A devastating case of a Balamuthia mandrillaris pediatric brain infection.

Balamuthia mandrillaris is an amoeba that causes an uncommon but deadly encephalitis, referred to as granulomatous amoebic encephalitis (GAE). The highest incidence reported worldwide has occurred in America, and within the United States, it has been highest in the Southwest affecting predominantly children and young men of Hispanic ethnicity. Clinical presentation of GAE includes fever, headache, nausea, vomiting, lethargy, irritability, stiff neck, hallucinations, photophobia, and seizures. Our patient was a Hispanic male child living in Arizona. The patient presented at 3 years of age for severe encephalitis. Symptoms included difficulty with balance, gait, and sitting up and seizure-like activity. Initial CT showed an area of decreased density consistent with edema in the right frontal and left frontoparietal lobes. Rapid progression was seen on further imaging over the length of the patient's hospital stay revealing diffusion restriction, necrosis/blood products, edema, and hemorrhage. The patient expired three weeks after onset of symptoms and one week after admission to our institution. While there are multiple biochemical techniques that can test for B. mandrillaris, they are rarely employed for multiple reasons stemming from the rare occurrence of this infection. Because of the fatal nature of this infection, we propose (1) testing should be considered if a patient presents with progressing encephalitis on imaging and other pathogenic etiologies are ruled out and (2) the threshold to treat empirically should be low due to the fatal nature of the infection.

The anti-amoebic activity of Pinus densiflora leaf extract against the brain-eating amoeba Naegleria fowleri.

Naegleria fowleri invades the brain and causes a fatal primary amoebic meningoencephalitis (PAM). Despite its high mortality rate of approximately 97%, an effective therapeutic drug for PAM has not been developed. Approaches with miltefosine, amphotericin B, and other antimicrobials have been clinically attempted to treat PAM, but their therapeutic efficacy remains unclear. The development of an effective and safe therapeutic drug for PAM is urgently needed. In this study, we investigated the anti-amoebic activity of Pinus densiflora leaf extract (PLE) against N. fowleri. PLE induced significant morphological changes in N. fowleri trophozoites, resulting in the death of the amoeba. The IC50 of PLE on N. fowleri was 62.3±0.95 µg/ml. Alternatively, PLE did not significantly affect the viability of the rat glial cell line C6. Transcriptome analysis revealed differentially expressed genes (DEGs) between PLE-treated and non-treated amoebae. A total of 5,846 DEGs were identified, of which 2,189 were upregulated, and 3,657 were downregulated in the PLE-treated amoebae. The DEGs were categorized into biological process (1,742 genes), cellular component (1,237 genes), and molecular function (846 genes) based on the gene ontology analysis, indicating that PLE may have dramatically altered the biological and cellular functions of the amoeba and contributed to their death. These results suggest that PLE has anti-N. fowleri activity and may be considered as a potential candidate for the development of therapeutic drugs for PAM. It may also be used as a supplement compound to enhance the therapeutic efficacy of drugs currently used to treat PAM.

Concomitant Occurrence of Hepatitis A and a Pyogenic Liver Abscess: A Pediatric Case.

Pyogenic liver abscess (PLA) and hepatitis A are common in developing countries. As there is an overlap of clinical features, a diagnosis of dual infection can be missed. Here, we present the case of a five-year-old male who presented with abdominal pain, fever, and jaundice diagnosed as a complicated liver abscess with concurrent hepatitis A. To our knowledge, this is the first case where a PLA co-existed with hepatitis A. Simultaneous infection should be considered when a patient with liver abscess presents with jaundice, especially in areas where both diseases are endemic. Early diagnosis of both is crucial as PLA is a potentially fatal disease and co-infection with hepatitis A may worsen clinical outcomes.

Combating the causative agent of amoebic keratitis, Acanthamoeba castellanii, using Padina pavonica alcoholic extract: toxicokinetic and molecular docking approaches.

Natural products play a significant role in providing the current demand as antiparasitic agents, which offer an attractive approach for the discovery of novel drugs. The present study aimed to evaluate in vitro the potential impact of seaweed Padina pavonica (P. pavonica) extract in combating Acanthamoeba castellanii (A. castellanii). The phytochemical constituents of the extract were characterized by Gas chromatography-mass spectrometry. Six concentrations of the algal extract were used to evaluate its antiprotozoal activity at various incubation periods. Our results showed that the extract has significant inhibition against trophozoites and cysts viability, with complete inhibition at the high concentrations. The IC50 of P. pavonica extract was 4.56 and 4.89 µg/mL for trophozoites and cysts, respectively, at 24 h. Morphological alterations of A. castellanii trophozoites/cysts treated with the extract were assessed using inverted and scanning electron microscopes and showed severe damage features upon treatment with the extract at different concentrations. Molecular Docking of extracted compounds against Acanthamoeba cytochrome P450 monooxygenase (AcCYP51) was performed using Autodock vina1.5.6. A pharmacokinetic study using SwissADME was also conducted to investigate the potentiality of the identified bioactive compounds from Padina extract to be orally active drug candidates. In conclusion, this study highlights the in vitro amoebicidal activity of P. pavonica extract against A. castellanii adults and cysts and suggests potential AcCYP51 inhibition.

Exploring therapeutic approaches against Naegleria fowleri infections through the COVID box.

Naegleria fowleri, known as the brain-eating amoeba, is the pathogen that causes the primary amoebic meningoencephalitis (PAM), a severe neurodegenerative disease with a fatality rate exceeding 95%. Moreover, PAM cases commonly involved previous activities in warm freshwater bodies that allow amoebae-containing water through the nasal passages. Hence, awareness among healthcare professionals and the general public are the key to contribute to a higher and faster number of diagnoses worldwide. Current treatment options for PAM, such as amphotericin B and miltefosine, are limited by potential cytotoxic effects. In this context, the repurposing of existing compounds has emerged as a promising strategy. In this study, the evaluation of the COVID Box which contains 160 compounds demonstrated significant in vitro amoebicidal activity against two type strains of N. fowleri. From these compounds, terconazole, clemastine, ABT-239 and PD-144418 showed a higher selectivity against the parasite compared to the remaining products. In addition, programmed cell death assays were conducted with these four compounds, unveiling compatible metabolic events in treated amoebae. These compounds exhibited chromatin condensation and alterations in cell membrane permeability, indicating their potential to induce programmed cell death. Assessment of mitochondrial membrane potential disruption and a significant reduction in ATP production emphasized the impact of these compounds on the mitochondria, with the identification of increased ROS production underscoring their potential as effective treatment options. This study emphasizes the potential of the mentioned COVID Box compounds against N. fowleri, providing a path for enhanced PAM therapies.

Anti-Acanthamoeba metallopharmaceuticals: Amoebicidal activity and synergistic effect of copper(II) coordination compound.

Acanthamoeba spp. emerged as a clinically important pathogen related to amoebic keratitis. It is among the main causes of corneal transplantation and vision loss in ophthalmology. The treatment protocols have a low cure rate, high toxicity, and need for drug combination. Transition metal compounds have shown promising antiprotozoal effects. This study evaluates the amoebicidal activity of copper(II) coordination compounds in combination with chlorhexidine and the cytotoxicity to topical ocular application. These copper(II) coordination compounds were screened against Acanthamoeba castellanii trophozoites (ATCC 50492). The cytotoxicity on rabbit corneal cell line (ATCC-CCL 60) was performed. The compounds showed high amoebicidal potential, with inhibition of trophozoite viability above 80%. The Cp12 and Cp13 compounds showed Minimal Inhibitory Amoebicidal Concentration (MIAC) at 200 µM and mean inhibitory concentration (IC50) values lower than 10 µM. Against the cysts, Cp12 showed a reduction in viability (48%) in the longest incubation period. A synergistic effect for Cp12 with chlorhexidine was observed. The compounds have a dose-dependent effect against rabbit corneal cells. Compound Cp12 has potential for future application in developing ophthalmic formulations against Acanthamoeba keratitis and its use in multipurpose solutions is highlighted.

Noninvasive diagnostic biomarkers, genomic profiling, and advanced microscopic imaging in the early detection and characterization of Naegleria fowleri infections leading to primary amebic meningoencephalitis (PAM).

This review delves into the strategies for early detection and characterization of Naegleria fowleri infections leading to primary amoebic meningoencephalitis (PAM). The study provides an in-depth analysis of current diagnostic approaches, including cerebrospinal fluid analysis, brain tissue examination, immunostaining techniques, and culture methods, elucidating their strengths and limitations. It explores the geographical distribution of N. fowleri, with a focus on regions near the equator, and environmental factors contributing to its prevalence. The review emphasizes the crucial role of early detection in PAM management, discussing the benefits of timely identification in treatment, personalized care, and prevention strategies. Genomic profiling techniques, such as conventional PCR, nested PCR, multiplex PCR, and real-time PCR, are thoroughly examined as essential tools for accurate and prompt diagnosis. Additionally, the study explores advanced microscopic imaging techniques to characterize N. fowleri's morphology and behavior at different infection stages, enhancing our understanding of its life cycle and pathogenic mechanisms. In conclusion, this review underscores the potential of these strategies to improve our ability to detect, understand, and combat N. fowleri infections, ultimately leading to better patient outcomes and enhanced public health protection.

Enteropathogenic Escherichia coli modulates the virulence and pathogenicity of Entamoeba dispar.

Amoebiasis is a disease caused by Entamoeba histolytica, affecting the large intestine of humans and occasionally leading to extra-intestinal lesions. Entamoeba dispar is another amoeba species considered commensal, although it has been identified in patients presenting with dysenteric and nondysenteric colitis, as well as amoebic liver abscess. Amoebic virulence factors are essential for the invasion and development of lesions. There is evidence showing that the association of enterobacteria with trophozoites contributes to increased gene expression of amoebic virulence factors. Enteropathogenic Escherichia coli is an important bacterium causing diarrhea, with high incidence rates in the world population, allowing it to interact with Entamoeba sp. in the same host. In this context, this study aims to evaluate the influence of enteropathogenic Escherichia coli on ACFN and ADO Entamoeba dispar strains by quantifying the gene expression of virulence factors, including galactose/N-acetyl-D-galactosamine-binding lectin, cysteine proteinase 2, and amoebapores A and C. Additionally, the study assesses the progression and morphological aspect of amoebic liver abscess and the profile of inflammatory cells. Our results demonstrated that the interaction between EPEC and ACFN Entamoeba dispar strains was able to increase the gene expression of virulence factors, as well as the lesion area and the activity of the inflammatory infiltrate. However, the association with the ADO strain did not influence the gene expression of virulence factors. Together, our findings indicate that the interaction between EPEC, ACFN, and ADO Entamoeba dispar strains resulted in differences in vitro and in vivo gene expression of Gal/GalNAc-binding lectin and CP2, in enzymatic activities of MPO, NAG, and EPO, and consequently, in the ability to cause lesions.

A case report of Balamuthia mandrillaris encephalitis.

Balamuthia amoebic encephalitis (BAE) is a rare and severe parasitic infection of the central nervous system. Its delayed diagnosis and treatment are often due to the lack of specific clinical manifestations and its poor prognosis. Reported mortality rates reach around 95%. The Balamuthia mandrillaris is also known as the "brain-eating amoeba." Recently, the use of metagenomic next-generation sequencing (mNGS) in clinical settings has led to an increase in BAE diagnoses. A case report detailing the use of mNGS to diagnose granulomatous encephalitis caused by the Baramsi amoeba has improved clinicians' understanding of this disease and helped reduce misdiagnoses and missed diagnoses.

Isolation and habitat analysis of Balamuthia mandrillaris from soil.

Balamuthia mandrillaris is a free-living amoeba that causes meningoencephalitis in mammals. Over 200 cases of infection were reported worldwide, with a fatality rate of over 95%. A clear route of infection was unknown for a long time until a girl died of granulomatous amoebic encephalitis (GAE) in California, USA, in 2003 due to infection with B. mandrillaris detected in a potted plant. Since then, epidemiological studies were conducted worldwide to detect B. mandrillaris in soil and other environmental samples. We previously reported the isolation of B. mandrillaris from the soil in Japan; however, the existing B. mandrillaris culture method with BM3 medium and COS-7 cells was unsuccessful. Therefore, in this study, we aimed to conduct soil analysis to determine the growth conditions of B. mandrillaris. B. mandrillaris-positive soils were defined as soils from which B. mandrillaris was isolated and environmental DNA was PCR-positive. Soils inhabited by B. mandrillaris were alkaline, with high electrical conductivity and characteristics of nutrient-rich soils of loam and clay loam. The results of this study suggest a possible reason for the high prevalence of GAE caused by B. mandrillaris among individuals employed in agriculture-related occupations.

Immunogens in Balamuthia mandrillaris: a proteomic exploration.

Balamuthia mandrillaris is the causative agent of granulomatous amoebic encephalitis, a rare and often fatal infection affecting the central nervous system. The amoeba is isolated from diverse environmental sources and can cause severe infections in both immunocompromised and immunocompetent individuals. Given the limited understanding of B. mandrillaris, our research aimed to explore its protein profile, identifying potential immunogens crucial for early granulomatous amoebic encephalitis diagnosis. Cultures of B. mandrillaris and other amoebas were grown under axenic conditions, and total amoebic extracts were obtained. Proteomic analyses, including two-dimensional electrophoresis and mass spectrometry, were performed. A 50-kDa band showed a robust recognition of antibodies from immunized BALB/c mice; peptides contained in this band were matched with elongation factor-1 alpha, which emerged as a putative key immunogen. Besides, lectin blotting revealed the presence of glycoproteins in B. mandrillaris, and confocal microscopy demonstrated the focal distribution of the 50-kDa band throughout trophozoites. Cumulatively, these observations suggest the participation of the 50-kDa band in adhesion and recognition mechanisms. Thus, these collective findings demonstrate some protein characteristics of B. mandrillaris, opening avenues for understanding its pathogenicity and developing diagnostic and therapeutic strategies.

Amoebic colitis-A diagnostic challenge on endoscopy: Case report.

Amoebic colitis remains a diagnostic challenge on colonoscopy given that its features resemble that of inflammatory bowel disease. We describe a similar case of a 66 years old male patient with multiple comorbidities including morbid obesity, end-stage renal disease requiring haemodialysis, IHD with PCI, T2DM, HTN and new onset dry cough for which he received a short course of steroids. He presented to the colorectal clinic with bleeding and mucus discharge per-rectum with no other symptoms related to bowel and non-specific colonoscopy findings with amoebic colitis only confirmed on histological diagnosis. The patient was commenced on a course of Metronidazole and followed up on first and sixth month after treatment where he remained well and reported no further symptoms.

Metagenomic next-generation sequencing for diagnosis of fatal Balamuthia amoebic encephalitis.

INTRODUCTION: Balamuthia amoebic encephalitis (BAE), caused by Balamuthia mandrillaris, is a rare and life-threatening infectious disease with no specific and effective treatments available. The diagnosis of BAE at an early stage is difficult because of the non-specific clinical manifestations and neuroimaging. CASE DESCRIPTION: A 52-year-old male patient, who had no previous history of skin lesions, presented to the emergency department with an acute headache, walking difficulties, and disturbance of consciousness. The patient underwent a series of examinations, including regular cerebrospinal fluid (CSF) studies and magnetic resonance imaging, and tuberculous meningoencephalitis was suspected. Despite being treated with anti-TB drugs, no clinical improvement was observed in the patient. Following corticosteroid therapy, the patient developed a rapid deterioration in consciousness with dilated pupils. Metagenomic next-generation sequencing (mNGS) revealed an unexpected central nervous system (CNS) amoebic infection, and the patient died soon after the confirmed diagnosis. CONCLUSION: This study highlights the application of mNGS for the diagnosis of patients with suspected encephalitis or meningitis, especially those caused by rare opportunistic infections.

Comparative Incidence of Pyogenic and Amoebic Liver Abscesses in Trauma and Emergency Patients at Indira Gandhi Institute of Medical Sciences (IGIMS), Patna: A One-Year Observational Study.

BACKGROUND: Liver abscesses, particularly pyogenic and amoebic types, pose a significant healthcare challenge, especially in developing countries. Accurate differentiation and effective treatment of these abscess types are crucial in emergency medical settings. This study aims to analyze the incidence, clinical characteristics, and treatment outcomes of pyogenic and amoebic liver abscesses in a trauma and emergency department setting. METHODS: Conducted at the Indira Gandhi Institute of Medical Sciences (IGIMS), Patna, this one-year observational study involved 100 patients diagnosed with liver abscesses. The study employed a comprehensive approach, examining incidence rates, demographic trends, clinical presentations, treatment modalities, and outcomes, including recurrence rates. RESULTS: The study observed a higher incidence of pyogenic liver abscesses, accounting for 60% of cases (n=60), compared to amoebic liver abscesses, which constituted 40% (n=40). In terms of demographics, pyogenic liver abscesses were more prevalent among older males, with the average age being 48 years, and 70% (n=42) of the patients were male. In contrast, amoebic liver abscess patients had an average age of 42 years, with 60% (n=24) being male. Key clinical findings revealed that pyogenic liver abscess cases (n=60) had higher white blood cell counts and elevated liver enzyme levels than those with amoebic liver abscesses (n=40). The treatment outcomes indicated high success rates for both types of liver abscesses. Pyogenic liver abscesses had a success rate of 90% (n=54), while amoebic liver abscesses showed a slightly higher success rate at 95% (n=38). However, there was a notable difference in recurrence rates: pyogenic liver abscesses had a recurrence rate of 8.3% (n=5), whereas amoebic liver abscesses had a lower recurrence rate of 2.5% (n=1). The logistic regression analysis conducted to identify potential predictors of treatment success did not reveal any statistically significant factors across both types of liver abscesses. CONCLUSION: The study highlights a higher incidence of pyogenic liver abscesses in an urban Indian healthcare setting and the complexity of predicting treatment outcomes based on demographic and clinical factors. The findings emphasize the need for nuanced clinical approaches and vigilant post-treatment monitoring, especially for pyogenic liver abscesses. They also underscore the importance of further research to explore additional variables influencing liver abscess treatment outcomes.

Real-time loop-mediated isothermal amplification (real-time LAMP) assay for rapid diagnosis of amoebic liver abscess.

Among the parasitic diseases, amoebic liver abscess (ALA) ranks second to malaria in terms of mortality. Due to the poor sensitivity of conventional diagnostic methods, there is a need for the development of effective and rapid diagnostic methods for ALA. Thus, the purpose of this work was to develop a real-time loop-mediated isothermal amplification (RT-LAMP) assay specific to Entamoeba histolytica. Further, we compared the performance of real-time LAMP with conventional and real-time PCR (RT-PCR) targeting 18S small subunit ribosomal RNA (18S SSU rRNA) gene of E. histolytica in patients with ALA. A total of 126 liver samples were obtained for the study. Of these, 96 aspirated pus samples were obtained from patients suffering from an ALA (serology confirmed, anti-amoebic immunoglobulin IgG positive), 19 aspirated pus samples from patients with pyogenic liver abscess (PLA, 16S RNA gene positive) and 11 autopsy liver tissues. The results showed that the DNA of E. histolytica was detected in 81 samples by conventional PCR, 93 by RT-PCR and 95 by RT-LAMP. The analytical sensitivity of the RT-LAMP assay was much higher than the other two techniques. RT-LAMP assay was able to amplify up to one copy of the targeted gene of E. histolytica while conventional PCR and RT-PCR could amplify up to 103 and 102 copies of the targeted gene of E. histolytica, respectively. In conclusion, RT-LAMP proved to be a sensitive, specific and rapid test which can be utilised as an effective tool for the diagnosis of ALA.

Novel diagnostic approach for amoebic liver abscess using cell free (cf) DNA: a prospective study.

BACKGROUND: Amoebic liver abscess (ALA) is commonly seen in tropical countries and diagnosis of ALA relies mainly on non-specific serological and imaging techniques as well as PCR from pus. OBJECTIVE: This study evaluated the potential of using cell free DNA (cfDNA) from serum and urine for diagnosing ALA. METHODS: We prospectively evaluated quantitative PCR (qPCR) for detection of cf DNA in serum and urine sample in all liver abscess patients. The samples were collected from patients reporting to emergency ward of Postgraduate Institute of Medical Education and Research, Chandigarh, India with symptoms suggestive of liver abscess. Real time PCR was done to detect cf DNA in serum and urine by targeting 99-bp unit of small subunit rRNA of Entamoeba histolytica and conventional PCR for pus. RESULTS: A total 113 samples (serum and urine) and 100 pus samples were analysed. A total of 62 ALA patients were confirmed; with maximum 57 patients detected by qPCR for cfDNA in the serum, 55 patients by PCR on pus aspirate and 50 ALA patients by qPCR for cfDNA in urine sample. Therefore, the sensitivity of qPCR for detection of cf DNA in serum was 91.94% and for urine was 80.65%. CONCLUSION: A total of 11.2% of ALA patients were diagnosed only through detection of E. histolytica cf DNA in their serum and urine. Detection of cfDNA from serum, urine of ALA has a potential role in future especially for developing countries as it is a rapid, sensitive and patient friendly diagnostic approach.

Liver abscess with extension into the Inferior Vena Cava: Case series of a rare complication.

Liver abscess is a common disease, however, it is a rare cause of venous thrombosis. The incidence of amoebic and pyogenic liver abscess in developing countries is high, however, the occurrence of inferior vena cava thrombosis secondary to liver abscess is a very rare and life-threatening complication. Computed tomography (CT) scan is an ideal investigation tool for diagnosing the various complications associated with liver abscess. Here we describe 3 cases of liver abscess in patients who developed a rare vascular complication of inferior vena cava. Liver abscesses should be considered as a rare cause of IVC thrombosis in the correct clinical context. All the patients were managed with ultrasound-guided pigtail insertion and antimicrobial agents.

The Challenging Path to Diagnosing Extraintestinal Amoebiasis: A Case Report of an HIV-Infected Patient.

Background: Amoebiasis, an infectious disease caused by the parasitic protozoan E. histolytica, is easily misdiagnosed due to its declining incidence and atypical symptoms. Case Presentation: A 31-year-old male presented to the hospital with dyspnea and inability to lie flat. Imaging studies indicated a large amount of pleural effusion on the right side and multiple huge cysts in the liver. The patient underwent liver tumor resection surgery at another hospital due to suspected malignancy, but no evidence of relevant malignant tumors was found in the pathological examination. Subsequently, we performed metagenomic next-generation sequencing on the liver drainage fluid and obtained liver pathology slides from the hospital where the surgery was performed at that time. Both of them confirmed the diagnosis of amoebic infection. Empirical treatment with metronidazole was initiated before the diagnosis was confirmed, along with symptomatic treatments such as thoracic drainage and liver drainage. Eventually, the patient's condition improved and he was discharged smoothly. Conclusion: In order to avoid misdiagnosis of amoebiasis, thoroughly inquiring about the patient's medical history, shifting perspectives and continuing investigating are necessary when one diagnostic approach proves ineffective. Besides, interdisciplinary collaboration and persistent efforts are crucial for accurate diagnosis.