Practical Guidance for Clinical Microbiology Laboratories: Laboratory Diagnosis of Parasites from the Gastrointestinal Tract

This Practical Guidance for Clinical Microbiology document on the laboratory diagnosis of parasites from the gastrointestinal tract provides practical information for the recovery and identification of relevant human parasites. The document is based on a comprehensive literature review and expert consensus on relevant diagnostic methods. However, it does not include didactic information on human parasite life cycles, organism morphology, clinical disease, pathogenesis, treatment, or epidemiology and prevention. As greater emphasis is placed on neglected tropical diseases, it becomes highly probable that patients with gastrointestinal parasitic infections will become more widely recognized in areas where parasites are endemic and not endemic. Generally, these methods are nonautomated and require extensive bench experience for accurate performance and interpretation.

Malignant Transformation of Hymenolepis nana in a Human Host.

Neoplasms occur naturally in invertebrates but are not known to develop in tapeworms. We observed nests of monomorphic, undifferentiated cells in samples from lymph-node and lung biopsies in a man infected with the human immunodeficiency virus (HIV). The morphologic features and invasive behavior of the cells were characteristic of cancer, but their small size suggested a nonhuman origin. A polymerase-chain-reaction (PCR) assay targeting eukaryotes identified Hymenolepis nana DNA. Although the cells were unrecognizable as tapeworm tissue, immunohistochemical staining and probe hybridization labeled the cells in situ. Comparative deep sequencing identified H. nana structural genomic variants that are compatible with mutations described in cancer. Invasion of human tissue by abnormal, proliferating, genetically altered tapeworm cells is a novel disease mechanism that links infection and cancer.

Three Cases of Neurologic Syndrome Caused by Donor-Derived Microsporidiosis.

In April 2014, a kidney transplant recipient in the United States experienced headache, diplopia, and confusion, followed by neurologic decline and death. An investigation to evaluate the possibility of donor-derived infection determined that 3 patients had received 4 organs (kidney, liver, heart/kidney) from the same donor. The liver recipient experienced tremor and gait instability; the heart/kidney and contralateral kidney recipients were hospitalized with encephalitis. None experienced gastrointestinal symptoms. Encephalitozoon cuniculi was detected by tissue PCR in the central nervous system of the deceased kidney recipient and in renal allograft tissue from both kidney recipients. Urine PCR was positive for E. cuniculi in the 2 surviving recipients. Donor serum was positive for E. cuniculi antibodies. E. cuniculi was transmitted to 3 recipients from 1 donor. This rare presentation of disseminated disease resulted in diagnostic delays. Clinicians should consider donor-derived microsporidial infection in organ recipients with unexplained encephalitis, even when gastrointestinal manifestations are absent.

Use of the Novel Therapeutic Agent Miltefosine for the Treatment of Primary Amebic Meningoencephalitis: Report of 1 Fatal and 1 Surviving Case.

Primary amebic meningoencephalitis (PAM) is a fulminant central nervous system infection caused by the thermophilic free-living ameba Naegleria fowleri. Few survivals have been documented and adequate treatment is lacking. We report 2 PAM cases, 1 fatal and 1 surviving, treated with the novel antiparasitic agent miltefosine.

Transmission of Balamuthia mandrillaris by Organ Transplantation.

BACKGROUND: During 2009 and 2010, 2 clusters of organ transplant-transmitted Balamuthia mandrillaris, a free-living ameba, were detected by recognition of severe unexpected illness in multiple recipients from the same donor. METHODS: We investigated all recipients and the 2 donors through interview, medical record review, and testing of available specimens retrospectively. Surviving recipients were tested and treated prospectively. RESULTS: In the 2009 cluster of illness, 2 kidney recipients were infected and 1 died. The donor had Balamuthia encephalitis confirmed on autopsy. In the 2010 cluster, the liver and kidney-pancreas recipients developed Balamuthia encephalitis and died. The donor had a clinical syndrome consistent with Balamuthia infection and serologic evidence of infection. In both clusters, the 2 asymptomatic recipients were treated expectantly and survived; 1 asymptomatic recipient in each cluster had serologic evidence of exposure that decreased over time. Both donors had been presumptively diagnosed with other neurologic diseases prior to organ procurement. CONCLUSIONS: Balamuthia can be transmitted through organ transplantation with an observed incubation time of 17-24 days. Clinicians should be aware of Balamuthia as a cause of encephalitis with high rate of fatality, and should notify public health departments and evaluate transplant recipients from donors with signs of possible encephalitis to facilitate early diagnosis and targeted treatment. Organ procurement organizations and transplant centers should be aware of the potential for Balamuthia infection in donors with possible encephalitis and also assess donors carefully for signs of neurologic infection that may have been misdiagnosed as stroke or as noninfectious forms of encephalitis.

Disseminated Balamuthia mandrillaris Infection.

Balamuthia mandrillaris is a rare cause of human infection, but when infections do occur, they result in high rates of morbidity and mortality. A case of disseminated Balamuthia infection is presented. Early diagnosis and initiation of recommended therapy are essential for increased chances of successful outcomes.

In vitro growth, cytopathic effects and clearance of monolayers by clinical isolates of Balamuthia mandrillaris in human skin cell cultures.

Balamuthia mandrillaris is a free-living ameba (FLA) that has been isolated or its DNA identified in soil, dust and water. It causes a fatal central nervous system infection in humans and animals. Although it is environmental as Acanthamoeba and Naegleria fowleri, the two other free-living amebae that also cause CNS infections in humans and other animals, Balamuthia does not feed on bacteria as the other FLA. In the laboratory, it can be grown on a variety of mammalian cell cultures. In this study we examined the ability of three different Balamuthia isolates to grow on several different human skin cell cultures including the WT/A keratinocyte cell cultures. A corneal isolate of Acanthamoeba castellanii was used for comparison.

Microsporidiosis acquired through solid organ transplantation: a public health investigation.

BACKGROUND: Encephalitozoon cuniculi, a microsporidial species most commonly recognized as a cause of renal, respiratory, and central nervous system infections in immunosuppressed patients, was identified as the cause of a temporally associated cluster of febrile illness among 3 solid organ transplant recipients from a common donor. OBJECTIVE: To confirm the source of the illness, assess donor and recipient risk factors, and provide therapy recommendations for ill recipients. DESIGN: Public health investigation. SETTING: Two transplant hospitals and community interview with the deceased donor's family. PATIENTS: Three transplant recipients and the organ donor. MEASUREMENTS: Specimens were tested for microsporidia by using culture, immunofluorescent antibody, polymerase chain reaction,immunohistochemistry, and electron microscopy. Donor medical records were reviewed and a questionnaire was developed to assess for microsporidial infection. RESULTS: Kidneys and lungs were procured from the deceased donor and transplanted to 3 recipients who became ill with fever 7 to 10 weeks after the transplant. Results of urine culture, serologic,and polymerase chain reaction testing were positive for E. cuniculi of genotype III in each recipient; the organism was also identified in biopsy or autopsy specimens in all recipients. The donor had positive serologic test results for E. cuniculi. Surviving recipients received albendazole. Donor assessment did not identify factors for suspected E. cuniculi infection. LIMITATION: Inability to detect organism by culture or polymerase chain reaction in donor due to lack of autopsy specimens. CONCLUSION: Microsporidiosis is now recognized as an emerging transplant-associated disease and should be considered in febrile transplant recipients when tests for routinely encountered agents are unrevealing. Donor-derived disease is critical to assess when multiple recipients from a common donor are ill.

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.

Serologic survey for exposure following fatal Balamuthia mandrillaris infection.

Granulomatous amebic encephalitis (GAE) from Balamuthia mandrillaris, a free-living ameba, has a case fatality rate exceeding 90% among recognized cases in the USA. In August 2010, a GAE cluster occurred following transplantation of infected organs from a previously healthy landscaper in Tucson, AZ, USA, who died from a suspected stroke. As B. mandrillaris is thought to be transmitted through soil, a serologic survey of landscapers and a comparison group of blood donors in southern Arizona was performed. Three (3.6%) of 83 serum samples from landscapers and 11 (2.5%) of 441 serum samples from blood donors were seropositive (p = 0.47). On multivariable analysis, county of residence was associated with seropositivity, whereas age, sex, and ethnicity were not. Exposure to B. mandrillaris, previously unexamined in North America, appears to be far more common than GAE in Southern Arizona. Risk factors for disease progression and the ameba's geographic range should be examined.

Balamuthia mandrillaris: in vitro interactions with selected protozoa and algae.

Although Balamuthia mandrillaris was identified more than two decades ago as an agent of fatal granulomatous encephalitis in humans and other animals, little is known about its ecological niche, biological behavior in the environment, food preferences and predators, if any. When infecting humans or other animals, Balamuthia feeds on tissues; and in vitro culture, it feeds on mammalian cells (monkey kidney cells, human lung fibroblasts, and human microvascular endothelial cells). According to recent reports, it is believed that Balamuthia feeds on small amebae, for example, Acanthamoeba that are present in its ecological niche. To test this hypothesis, we associated Balamuthia on a one-on-one basis with selected protozoa and algae. We videotaped the behavior of Balamuthia in the presence of a potential prey, its ability to hunt and attack its food, and the time required to eat and cause damage to the target cell by direct contact. We found that B. mandrillaris ingested trophozoites of Naegleria fowleri, Naegleria gruberi, Acanthamoeba spp., Trypanosoma cruzi epimastigotes, Toxoplasma gondii tachyzoites, and Giardia. However, it did not feed on Acanthamoeba cysts or algae. Balamuthia caused cytolysis of T. cruzi epimastigotes and T. gondii tachyzoites by direct contact. Balamuthia trophozoites and cysts were, however, eaten by Paramecium sp.

Characterization of a new pathogenic Acanthamoeba Species, A. byersi n. sp., isolated from a human with fatal amoebic encephalitis.

Acanthamoeba spp. are free-living amoebae that are ubiquitous in natural environments. They can cause cutaneous, nasopharyngeal, and disseminated infection, leading to granulomatous amebic encephalitis (GAE) in immunocompromised individuals. In addition, they can cause amoebic keratitis in contact lens wearers. Acanthamoeba GAE is almost always fatal because of difficulty and delay in diagnosis and lack of optimal antimicrobial therapy. Here, we report the description of an unusual strain isolated from skin and brain of a GAE patient. The amoebae displayed large trophozoites and star-shaped cysts, characteristics for acanthamoebas belonging to morphology Group 1. However, its unique morphology and growth characteristics differentiated this new strain from other Group 1 species. DNA sequence analysis, secondary structure prediction, and phylogenetic analysis of the 18S rRNA gene confirmed that this new strain belonged to Group 1, but that it was distinct from the other sequence types within that group. Thus, we hereby propose the establishment of a new species, Acanthamoeba byersi n. sp. as well as a new sequence type, T18, for this new strain. To our knowledge, this is the first report of a Group 1 Acanthamoeba that is indisputably pathogenic in humans.

The mitochondrial genome and a 60-kb nuclear DNA segment from Naegleria fowleri, the causative agent of primary amoebic meningoencephalitis.

Naegleria fowleri is a unicellular eukaryote causing primary amoebic meningoencephalitis, a neuropathic disease killing 99% of those infected, usually within 7-14 days. Naegleria fowleri is found globally in regions including the US and Australia. The genome of the related nonpathogenic species Naegleria gruberi has been sequenced, but the genetic basis for N. fowleri pathogenicity is unclear. To generate such insight, we sequenced and assembled the mitochondrial genome and a 60-kb segment of nuclear genome from N. fowleri. The mitochondrial genome is highly similar to its counterpart in N. gruberi in gene complement and organization, while distinct lack of synteny is observed for the nuclear segments. Even in this short (60-kb) segment, we identified examples of potential factors for pathogenesis, including ten novel N. fowleri-specific genes. We also identified a homolog of cathepsin B; proteases proposed to be involved in the pathogenesis of diverse eukaryotic pathogens, including N. fowleri. Finally, we demonstrate a likely case of horizontal gene transfer between N. fowleri and two unrelated amoebae, one of which causes granulomatous amoebic encephalitis. This initial look into the N. fowleri nuclear genome has revealed several examples of potential pathogenesis factors, improving our understanding of a neglected pathogen of increasing global importance.

Diagnostic challenges in Balamuthia mandrillaris infections.

Balamuthia mandrillaris is an emerging cause of subacute granulomatous amebic encephalitis (GAE). The diagnosis of this infection has proven to be difficult and is usually made postmortem. Early recognition and treatment may offer some benefit. This report describes a previously healthy woman who died from GAE due to B. mandrillaris.

Primary amebic meningoencephalitis in Florida: a case report and epidemiological review of Florida cases.

Primary amebic meningoencephalitis (PAM) is a rare but nearly always fatal infection of the central nervous system caused by the thermophilic, free-living ameba Naegleria fowleri. Since its first description in 1965 through 2010, 118 cases have been reported in the U.S.; all cases are related to environmental exposure to warm freshwater; most have occurred in children and adolescents and are associated with recreational water activities, such as swimming, diving, or playing in freshwater lakes, ponds, or rivers. Over one-fourth of all national PAM cases have occurred in Florida. The authors describe here a fatal case of PAM in a resident of northeast Florida and the ensuing environmental and public health investigation; they also provide a review of all cases of PAM in Florida from 1962 to 2010 and discuss public health responses to PAM in Florida, highlighting opportunities for positive collaboration between state and local environmental health specialists, epidemiologists, and the Centers for Disease Control and Prevention.

Fatal Naegleria fowleri infection acquired in Minnesota: possible expanded range of a deadly thermophilic organism.

BACKGROUND: Primary amebic meningoencephalitis (PAM), caused by the free-living ameba Naegleria fowleri, has historically been associated with warm freshwater exposures at lower latitudes of the United States. In August 2010, a Minnesota resident, aged 7 years, died of rapidly progressive meningoencephalitis after local freshwater exposures, with no history of travel outside the state. PAM was suspected on the basis of amebae observed in cerebrospinal fluid. METHODS: Water and sediment samples were collected at locations where the patient swam during the 2 weeks preceding illness onset. Patient and environmental samples were tested for N. fowleri with use of culture and real-time polymerase chain reaction (PCR); isolates were genotyped. Historic local ambient temperature data were obtained. RESULTS: N. fowleri isolated from a specimen of the patient's brain and from water and sediment samples was confirmed using PCR as N. fowleri genotype 3. Surface water temperatures at the times of collection of the positive environmental samples ranged from 22.1°C to 24.5°C. August 2010 average air temperature near the exposure site was 25°C, 3.6°C above normal and the third warmest for August in the Minneapolis area since 1891. CONCLUSIONS: This first reported case of PAM acquired in Minnesota occurred 550 miles north of the previously reported northernmost case in the Americas. Clinicians should be aware that N. fowleri-associated PAM can occur in areas at much higher latitude than previously described. Local weather patterns and long-term climate change could impact the frequency of PAM.

Primary amebic meningoencephalitis deaths associated with sinus irrigation using contaminated tap water.

BACKGROUND: Naegleria fowleri is a climate-sensitive, thermophilic ameba found in the environment, including warm, freshwater lakes and rivers. Primary amebic meningoencephalitis (PAM), which is almost universally fatal, occurs when N. fowleri-containing water enters the nose, typically during swimming, and N. fowleri migrates to the brain via the olfactory nerve. In 2011, 2 adults died in Louisiana hospitals of infectious meningoencephalitis after brief illnesses. METHODS: Clinical and environmental testing and case investigations were initiated to determine the cause of death and to identify the exposures. RESULTS: Both patients had diagnoses of PAM. Their only reported water exposures were tap water used for household activities, including regular sinus irrigation with neti pots. Water samples, tap swab samples, and neti pots were collected from both households and tested; N. fowleri were identified in water samples from both homes. CONCLUSIONS: These are the first reported PAM cases in the United States associated with the presence of N. fowleri in household plumbing served by treated municipal water supplies and the first reports of PAM potentially associated with the use of a nasal irrigation device. These cases occurred in the context of an expanding geographic range for PAM beyond southern tier states with recent case reports from Minnesota, Kansas, and Virginia. These infections introduce an additional consideration for physicians recommending nasal irrigation and demonstrate the importance of using appropriate water (distilled, boiled, filtered) for nasal irrigation. Furthermore, the changing epidemiology of PAM highlights the importance of raising awareness about this disease among physicians treating persons showing meningitislike symptoms.

Balamuthia mandrillaris and Acanthamoeba amebic encephalitis with neurotoxoplasmosis coinfection in a patient with advanced HIV infection.

We describe a patient with advanced HIV infection and Balamuthia mandrillaris and Acanthamoeba amebic encephalitis with Toxoplasma gondii coinfection. A multidisciplinary effort and state-of-the-art diagnostic techniques were required for diagnosis. Our patient is the first reported case of an HIV-infected person with dual Balamuthia mandrillaris and Acanthamoeba amebic encephalitis with neurotoxoplasmosis coinfection.

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.