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Microsporidia are a group of obligate intracellular parasites that naturally infect domestic and wild animals. Human microsporidiosis is an increasingly recognized multisystem opportunistic infection. The clinical manifestations are diverse with diarrhea being the most common presenting symptom. We present a 52-year-old woman with a history of amyopathic dermatomyositis complicated by interstitial lung disease managed with mycophenolate mofetil and hydroxychloroquine who presented with a 7-month history of recurrent subcutaneous nodules as well as intermittent diarrhea and chronic sinusitis. A punch biopsy showed superficial and deep lymphocytic and granulomatous dermatitis with focal necrosis. Tissue stains for microorganisms revealed oval 1 to 3 μm spores within the necrotic areas in multiple tissue stains. Additional studies at the Centers for Disease Control and Prevention confirmed cutaneous microsporidiosis. This case is one of very few confirmed examples of cutaneous microsporidiosis reported in the literature.

For complex clinical cases where a parasitic infection is suspected, it can be difficult for clinicians to recommend an appropriate laboratory test. These tests are usually pathogen-specific and require a certain degree of suspicion for the precise etiology. A recently described assay, the universal parasite diagnostic (UPDx) can potentially provide a diagnosis of any parasite present in a specimen. Using primers that amplify DNA from all eukaryotes, UPDx differentiates several parasitic infections in blood by amplicon-based next-generation sequencing (NGS) of the 18S rDNA locus. As the state’s public health reference laboratory, the Parasitology Laboratory at the Wadsworth Center (Albany, NY) receives specimens from patients who have potentially encountered a wide variety of parasites. As such, the ability to differentiate several blood parasites using a single assay is of interest. We assessed UPDx for its ability to confirm parasitic infections for 20 specimens that were previously identified by real-time PCR (RT-PCR). This included specimens positive for Babesia microti, Trypanosoma cruzi, Leishmania tropica, various Plasmodium species, and specimens comprising mixed Plasmodium sp. infections. Results obtained using UPDx were largely concordant with the RT-PCR assays. A T. cruzi positive specimen was negative by UPDx and for two mixed Plasmodium sp. infections only one species was detected. The results obtained for other specimens were concordant. We conclude that UPDx shows promise for the detection of blood parasites in diagnostic laboratories. As NGS becomes cheaper, assays like UPDx will become increasingly amenable to use in clinical settings.

Answer: d. Spores undergo gemination resulting in extrusion of polar tubule into the host cell. Via the polar tubule, the infective sporoplasm is released into the host cell, which undergoes various stages of development. Answer: a. Electron microscopy. Transmission electron microscopy helps in

Background Angiostrongylus cantonensis (Ac), or the rat lungworm, is a major cause of eosinophilic meningitis. Humans are infected by ingesting the 3rd stage larvae from primary hosts, snails, and slugs, or paratenic hosts. The currently used molecular test is a qPCR assay targeting the ITS1 rDNA region (ITS1) of Ac. Methods In silico design of a more sensitive qPCR assay was performed based on tandem repeats predicted to be the most abundant by the RepeatExplorer algorithm. Genomic DNA (gDNA) of Ac were used to determine the analytical sensitivity and specificity of the best primer/probe combination. This assay was then applied to clinical and environmental samples. Results The limit of detection of the best performing assay, AcanR3990, was 1 fg (the DNA equivalent of 1/100 000 dilution of a single 3rd stage larvae). Out of 127 CDC archived CSF samples from varied geographic locations, the AcanR3990 qPCR detected the presence of Ac in 49/49 ITS1 confirmed angiostrongyliasis patients, along with 15/73 samples previously negative by ITS1 qPCR despite strong clinical suspicion for angiostrongyliasis. Intermediate hosts (gastropods) and an accidental host, a symptomatic horse, were also tested with similar improvement in detection observed. AcanR3990 qPCR did not cross-react in 5 CSF from patients with proven neurocysticercosis, toxocariasis, gnathostomiasis, and baylisascariasis. AcanR3990 qPCR failed to amplify genomic DNA from the other related Angiostrongylus species tested except for Angiostrongylus mackerrasae (Am), a neurotropic species limited to Australia that would be expected to present with a clinical syndrome indistinguishable from Ac. Conclusion These results suggest AcanR3990 qPCR assay is highly sensitive and specific with potential wide applicability as a One Health detection method for Ac and Am.

Angiostrongyliasis is caused by infection and migration to the brain of larvae of the parasitic nematode Angiostrongylus cantonensis, or rat lungworm. Adult A. cantonensis reside in the lungs of the definitive wild rodent host, where they produce larvae passed in feces, which are then ingested by snails and slugs (gastropods). Human infection typically occurs when gastropods containing mature larvae are inadvertently ingested by humans. Although human infection often is asymptomatic or involves transient mild symptoms, larval migration to the brain can lead to eosinophilic meningitis, focal neurologic deficits, coma, and death. The majority of cases of human angiostrongyliasis occur in Asia and the Pacific Islands, including Hawaii, but autochthonous and imported cases have been reported in the continental United States (1,2), underscoring the importance of provider recognition to ensure prompt identification and treatment. The epidemiologic and clinical features of 12 angiostrongyliasis cases in the continental United States were analyzed. These cases were identified through A. cantonensis polymerase chain reaction (PCR) testing (3) of cerebrospinal fluid (CSF) submitted to CDC from within the continental United States. Six cases were likely a result of autochthonous transmission in the southern United States. All 12 patients had CSF pleocytosis and eosinophilia, consistent with eosinophilic meningitis. Health care providers need to be aware of the possibility of angiostrongyliasis in patients with eosinophilic meningitis, especially in residents in the southern United States or persons who have traveled outside the continental United States and have a history of ingestion of gastropods or contaminated raw vegetables.

Infection by the rat lungworm Angiostrongylus cantonensis represents the most common cause of infectious eosinophilic meningitis in humans, causing central nervous system (CNS) angiostrongyliasis. Most of CNS angiostrongyliasis cases were described in Asia, Pacific Basin, Australia, and some limited parts of Africa and America. CNS angiostrongyliasis has been reported in the Caribbean but never in the Lesser Antilles. The primary objectives of this study were to depict the first case of CNS angiostrongyliasis in the Lesser Antilles and investigate the environmental presence of A. cantonensis in Guadeloupe, Lesser Antilles. In December 2013, a suspected case of CNS angiostrongyliasis in an 8-month-old infant in Guadeloupe was investigated by real-time polymerase chain reaction (PCR) testing on cerebral spinal fluid (CSF). The environmental investigation was performed by collecting Achatina fulica molluscs from different parts of Guadeloupe and testing the occurrence of A. cantonensis by real-time PCR. CSF from the suspected case of angiostrongyliasis was positive for A. cantonensis by real-time PCR. Among 34 collected snails for environmental investigation, 32.4% were positive for A. cantonensis. In conclusion, we report the first laboratory-confirmed case of CNS-angiostrongyliasis in the Lesser Antilles. We identified the presence and high prevalence of A. cantonensis in A. fulica in Guadeloupe. These results highlight the need to increase awareness of this disease and implement public health programs in the region to prevent human cases of angiostrongyliasis and improve management of eosinophilic meningitis patients.

Babesiosis is an emerging tick-borne disease caused by apicomplexan parasites of the genus Babesia. Most human infections in the United States are caused by Babesia microti, but other infection-causing Babesia parasites have been documented as well. Polymerase chain reaction (PCR)–based methods can be used to identify this parasite to the species level. In this study, published real-time PCR assays for the specific detection of B. microti were evaluated against conventional PCR for their analytical performance. All evaluated real-time PCR assays had comparable dynamic range and amplification efficiency, but the sensitivity and specificity varied. The best performing test, a TaqMan assay targeting the 18S ribosomal RNA gene, was further evaluated for diagnostic performance using blood specimens submitted to the Centers for Disease Control and Prevention for parasite detection and was found to have 100% sensitivity and specificity. In conclusion, the 18S TaqMan real-time PCR assay is a sensitive, specific, and rapid method for identification of B. microti among cases of babesiosis in the United States.

International audience; We report a case of eosinophilic meningitis complicated by transverse myelitis caused by Angiostrongylus cantonensis in a 10-year-old boy from Brazil who had traveled to Suriname. We confirmed diagnosis by serology and real-time PCR in the cerebrospinal fluid. The medical community should be aware of angiostrongyliasis in the Guiana Shield.

Microsporidiosis is an emerging opportunistic infection in immunocompromised patients. We report a case of fatal disseminated Anncaliia algerae infection in a profoundly immunosuppressed pancreas and kidney transplant recipient.

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.

BACKGROUND Babesiosis is an emerging tick-borne infection in humans. The increasing numbers of reported cases of transfusion-associated babesiosis (TAB), primarily caused by Babesia microti, represents a concern for the safety of the US blood supply. STUDY DESIGN AND METHODS This study investigated kinetics of parasitemia and innate immune responses and dynamics of antibody responses during B. microti infection in rhesus macaques (RMs) using blood smears, quantitative polymerase chain reaction (qPCR), flow cytometry, and indirect fluorescent antibody testing. A total of six monkeys were transfused with either hamster or monkey-passaged B. microti–infected red blood cells (two and four monkeys, respectively) simulating TAB. RESULTS The prepatent period in monkeys inoculated with hamster-passaged B. microti was 35 days compared with 4 days in monkeys transfused with monkey-passaged B. microti; the latter monkeys also had markedly higher parasitemia levels. The duration of the window period from the first detected parasitemia by qPCR analysis to the first detected antibody response ranged from 10 to 17 days. Antibody responses fluctuated during the course of the infection. Innate responses assessed by the frequencies of monocytes and activated B cells correlated with the kinetics and magnitude of parasitemia. On Day 14, additional activation peaks were noted for CD14+CD16+ and CD14–CD16+ monocytes and for CD11c+ myeloid dendritic cells, but only in animals transfused with monkey-passaged B. microti. Parasitemia persisted in these immunocompetent animals, similar to human infection. CONCLUSION The results suggest that transfusion-associated transmission of B. microti leads to rapid onset of parasitemia (Day 4) in RMs, detectable antibody response 14 days later, and persistent parasitemia.

CASE DESCRIPTION A 22-year-old male gorilla (Gorilla gorilla gorilla) housed in a zoo was evaluated for signs of lethargy, head-holding, and cervical stiffness followed by development of neurologic abnormalities including signs of depression, lip droop, and tremors. CLINICAL FINDINGS Physical examination under general anesthesia revealed a tooth root abscess and suboptimal body condition. A CBC and serum biochemical analysis revealed mild anemia, neutrophilia and eosinopenia consistent with a stress leukogram, and signs consistent with dehydration. Subsequent CSF analysis revealed lymphocytic pleocytosis and markedly increased total protein concentration. TREATMENT AND OUTCOME Despite treatment with antimicrobials, steroids, and additional supportive care measures, the gorilla's condition progressed to an obtunded mentation with grand mal seizures over the course of 10 days. Therefore, the animal was euthanized and necropsy was performed. Multifocal areas of malacia and hemorrhage were scattered throughout the brain; on histologic examination, these areas consisted of necrosis and hemorrhage associated with mixed inflammation, vascular necrosis, and intralesional amoebic trophozoites. Tan foci were also present in the kidneys and pancreas. Immunohistochemical testing positively labeled free-living amoebae within the brain, kidneys, eyes, pancreas, heart, and pulmonary capillaries. Subsequent PCR assay of CSF and frozen kidney samples identified the organism as Balamuthia mandrillaris, confirming a diagnosis of amoebic meningoencephalitis. CLINICAL RELEVANCE Infection with B mandrillaris has been reported to account for 2.8% of captive gorilla deaths in North America over the past 19 years. Clinicians working with gorillas should have a high index of suspicion for this diagnosis when evaluating and treating animals with signs of centrally localized neurologic disease.

Angiostrongylus cantonensis, the rat lungworm, is the most common infectious cause of eosinophilic meningoencephalitis worldwide. This parasite is endemic to Southeast Asia and the Pacific Islands, and its global distribution is increasing. We report A. cantonensis meningoencephalitis in a 12-month-old boy in Tennessee, USA, who had not traveled outside of southwestern Tennessee or northwestern Mississippi.

Anncaliia algerae myositis is a life-threatening, emerging microsporidiosis among immunocompromised hosts. We report a case of disseminated A algerae infection in a man previously treated with alemtuzumab. Due to failure of albendazole-based therapy, fumagillin was added as a novel approach to management, with a good clinical response and patient survival.

We report a case of disseminated microsporidiosis in a patient with multiple myeloma who had received an allogeneic stem cell transplant requiring substantial immunosuppression. The causative organism was identified as Tubulinosema acridophagus, confirming this genus of microsporidia as a novel human pathogen.

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.

We report a 62-year-old male who presented status post lung transplantation with subcutaneous nodules. One week later, he showed signs of altered mental status; brain imaging demonstrated mass effect in the cerebellum and meningoencephalitis. In spite of treatment with a broad range of antimicrobials, he died. A punch biopsy of the skin lesions showed a superficial and deep mixed inflammatory infiltrate admixed with large mononuclear cells. A diagnosis of cutaneous amoebiasis was made and the amoebae were identified as Acanthamoeba spp. based on immunofluorescent stains and PCR assays. Cutaneous Acanthamoebiasis is a rare infection in immunocompro- mised patients, particularly organ recipients. It is important that this entity is included in the differential diagnosis of immunocompromised patients who have cutaneous infections that are not responding to antibiotics. An early diagnosis is crucial since cutaneous Acanthamoebiasis can disseminate to the central nervous system and cause granulomatous amoebic encephalitis (GAE), which is usually fatal.

Paravahlkampfia francinae n. sp., a new species of the free-living amoeba genus Paravahlkampfia, designated as CDC:V595, was isolated from the cerebrospinal fluid of a patient with headache, sore throat, and vomiting, typical symptoms of primary amoebic meningoencephalitis (PAM) caused by Naegleria fowleri. The isolate grew at 33 degrees C, 37 degrees C, 40 degrees C, and 42 degrees C and destroyed mammalian cell cultures. However, it did not kill young mice upon intranasal inoculation. P. francinae does not produce flagellates and does not grow on agar plates coated with Gram-negative bacteria such as Escherichia coli, the usual food source of Paravahlkampfia ustiana, the type species of the genus. The trophozoite at light microscopy exhibited eruptive locomotion and possessed a single vesicular nucleus. Ultrastructurally, the trophozoites had numerous mitochondria with discoidal cristae but did not have a Golgi apparatus. The trophozoites differentiated into cysts after consuming most of the monolayer. The cyst had an inner well-differentiated endocyst and an outer thin, wrinkled, and wavy ectocyst with no pores. During excystation trophozoites ruptured the cyst wall and emerged from the cysts. A unique feature seen in the cysts was the presence of bacterial endosymbionts, both in the endoplasm and within the cyst wall. Full-length sequencing analysis of the 18S and 5.8S RNA genes of P. francinae showed that they were distinct from those of other Paravahlkampfia species. The patient recovered within a few days indicating that some of the previously reported cases of PAM that survived may have been due to P. francinae.

Acanthamoebae are free-living amoebae found in the environment, including soil, freshwater, brackish water, seawater, hot tubs, and Jacuzzis. Acanthamoeba species can cause keratitis, a painful vision-threatening infection of the cornea, and fatal granulomatous encephalitis in humans. More than 20 species of Acanthamoeba belonging to morphological groups I, II, and III distributed in 15 genotypes have been described. Among these, Acanthamoeba castellanii , A . polyphaga , and A. hatchetti are frequently identified as causing Acanthamoeba keratitis (AK). Improper contact lens care and contact with nonsterile water while wearing contact lenses are known risk factors for AK. During a recent multistate outbreak, AK was found to be associated with the use of Advanced Medical Optics Complete MoisturePlus multipurpose contact lens solution, which was hypothesized to have had insufficient anti- Acanthamoeba activity. As part of the investigation of that outbreak, we compared the efficacies of 11 different contact lens solutions against cysts of A. castellanii , A. polyphaga , and A. hatchetti (the isolates of all species were genotype T4), which were isolated in 2007 from specimens obtained during the outbreak investigation. The data, generated with A. castellanii , A. polyphaga , and A. hatchetti cysts, suggest that the two contact lens solutions containing hydrogen peroxide were the only solutions that showed any disinfection ability, with 0% and 66% growth, respectively, being detected with A. castellanii and 0% and 33% growth, respectively, being detected with A. polyphaga . There was no statistically significant difference in disinfection efficacy between the 11 solutions for A. hatchetti .

Pathogenic free-living amoebae, such as Acanthamoeba species, Balamuthia mandrillaris, and Naegleria fowleri, are known to cause infections of the central nervous system in human and other animals. In 2001, a case of human encephalitis was reported that was caused by another amoeba with morphological features suggestive of Sappinia. The amoeba originally identified as Sappinia diploidea was identified, most likely as S. pedata, by use of newly developed real-time polymerase chain reaction assays. This amoeba had previously been found only in environmental sources, such as soil and tree bark. The results illustrate the potential for other free-living amoebae, which are not normally associated with human disease, to cause occasional infections.

The semi-slug Parmarion cf. martensi Simroth, 1893, was first discovered on O‘ahu, Hawai‘i, in 1996 and then on the island of Hawai‘i in 2004. This species, which is probably native to Southeast Asia, is abundant in eastern Hawai‘i Island, reportedly displacing the Cuban slug, Veronicella cubensis (Pfeiffer, 1840), in some areas. A survey in July–August 2005 found P. cf. martensi primarily in the lower Puna area of Hawai‘i Island, with an isolated population in Kailua-Kona (western Hawai‘i Island). It is now established in commercial papaya plantations, and survey participants reported it as a pest of lettuce and papaya in home gardens. Survey respondents considered P. cf. martensi a pest also because of its tendency to climb on structures where it deposits its feces and because of its potential to transmit disease. Individuals of this species were found to carry large numbers of infective third-stage larvae of the nematode Angiostrongylus cantonensis (Chen, 1935), the causative agent of human an...

Angiostrongylus cantonensis is the most common infectious cause of eosinophilic meningitis. Timely diagnosis of these infections is difficult, partly because reliable laboratory diagnostic methods are unavailable. The aim of this study was to evaluate the usefulness of a real-time polymerase chain reaction (PCR) assay for the detection of A. cantonensis DNA in human cerebrospinal fluid (CSF) specimens. A total of 49 CSF specimens from 33 patients with eosinophilic meningitis were included: A. cantonensis DNA was detected in 32 CSF specimens, from 22 patients. Four patients had intermittently positive and negative real-time PCR results on subsequent samples, indicating that the level of A. cantonensis DNA present in CSF may fluctuate during the course of the illness. Immunodiagnosis and/or supplemental PCR testing supported the real-time PCR findings for 30 patients. On the basis of these observations, this real-time PCR assay can be useful to detect A. cantonensis in the CSF from patients with eosinophilic meningitis.

Naegleria fowleri is a thermophilic, free-living ameba that causes primary amebic meningoencephalitis. The infections are nearly always fatal. We present the third well-documented survivor of this infection in North America. The patient’s survival most likely resulted from a variety of factors: early identification and treatment, use of a combination of antimicrobial agents (including miltefosine), and management of elevated intracranial pressure based on the principles of traumatic brain injury.

Naegleria fowleri is a climate-sensitive, thermophilic ameba found in 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 migrates to the brain via the olfactory nerve. In August 2013, a 4-year-old boy died of meningoencephalitis of unknown etiology in a Louisiana hospital.Clinical and environmental testing and a case investigation were initiated to determine the cause of death and to identify potential exposures.Based on testing of cerebrospinal fluid and brain specimens, the child was diagnosed with PAM. His only reported water exposure was tap water; in particular, tap water that was used to supply water to a lawn water slide on which the child had played extensively prior to becoming ill. Water samples were collected from both the home and the water distribution system that supplied the home and tested; N. fowleri was identified in water samples from both the home and the water distribution system.This case is the first reported PAM death associated with culturable N. fowleri in tap water from a US treated drinking water system. This case occurred in the context of an expanding geographic range for PAM beyond southern states, with recent case reports from Minnesota, Kansas, and Indiana. This case also highlights the role of adequate disinfection throughout drinking water distribution systems and the importance of maintaining vigilance when operating drinking water systems using source waters with elevated temperatures.

The aim of the study was to determine the effect of clarithromycin treatment on resistance development in the commensal throat flora. Alpha-haemolytic streptococci and Neisseria spp. were isolated from patients receiving clarithromycin for eradication of Helicobacter pylori. The treatment resulted in an immediate increase in the number of macrolide-resistant streptococci, which remained for one year after treatment, but declined to background level three years later. The most prevalent resistance gene was mef(A). Neisseria spp. were less affected by the treatment: the number of resistant isolates increased in only in one case during treatment. In conclusion, a one-week standard therapy with clarithromycin selects for an increased prevalence of macrolide-resistant streptococci that persisted for more than one year.

Acute Chagas disease is rarely recognized, and the risk for acquiring the disease is undefined in travelers to Central America. We describe a case of acute Chagas disease in a traveler to Costa Rica and highlight the need for increased awareness of this infection in travelers to Chagas-endemic areas.

A 10-month-old, female African pygmy falcon ( Polihierax semitorquatus) hatched and housed at the San Diego Zoo developed neurologic signs and died from a cerebral infection with the rat lungworm Angiostrongylus cantonensis. There was an associated mild nonsuppurative meningoencephalitis. This infection was diagnosed on histology and confirmed by detection of species-specific A. cantonensis DNA in formalin-fixed and frozen brain tissue by a polymerase chain reaction assay. To the authors’ knowledge, this infection has not previously been reported in a bird in the United States and has not been known to be naturally acquired in any species in this region of the world. The source of the infection was not definitively determined but was possibly feeder geckos ( Hemidactylus frenatus) imported from Southeast Asia where the parasite is endemic.

Sulphonamide resistance in some clinical isolates of Neisseria meningitidis is associated with an insertion in the chromosomal folP gene leading to the addition of two amino acids, serine and glycine, in the drug target enzyme dihydropteroate synthase (DHPS). Removal of the insertion resulted in a markedly higher K m for the substrate p-aminobenzoic acid and a markedly lower K m for 2-amino-4-hydroxy-6-(hydroxymethyl)-7,8-dihydropteridine pyrophosphate. In the same isolates an additional important difference, compared to wild-type enzymes, was found at amino acid position 68, which is a proline in most DHPS enzymes, but is serine in one and leucine in another clinical isolate of sulphonamide-resistant N. meningitidis. The alteration at position 68 was found to affect mainly the level of sulphonamide resistance and had only a minor effect on the K m for the substrates. Introduction of the serine-glycine dipeptide at position 194 and a proline to serine substitution at position 68 in DHPS from normal, susceptible N. meningitidis failed to produce a functional sulphonamide-resistant enzyme. The conclusion of this study is that it is not possibile to change a normal chromosomally encoded DHPS of N. meningitidis to a sulphonamide-resistant one simply by an insertion of serine and glycine as seen in clinical isolates. It is likely that the resistance gene found in clinical isolates has evolved in another bacterial species where a combination of other amino acid changes may have contributed to produce a functionally resistant enzyme. This new resistance gene may have then been introduced into N. meningitidis by natural transformation.

Primary amebic meningoencephalitis (PAM) caused by the free-living ameba Naegleria fowleri is a rare but rapidly fatal disease of the central nervous system (CNS) affecting predominantly young, previously healthy persons. No effective chemotherapeutic prophylaxis or treatment has been identified. Recently, three transplant-associated clusters of encephalitis caused by another free-living ameba, Balamuthia mandrillaris, have occurred, prompting questions regarding the suitability of extra-CNS solid organ transplantation from donors with PAM. During 1995–2012, 21 transplant recipients of solid organs donated by five patients with fatal cases of PAM were reported in the United States. None of the recipients developed PAM and several recipients tested negative for N. fowleri by serology. However, historical PAM case reports and animal experiments with N. fowleri, combined with new post-mortem findings from four PAM patients, suggest that extra-CNS dissemination of N. fowleri can occur and might pose a risk for disease transmission via transplantation. The risks of transplantation with an organ possibly harboring N. fowleri should be carefully weighed for each individual recipient against the potentially greater risk of delaying transplantation while waiting for another suitable organ. In this article we present a case series and review existing data to inform such risk assessments.

Since an initial case in 2006, we noted multiple patients undergoing heart transplantation (HTx) for Chagas cardiomyopathy (CC) at our transplant program. The clinical characteristics, laboratory results and outcomes of patients with CC undergoing HTx in the United States have not been reported previously. In 2010, we implemented a systematic screening and management program for patients undergoing HTx for CC. Before HTx, all patients with idiopathic dilated cardiomyopathy who were born in a Chagas disease endemic country were screened for Trypanosoma cruzi (TC) infection with serology. After HTx, monitoring for TC reactivation was performed using clinical visits, echocardiography, endomyocardial biopsy and serial whole blood polymerase chain reaction (PCR) testing. Between June 2006 and January 2012, 11 patients underwent HTx for CC. One patient was empirically treated due to the presence of TC amastigotes in explanted cardiac tissue. Two patients experienced allograft dysfunction due to TC reactivation and three patients experienced subclinical reactivation (positive PCR results), which were treated. Chagas disease is a common cause of dilated cardiomyopathy in patients from endemic countries undergoing HTx at a transplant program in the United States. Reactivation is common after transplantation and can cause adverse outcomes.

Although Trypanosoma cruzi, the parasite that causes Chagas disease, can be transmitted via organ transplantation, liver and kidney transplantation from infected donors may be feasible. We describe the outcomes of 32 transplant recipients who received organs from 14 T. cruzi seropositive donors in the United States from 2001 to 2011. Transmission was confirmed in 9 recipients from 6 donors, including 3 of 4 (75%) heart transplant recipients, 2 of 10 (20%) liver recipients and 2 of 15 (13%) kidney recipients. Recommended monitoring posttransplant consisted of regular testing by PCR, hemoculture, and serology. Thirteen recipients had no or incomplete monitoring; transmission was confirmed in five of these recipients. Four of the five recipients had symptomatic disease and all four died although death was directly related to Chagas disease in only one. Nineteen recipients had partial or complete monitoring for T. cruzi infection with weekly testing by PCR, hemoculture and serology; transmission was confirmed in 4 of 19 recipients with no cases of symptomatic disease. Our results suggest that liver and kidney transplantation from T. cruzi seropositive donors may be feasible when the recommended monitoring schedule for T. cruzi infection is followed and prompt therapy with benznidazole can be administered.

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.

A 38-year-old male immunocompetent soldier developed generalized seizures. He underwent surgical debulking and a progressive demyelinating pseudotumor was identified. Serology and molecular testing confirmed a diagnosis of granulomatous amoebic encephalitis caused by Acanthamoeba sp. in this immunocompetent male. The patient was treated with oral voriconazole and miltefosine with Acanthamoeba titers returning to control levels and serial imaging demonstrating resolution of the residual lesion.

Background The laboratory diagnosis of Chagas disease is challenging because the usefulness of different diagnostic tests will depend on the stage of the disease. Serology is the preferred method for patients in the chronic phase, whereas PCR can be successfully used to diagnose acute and congenital cases. Here we present data using a combination of three TaqMan PCR assays to detect T. cruzi DNA in clinical specimens. Methods/Principal Findings Included in the analysis were DNA extracted from 320 EDTA blood specimens, 18 heart tissue specimens, 6 umbilical cord blood specimens, 2 skin tissue specimens and 3 CSF specimens. For the blood specimens both whole blood and buffy coat fraction were analyzed. The specimens were from patients living in the USA, with suspected exposure to T. cruzi through organ transplantation, contact with triatomine bugs or laboratory accidents, and from immunosuppressed patients with suspected Chagas disease reactivation. Real-time PCR was successfully used to diagnose acute and Chagas disease reactivation in 20 patients, including one case of organ-transmitted infection and one congenital case. Analysis of buffy coat fractions of EDTA blood led to faster diagnosis in six of these patients compared to whole blood analysis. The three real-time PCR assays produced identical results for 94% of the specimens. The major reason for discrepant results was variable sensitivity among the assays, but two of the real-time PCR assays also produced four false positive results. Conclusions/Significance These data strongly indicate that at least two PCR assays with different performances should be combined to increase the accuracy. This evaluation also highlights the benefit of extracting DNA from the blood specimen's buffy coat to increase the sensitivity of PCR analysis., Author Summary Chagas disease is endemic in several Latin American countries and affects approximately 8 to 11 million people. The protozoan parasite, Trypanosoma cruzi, is the agent of Chagas disease, a zoonotic disease that can be transmitted to humans by blood-sucking triatomine bugs. Other routes of infection include congenital transmission, blood transfusion, organ transplantation, accidental inoculation of the parasite during laboratory research and by consuming food and juice contaminated with the parasite. This study focused on the evaluation of three quantitative PCR (QPCR) assays for the diagnosis of Chagas disease. The evaluation was based on the analysis of 349 specimens submitted for confirmatory diagnosis of Chagas disease to the Centers for Disease Control and Prevention from 2008 to 2010. By using such assays we were able to diagnose acute and Chagas disease reactivation in 20 patients, including one case of organ-transmitted infection and one congenital case. The paper also highlights the benefit of extracting DNA from the blood specimen's buffy coat to increase the sensitivity of diagnostic PCR analysis. The results obtained in this study strongly indicate that at least two QPCR assays with different performance characteristics should be combined to increase diagnostic accuracy.

To the Editor: Protozoan pathogens, including Entamoeba histolytica, Giardia, Cryptosporidium, Cyclospora, Cystoisospora, and microsporidia such as Enterocytozoon bieneusi, are well-known agents of diarrhea and a major public health problem in developing countries. Infection with Cyclospora cayetanensis and E. bieneusi can occur in immunocompromised and immunocompetent persons. Severe diarrhea and weight loss along with anorexia, nausea, and low-grade fever occur in immunocompromised persons, particularly those with HIV/AIDS and transplant recipients who are taking immunosuppressive drugs (1,2). However, transient diarrhea occurs in immunocompetent persons, notably in travelers returning from countries with poor hygienic standards (1–3). We report on a kidney transplant recipient who had uncontrollable diarrhea and weight loss in whom C. cayetanensis and E. bieneusi were detected in biopsy specimens; the diarrhea resolved after treatment with drugs that act specifically on these 2 parasites. The patient was a 55-year-old man from the Dominican Republic living in New York, NY, USA; he had a history of long-term diabetes, coronary disease, and alcoholism. He had undergone a cadaveric renal transplant 14 months earlier and had an uneventful posttransplant course. After returning from visiting family in the Dominican Republic, he sought treatment for acute, profuse watery diarrhea in early November, 2009. He had >10 watery bowel movements daily that were associated with a 20-lb weight loss. His symptoms persisted for 2 months, and he required 2 hospitalizations for the diarrhea. Results of 4 repeat fecal specimen tests (routine diagnostic microscopy and culture) were negative for parasites. Colonoscopy findings were normal; because of evidence of leukocytes in the feces and elevated fecal fat level, however, he received empirically prescribed metronidazole. Because his diarrhea and weight loss persisted, an upper endoscopy was performed, which revealed the presence of microsporidia. He then received albendazole for 3 weeks without substantial benefit. The biopsy specimens were sent to the Centers for Disease Control and Prevention (Atlanta, GA, USA) for further analysis. Biopsy slides were stained with hematoxylin and eosin and with Gram chromotrope (4) and examined by microscopy. The Gram chromotrope–stained slide revealed oval spores, pinkish-red in color, measuring ≈1 µm (5). These spores were supra nuclear in position and were consistent with E. bieneusi (Figure, panel A). The tissue sections were scraped from the slides, DNA was extracted, and conventional PCR was performed by using E. bieneusi–specific primers as described (5); the sizes of the amplified product in the tissue DNA specimen and in the E. bieneusi control specimen were identical (Figure, panel B), confirming the presence of E. bieneusi. On further microscopic examination of the Gram chromotrope and the hematoxylin and eosin–stained slides, oval bodies (8–10 µm) were seen. A few of these oval bodies exhibited 4 spindle-shaped structures which were identified provisionally as merozoites of a coccidian parasite (Figure, panel C). Others had morula-like internal structure (Figure, panel D). We hypothesized that the coccidian parasite could either be C. cayetanensis or Cystoisospora hominis. Because the parasites, in various stages, were just beneath the surface of the epithelium, rather than deep within the epithelium, we believed this organism to be a Cyclospora sp. rather than a Cystoisospora sp. We searched the serum bank of the Division of Parasitic Diseases, Centers for Disease Control and Prevention, and identified a serum sample from a person with a case of C. cayetanensis cyclosporiasis. An indirect immunofluorescence test was performed by using this serum on a deparaffinized section of the tissue biopsy specimen. Different stages of the coccidian organism were labeled brightly and produced apple-green fluorescence against a red counterstain (Eriochrome Black T), indicating that the parasite could possibly be a Cyclospora sp. (Figure, panels E, F). We considered that the Cyclospora-positive serum sample obtained from this particular patient may not be species-specific, since he might have also been infected with Cystoisospora. Therefore, we performed a real-time PCR assay that can distinguish C. cayetanensis from other coccidian parasites to identify the parasite definitively (3). DNA recovered from tissue in paraffin sections was successfully amplified and detected with this assay (data not shown), confirming the presence of C. cayetanensis. Figure Tissue specimens from a kidney transplant recipient with concurrent parasitic infections after traveling to the Dominican Republic. A) Tissue section stained with Gram chromotrope. Note the apical location of a cluster of Enterocytozoon bieneusi spores ... The patient’s illness was treated with albendazole for E. bieneusi infection and with trimethoprim and sulfamethoxazole for C. cayetanensis infection. The patient’s diarrhea subsided after 1 week, and several subsequent fecal samples were negative for microsporidia spores and Cyclospora oocysts. His immunosuppressive medications were reduced, and he remained diarrhea-free for the following 3-year period of April 2010 to April 2013.

Background Horizontal gene transfer is an important source of genetic variation among Neisseria species and has contributed to the spread of resistance to penicillin and sulfonamide drugs in the pathogen Neisseria meningitidis. Sulfonamide resistance in Neisseria meningitidis is mediated by altered chromosomal folP genes. At least some folP alleles conferring resistance have been horizontally acquired from other species, presumably from commensal Neisseriae. In this work, the DNA sequence surrounding folP in commensal Neisseria species was determined and compared to corresponding regions in pathogenic Neisseriae, in order to elucidate the potential for inter-species DNA transfer within this region. Results The upstream region of folP displayed differences in gene order between species, including an insertion of a complete Correia element in Neisseria lactamica and an inversion of a larger genomic segment in Neisseria sicca, Neisseria subflava and Neisseria mucosa. The latter species also had DNA uptake signal sequences (DUS) in this region that were one base different from the DUS in pathogenic Neisseriae. Another interesting finding was evidence of a horizontal transfer event from Neisseria lactamica or Neisseria cinerea that introduced a novel folP allele to the meningococcal population. Conclusion Genetic recombination events immediately upstream of folP and horizontal transfer have resulted in sequence differences in the folP region between the Neisseria species. This variability could be a consequence of the selective pressure on this region exerted by the use of sulfonamide drugs.

A previously healthy 13-year-old male developed acute onset of a bi-temporal headache. The headache persisted, and two weeks later, he was diagnosed with sinusitis and was given amoxicillin. On illness day 17, he experienced worsening headache, slurred speech, transient left facial droop, right hand numbness, and dizziness. At a local emergency department (ED), magnetic resonance imaging (MRI) of the brain showed no significant abnormalities, though images were obscured by artifact from his orthodontic hardware. He was discharged without a specific diagnosis after his neurologic symptoms had resolved. On illness day 19, the patient had return of hand tingling and slurred speech and presented to the Children’s Hospital Colorado ED. He was diagnosed with an atypical migraine and was treated with ketorolac, diphenhydramine, and prochlorperazine. His symptoms improved, and he was discharged. On illness day 20, the patient experienced severe right eye pain and recurrence of his headache. He returned to his local ED and was hospitalized after minimal clinical improvement with treatment for migraine. At that time, his Romberg sign was abnormal, he was ataxic, and he had diplopia. On illness day 22, he underwent a lumbar puncture (LP) because of concern of pseudotumor cerebri. The cerebral spinal fluid (CSF) showed white blood cell count (WBC) of 763/mm3 (with a differential of 80% lymphocytes, 15% monocytes, and 5% eosinophils), red blood cell count (RBC) of 2/mm3, protein of 49 mg/dL, and glucose of 54 mg/dL. Intravenous acyclovir was started but was discontinued when polymerase chain reaction assay (PCR) of the CSF for herpes simplex virus returned negative. Bacterial and fungal cultures of the CSF were negative. He was discharged home after a three-day hospitalization. Over the next several weeks, he remained stable but continued to have intermittent headaches and ongoing diplopia. Then, forty-seven days after initial onset of illness, the patient developed severe right eye pain and photophobia and returned to the local ED. Repeat LP showed an opening pressure of 44 cm H20, WBC of 347/mm3 (with a differential of 53% lymphocytes, 26% monocytes, 21% eosinophils), RBC of 3/mm3, protein of 127 of mg/dL, and glucose of 35 mg/dL. He was subsequently transferred to Children’s Hospital Colorado. On admission, the patient was afebrile and had normal vital signs. Physical examination showed papilledema with splinter hemorrhages, left esotropia with subjective diplopia, and no signs of meningismus. Complete blood count (CBC) showed WBC of 9,100/mm3 (with differential of 44% neutrophils, 33% lymphocytes, 11% monocytes, 12% eosinophils), hemoglobin of 16.1 g/dL, hematocrit of 47.1%, and platelet count of 320,000/mm3. Repeat lumbar puncture on day 50 of illness showed an opening pressure of 34–35 cm H20, WBC of 273/mm3 (with a differential of 46% lymphocytes, 46% eosinophils, 5% monocytes, 3% macrophages), RBC of 32/mm3, protein of 107 mg/dL, and glucose of 38 mg/dL. A brain MRI, performed after removal of the patient’s braces, was normal. Additional blood testing was negative, including: EBV PCR, Bartonella henselae IgG/IgM, Bartonella quintana IgG/IgM, Blastomyces antibody, Coccidioides IgG/IgM, Histoplasma antibody, HIV 1/2 antibodies, Cryptococcus antigen, Toxocara antibody, and Toxoplasma IgG/IgM. CSF acid-fast bacillus stain, cryptococcal antigen, and mycobacterial, fungal, aerobic and anaerobic cultures were also negative. Upon further questioning, the patient reported that he and his family had traveled to their vacation home in Kauai, Hawaii for the two weeks directly preceding the onset of symptoms. They had not stopped on any other Hawaiian island. During the trip, the family had found a dead rat in their hot tub, though the patient had not been in it. They had consumed organic lettuce from a local vendor and fresh produce from their herb garden. The patient denied exposure to seafood, mollusks or other exotic cuisine. The family had stopped in Los Angeles on their return trip home but had not ventured outside of the city. The patient denied any other travel, animal contact, or known insect bites. The patient’s exposure history prompted additional testing of the CSF, which confirmed the etiology of his eosinophilic meningitis.

Development of sulphonamide resistance in Neisseria meningitidis has been suggested to involve horizontal DNA-transfer from a commensal Neisseria species. In this study, we isolated commensal Neisseria from throat specimens and examined the isolates with respect to sulphonamide resistance. Three resistant clones were identified and the resistance phenotype could be explained by amino acid variations in their dihydropteroate synthase, the target molecule for sulphonamides. Some of these variations occurred in positions corresponding to previously detected variations in resistant N. meningitidis. Sulphonamide resistant commensal Neisseria were isolated from an environment not exposed to sulphonamides, suggesting that resistant Neisseria has become a natural part of the commensal throat flora.