a histopathological examination confirmed amebic infection of the cecum. The colonic ulcers subsided after anti-amebic treatment. This case highlights the need to consider the differential diagnosis of amebic colitis in patients presenting with manifestations of inflammatory bowel disease, especially in patients who live in or have traveled to endemic areas.

Crosstalk between Entamoeba histolytica and the human intestinal tract during amoebiasis.

The protozoan parasite Entamoeba histolytica is the microbial agent of amoebiasis - an infection that is endemic worldwide and is associated with high morbidity and mortality rates. As the disease develops, virulent E. histolytica deplete the mucus layer, interact with the intestinal epithelium, and then degrade the colonic mucosa and disrupt the extracellular matrix (ECM). Our research demonstrated that virulent parasites with an invasive phenotype display rapid, highly specific changes in their transcriptome (notably for essential factors involved in carbohydrate metabolism and the processing of glycosylated residues). Moreover, combined activation of parasite and host lytic enzymes leads to the destruction of the intestinal parenchyma. Together, these enzymes degrade the mucus layer and the ECM, and trigger the inflammatory response essential to the development of amoebiasis.

The Epidemiology and Clinical Features of Balamuthia mandrillaris Disease in the United States, 1974-2016.

BACKGROUND: Balamuthia mandrillaris is a free-living ameba that causes rare, nearly always fatal disease in humans and animals worldwide. B. mandrillaris has been isolated from soil, dust, and water. Initial entry of Balamuthia into the body is likely via the skin or lungs. To date, only individual case reports and small case series have been published. METHODS: The Centers for Disease Control and Prevention (CDC) maintains a free-living ameba (FLA) registry and laboratory. To be entered into the registry, a Balamuthia case must be laboratory-confirmed. Several sources were used to complete entries in the registry, including case report forms, CDC laboratory results, published case reports, and media information. SAS© version 9.3 software was used to calculate descriptive statistics and frequencies. RESULTS: We identified 109 case reports of Balamuthia disease between 1974 and 2016. Most (99%) had encephalitis. The median age was 36 years (range 4 months to 91 years). Males accounted for 68% of the case patients. California had the highest number of case reports, followed by Texas and Arizona. Hispanics constituted 55% for those with documented ethnicity. Exposure to soil was commonly reported. Among those with a known outcome, 90% of patients died. CONCLUSIONS: Balamuthia disease in the United States is characterized by a highly fatal encephalitis that affects patients of all ages. Hispanics were disproportionately affected. The southwest region of the United States reported the most cases. Clinician awareness of Balamuthia as a cause of encephalitis might lead to earlier diagnosis and initiation of treatment, resulting in better outcomes.

Development of a high- versus low-pathogenicity model of the free-living amoeba Naegleria fowleri.

Species in the genus Naegleria are free-living amoebae of the soil and warm fresh water. Although around 30 species have been recognized, Naegleria fowleri is the only one that causes primary amoebic meningoencephalitis (PAM) in humans. PAM is an acute and fast progressing disease affecting the central nervous system. Most of the patients die within 1-2 weeks of exposure to the infectious water source. The fact that N. fowleri causes such fast progressing and highly lethal infections has opened many questions regarding the relevant pathogenicity factors of the amoeba. In order to investigate the pathogenesis of N. fowleri under defined experimental conditions, we developed a novel high- versus low-pathogenicity model for this pathogen. We showed that the composition of the axenic growth media influenced growth behaviour and morphology, as well as in vitro cytotoxicity and in vivo pathogenicity of N. fowleri. Trophozoites maintained in Nelson's medium were highly pathogenic for mice, demonstrated rapid in vitro proliferation, characteristic expression of surface membrane vesicles and a small cell diameter, and killed target mouse fibroblasts by both contact-dependent and -independent destruction. In contrast, N. fowleri cultured in PYNFH medium exhibited a low pathogenicity, slower growth, increased cell size and contact-dependent target cell destruction. However, cultivation of the amoeba in PYNFH medium supplemented with liver hydrolysate (LH) resulted in trophozoites that were highly pathogenic in mice, and demonstrated an intermediate proliferation rate in vitro, diminished cell diameter and contact-dependent target cell destruction. Thus, in this model, the presence of LH resulted in increased proliferation of trophozoites in vitro and enhanced pathogenicity of N. fowleri in mice. However, neither in vitro cytotoxicity mechanisms nor the presence of membrane vesicles on the surface correlated with the pathologic potential of the amoeba. This indicated that the pathogenicity of N. fowleri remains a complex interaction between as-yet-unidentified cellular mechanisms.

Primary amebic meningoencephalitis: a case report and literature review.

Primary amebic meningoencephalitis (PAM) is a rare but nearly always fatal disease caused by infection with Naegleria fowleri, a thermophilic, free-living ameba found in freshwater environments. Cases of N. fowleri infection have been reported from many of the southern-tier states in the United States, with Florida and Texas disproportionately represented among them. Primary amebic meningoencephalitis presents clinically in a fashion that may be indistinguishable from bacterial and viral meningitis. Unfortunately, because the disease is so rare, PAM is often excluded from the differential diagnosis of children with meningitis resulting in delayed diagnostic and therapeutic efforts.Pediatric acute care practitioners in emergency departments, general pediatric wards, and critical care units, especially those practicing in the southern United States, should be familiar with the risk factors for acquisition of PAM, its clinical presentation, and the fact that common empiric treatment of bacterial meningitis will not treat N. fowleri. Herein, we present the case of an adolescent who died of PAM and review the (a) epidemiology, (b) pathophysiology, (c) available diagnostic modalities, (d) treatment options, and (e) outcomes of patients treated for N. fowleri infection of the central nervous system.

Primary amebic meningoencephalitis caused by Naegleria fowleri, Karachi, Pakistan.

We report 13 cases of Naegleria fowleri primary amebic meningoencephalitis in persons in Karachi, Pakistan, who had no history of aquatic activities. Infection likely occurred through ablution with tap water. An increase in primary amebic meningoencephalitis cases may be attributed to rising temperatures, reduced levels of chlorine in potable water, or deteriorating water distribution systems.

[Blastocystis hominis and nonpathogenic enteric protozoa in patients with pulmonary tuberculosis and those with HIV infection].

Blastocystis hominis and nonpathogenic enteric protozoa were diagnosed in 300 patients with pulmonary tuberculosis mainly of its infiltrative form and 500 with Stages II and III HIV infection; the patients received antituberculosis therapy (ATT) and antiretroviral therapy (ART), respectively. Control groups included 200 Tashkent dwellers and 350 patients with various noninfectious diseases of the gastrointestinal tract. Triple coproscopy was made. B. hominis was significantly more frequently detected in patients with pulmonary tuberculosis and those with HIV infection than in healthy individuals: in 53.6 +/- 2.9, 42.2 +/- 2.2, and 18.0 +/- 2.5, respectively (P < 0.01). Only did the tuberculosis or HIV-infected patients show a high intensity of B. hominis infection, which was accompanied by recurring diarrhea and nausea. The high activity of alanine aminotransferase and aspartate aminotransferase was observed in 20% of the patients with tuberculosis + blastocytosis; that of alkaline phosphatase was seen in 25%. The tuberculosis or HIV-infected patients were more frequently found to have Chylomastix mesnili, Jodamoeba butschlii, and Endolimax nana. The specific features of intestinal colonization seem to reflect changes in local immunity; the drugs included into ATT and ART have no substantial effects on the viability of protozoa.

Acanthamoeba affects the integrity of human brain microvascular endothelial cells and degrades the tight junction proteins.

Haematogenous spread is a key step in the development of Acanthamoeba granulomatous encephalitis, however it is not clear how circulating amoebae cross the blood-brain barrier to enter the CNS to produce disease. Using the primary human brain microvascular endothelial cells (HBMEC), which constitute the blood-brain barrier, here it is shown that Acanthamoeba abolishes the HBMEC transendothelial electrical resistance. Using traversal assays, it was observed that Acanthamoeba crosses the HBMEC monolayers. The primary interactions of Acanthamoeba with the HBMEC resulted in increased protein tyrosine phosphorylations and the activation of RhoA, suggesting host-parasite cross-talk. Furthermore, Western blot assays revealed that Acanthamoeba degraded occludin and zonula occludens-1 proteins in a Rho kinase-dependent manner. Overall, these findings suggest that Acanthamoeba affects the integrity of the monolayer and traverses the HBMEC by targeting the tight junction proteins.

Invasive amebiasis: a microcirculatory disorder?

The two current models of invasive amebiasis both hold that direct contact of toxic molecules and amebas with tissue produces the necrotic areas characteristic of this disorder. Whereas one model characterizes these toxic molecules as amebic products (e.g., lectins, amebapores, cysteine proteinases and other proteolytic enzymes), the other describes them as products of the inflammatory response (e.g., cytokines, nitric oxide, reactive oxygen intermediates and cytotoxic granules). Both these models can account for necrotic areas with many amebas present and with acute inflammation, but not those with few or no amebas present or with scarce inflammation. A new model poses that an inadequate immune response leads to a continuous and prolonged activation of endothelial cells (ECs) by amebas, amebic molecules and cytokines, which triggers the mechanisms leading to necrosis. Other toxic molecules later contribute to EC activation: nitric oxide, reactive oxygen intermediates, the activated complement and proteases. Hyperactivated endothelial cells continuously express adhesion molecules (e.g., ICAM-1 and E-selectin), pro-coagulant molecules (e.g., tissue factor, von Willebrand factor, and the plasminogen activator inhibitor), resulting in ever greater inflammation and thrombosis, which eventually reduces or blocks blood flow in some vessels and starves certain tissue areas of an adequate oxygen and nutrient supply. When necrotic areas first develop, they are surrounded by inflammatory cells due to the acute inflammation at this stage. However, these cells are starved of oxygen and essential nutrients by the same microcirculatory dysfunction. The increasing concentration of nitric oxide during amebiasis eventually has an anti-inflammatory and vasodilating effect, creating a new mechanism for the microcirculatory dysfunction. This local microcirculatory dysfunction can explain necrotic areas in the presence of many, few, or no amebas, with abundant or scarce inflammation.

Under the radar: balamuthia amebic encephalitis.

BACKGROUND: We present data from 9 years (1999-2008) of tests for Balamuthia mandrillaris, an agent of amebic encephalitis that were conducted as part of the California Encephalitis Project. METHODS: Specimens obtained from patients with encephalitis were sent to the California Encephalitis Project for diagnostic testing; a subset of these specimens were tested for Balamuthia species. Tests included indirect immunofluorescent staining of sections for amebae, fluorescent antibody staining and enzyme-linked immunosorbent assay for serum titers, and polymerase chain reaction for Balamuthia 16S mitochondrial DNA. Cerebrospinal fluid (CSF) samples obtained from patients with diverse types of encephalitis were also tested for a broad range of cytokines. RESULTS: Of >3500 cases referred to the California Encephalitis Project, 10 were found to be amebic encephalitis on the basis of serologic and CSF tests and examination of stained tissue sections. Most of these cases would have been described as "encephalitis of unknown origin" if it were not for the California Encephalitis Project. Nine of the 10 patients were male; ages ranged from 1.5 to 72 years. All patients had abnormal neuroimaging findings and abnormal CSF composition. The more common symptoms at presentation included headache, seizures, cranial nerve palsies, and lethargy. CSF specimens from patients with Balamuthia infection had significant elevations in the levels of cytokines IL-6 and IL-8, compared with specimens obtained from persons with viral or noninfectious encephalitides. CONCLUSIONS: Balamuthiasis is difficult to diagnose, and it is likely that cases go unrecognized because clinicians and laboratorians are unfamiliar with the disease. Alerting the medical community to this disease may lead to earlier diagnosis and improve the chances of survival.

Th-1/Th-2 cytokine pattern in human amoebic colitis.

Amoebiasis, caused by Entamoeba histolytica, is still considered a major health problem in developing countries. Since the immune response during human amoebiasis has not been clearly defined, we chose to evaluate cytokine production in patients suffering from amoebic colitis. A case-control association study was carried out on 62 subjects, including 31 patients with amoebic colitis and 31 healthy controls (age, sex and geographic region-matched). Serum levels of IL-12, IFN-gamma, IL-13 and IL-5 were measured by solid-phase sandwich enzyme linked immunosorbant assay. Serum levels of IFN-gamma, IL-12, IL-13 and IL-5 were higher in the patients with amoebic colitis than in healthy controls, but were only statistically increased for IL-5 (p = 0.04) and IL-13 (p = 0.014). Stratification of patients according to gender revealed that IL-13 was significantly elevated in men as compared to levels measured in women (p = 0.04). These findings suggest that E. histolytica induce a mixed Th-1/Th-2 response with a polarization toward Th-2 during the early stage of amoebiasis, which may aide in developing a clinical illness.

Characterization of brain inflammation during primary amoebic meningoencephalitis.

Naegleria fowleri is a free-living amoeba and the etiologic agent of primary amoebic meningoencephalitis (PAM). Trophozoites reach the brain by penetrating the olfactory epithelium, and invasion of the olfactory bulbs results in an intense inflammatory reaction. The contribution of the inflammatory response to brain damage in experimental PAM has not been delineated. Using both optical and electron microscopy, we analyzed the morphologic changes in the brain parenchyma due to inflammation during experimental PAM. Several N. fowleri trophozoites were observed in the olfactory bulbs 72 h post-inoculation, and the number of amoebae increased rapidly over the next 24 h. Eosinophils and neutrophils surrounding the amoebae were then noted at later times during infection. Electron microscopic examination of the increased numbers of neutrophils and the interactions with trophozoites indicated an active attempt to eliminate the amoebae. The extent of inflammation increased over time, with a predominant neutrophil response indicating important signs of damage and necrosis of the parenchyma. These data suggest a probable role of inflammation in tissue damage. To test the former hypothesis, we used CD38-/- knockout mice with deficiencies in chemotaxis to compare the rate of mortality with the parental strain, C57BL/6J. The results showed that inflammation and mortality were delayed in the knockout mice. Based on these results, we suggest that the host inflammatory response and polymorphonuclear cell lysis contribute to a great extent to the central nervous system tissue damage.

Fatal subacute necrotising brainstem encephalitis in a young man due to a rare parasitic (Balamuthia) infection.

We describe a case of brainstem inflammation in a young man which at first defied diagnosis. However, after his death, and notwithstanding our inability to find a cause at autopsy, we did not give up. After sending paraffin blocks to the Centers for Disease Control in Atlanta, Georgia, USA, they suggested the diagnosis of Balamuthia (amoebic) infection.

Amebíase e epidemiologia / Amebiasis and epidemiology

A infecção por Entamoeba histolytica, identificada há mais de 130 anos por Fedor A. Lesh, existe praticamente em todo o mundo e é hoje considerada uma DST - com relação à transmissão é a doença dos cinco efes: finger, feces, flies, fomites e fornication. A epidemiologia da amebíase na cidade do Rio de Janeiro (Brasil), estudada por um de nós (R.M.), parece confirmar o lugar de infecção no rol das doenças sexualmente transmissíveis. Epidemiologia é o estudo da ocorrência de uma doença - estudos epidemiológicos podem influenciar a vida de populações inteiras. O estudo de Framingham (EUA), a investigação de Sharr sobre a doença dos legionários e o trabalho de John Snow sobre a cólera são exemplos clássicos de estudos epidemiológicos que mudaram o comportamento e estilos de vida.

Entamoeba histolytica infection was identified more than 130 years ago, has worldwide occurrence and nowadays is considered a sexually transmitted disease (STD). Regarding transmission is considered as the five Fs disease: finger, feces, flies, fomites and fornication. Rio de Janeiro city amebiasis epidemiology was studied by one of us and seems to confirm its place on STD list. Epidemiology consists on the study of the disease occurrence - epidemiological studies can influence an entire population life. Framinghan (EUA), Legionnaire's disease Sharr investigation and John Snow cholera study are classic examples of epidemiological studies that changed behaviour and lifestyle.

Balamuthia amoebic encephalitis: an emerging disease with fatal consequences.

Balamuthia amoebic encephalitis (BAE), caused by the protozoan pathogen, Balamuthia mandrillaris, is a serious human disease with fatal consequences and a mortality rate of more than 95%. A key factor that contributes to the high mortality is the incomplete understanding of its pathogenesis and pathophysiology. The most distressing aspect is that the high level of mortality is due to lack of awareness combined with the lack of effective drugs. Early diagnosis followed by aggressive treatment may lead to cure. Several lines of evidence suggest that BAE develops as a result of haematogenous spread, but it is unclear how circulating amoebae enter the central nervous system and cause inflammation, blood-brain barrier disruption and neuronal injury. Recent studies have identified several parasite-host determinants for B. mandrillaris translocation of the blood-brain barrier, and host inflammatory markers that may be associated with neuronal injury. These determinants may provide important targets for the prevention and treatment of BAE. Here, we present a brief overview of the current understanding of the pathogenesis and pathophysiology of BAE, available diagnostic methods, possible therapeutic interventions and biology of the causative agent.

Fatal amebic encephalitis caused by Balamuthia mandrillaris in an immunocompetent host: a clinicopathological review of pathogenic free-living amebae in human hosts.

Encephalitis caused by Balamuthia amebic species is an increasingly recognized chronic granulomatous infectious process that may affect both immunocompetent and immunocompromised individuals. The course of the disease is insidious but fatal in most cases, mainly because of delayed diagnosis, difficulty in isolation and/or identification of the organism, and lack of well-established therapeutic regimens. We report a fatal case of Balamuthia mandrillaris chronic granulomatous encephalitis in an immunocompromised host and review the clinicopathologic characteristics of infections caused by this and other pathogenic free-living amebae.

Increased systemic vascular resistance in Atlantic salmon, Salmo salar L., affected with amoebic gill disease.

Previous investigations into the pathophysiology of amoebic gill disease (AGD) have suggested that there are probable cardiovascular effects associated with this disease. In the present study Atlantic salmon, Salmo salar L., were experimentally infected by cohabitation with diseased individuals. Two commonly used vasodilators, sodium nitroprusside (SNP) and captopril, the angiotensin-converting enzyme (ACE) inhibitor, were used as tools to investigate possible vasoconstriction and/or renin-angiotensin system (RAS) dysfunction in AGD-affected animals. Within the SNP trial, results showed that AGD-affected fish exhibited lowered cardiac output (Q), lowered cardiac stroke volume (V(S)) and a significantly elevated systemic vascular resistance (R(S)) compared with non-affected naïve counterparts. These effects were totally abolished following SNP administration (40 microg kg(-1)), however significant cardiovascular effects associated with SNP were not observed. Within the captopril trial, where AGD-affected fish were more diseased compared with the SNP trial, a significant hypertension was observed in AGD-affected fish. Captopril administration (10(-4) mol L(-1) at 1 mL kg(-1)) resulted in a significant drop in dorsal aortic pressure (P(DA)) for both AGD-affected and naïve control fish. In terms of peak individual responses, captopril administration effectively lowered P(DA) in both AGD-affected and naïve control groups equally. The drop in P(DA) following SNP administration however was significantly greater in AGD-affected fish potentially suggesting disease-related vasoconstriction. The lack of significant cardiovascular effects directly associated with both SNP and captopril administrations possibly relate to the 6 h recovery period following surgical procedures. However, while variable, these results do suggest that there are significant cardiovascular effects including vasoconstriction and hypertension associated with AGD.