Infections caused by Naegleria fowleri, Acanthamoeba spp., and Balamuthia mandrillaris occur throughout the world and pose many diagnostic challenges. To date, at least 440 cases of severe central nervous system infections caused by these amebas have been documented worldwide. Rapid and specific identification of these free-living amebas in clinical samples is of crucial importance for efficient case management. We have developed a triplex real-time TaqMan PCR assay that can simultaneously identify Acanthamoeba spp., B. mandrillaris, and N. fowleri in the same PCR vessel. The assay was validated with 22 well-characterized amebic strains harvested from cultures and nine clinical specimens that were previously characterized by in vitro culture and/or immunofluorescence assay. The triplex assay demonstrated high specificity and a rapid test completion time of less than 5 h from the reception of the specimen in the laboratory. This assay was able to detect one single ameba per sample analyzed, as determined with cerebrospinal fluid spiked with diluted cultured amebas. This assay could become useful for fast laboratory diagnostic assessment of amebic infections (caused by free-living amebas) in laboratories with adequate infrastructure to perform real-time PCR testing.

Water was the suspected vehicle of Toxoplasma gondii dissemination in a toxoplasmosis outbreak in Brazil. A case-control study and geographic mapping of cases were performed. T. gondii was isolated directly from the implicated water and genotyped as SAG 2 type I.

We compared a nested PCR assay and microscopic examination of Giemsa-stained blood films for detection and identification of Plasmodium spp. in blood specimens. PCR was more sensitive than microscopy and capable of identifying malaria parasites at the species level when microscopy was equivocal.

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.

Wild-caught synanthropic flies were tested for the presence of Cryptosporidium parvum and Giardia lamblia on their exoskeletons and in their digestive tracks by fluorescent in situ hybridization and fluorescein isothiocyanate (FITC)−conjugated monoclonal antibody (MAb) against Cryptosporidium and Giardia cell wall epitopes. The levels of C. parvum were positively correlated with the levels of G. lamblia, indicating a common source of contamination. The majority of oocysts and cysts were potentially viable (C. parvum ‫ס‬ 80% and G. lamblia ‫ס‬ 69%). More G. lamblia cysts occurred on the exoskeleton of the flies than within the digestive tracts; the opposite relationship was observed for C. parvum. No genotype other than C. parvum G2 was found to be associated with flies. Because filth flies carry viable C. parvum oocysts and G. lamblia cysts acquired naturally from unhygienic sources, they can be involved in the epidemiology of cryptosporidiosis and giardiasis. Fluorescent oligonucleotide probes used together with FITCconjugated MAb represent a convenient and cost-effective technique for rapid and specific identification of humaninfectious species of Cryptosporidium and Giardia mechanically transported by flies, and for the assessment of the viability of these pathogens.

Naegleria fowleri is a thermophilic free-living ameba that causes primary amebic meningoencephalitis. Infections are nearly always fatal. We present the third well-documented survivor of this infection in North America. Survival most likely resulted from a combination of early identification and treatment, use of a combination of antimicrobials including miltefosine and management of elevated intracranial pressure based on traumatic brain injury principles.

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.

Isoenzyme analysis of cultured parasites is the conventional approach for Leishmania species identification. Molecular approaches have the potential to be more sensitive and rapid. We designed PCR generic primers to amplify a segment of the rRNA internal transcribed spacer 2 (ITS2) from multiple Leishmania species. To validate the selected ITS2 fragment, we tested clinical specimens and compared the species results obtained by the molecular approach (PCR followed by DNA sequencing analysis) with those from the parasitologic approach (in vitro culture followed by isoenzyme analysis). Among the 159 patients with clinical specimens positive by both approaches, a total of eight Leishmania species were identified. The species results were concordant for all but two patients: for one patient, the results were Leishmania (Viannia) guyanensis by the molecular approach versus L. (V.) braziliensis by the parasitologic approach; for the other patient, the results were L. (Leishmania) tropica versus L. (L.) major, respectively. ITS2 PCR, followed by sequencing analysis, can be used to detect and discriminate among Leishmania species. The results confirmed our hypothesis that a region of the ITS2 gene can complement the characterization of Leishmania parasites at the species level. The approach we developed can be used as a diagnostic tool in reference laboratories with adequate infrastructure to perform molecular characterization of pathogens.Leishmaniasis encompasses multiple clinical syndromes, most notably, visceral, cutaneous, and mucosal forms (9,13,14,22,27,30). Leishmaniasis is endemic in focal areas in the tropics, subtropics, and southern Europe and is caused by species in the Leishmania and Viannia subgenera (14). Different species can be associated with diverse clinical manifestations and sequelae. Species identification can facilitate clinical management, such as decisions regarding whether/which treatment is indicated.Isoenzyme analysis of cultured parasites is the conventional diagnostic approach for Leishmania species identification. Molecular approaches have the potential to be more sensitive and rapid; e.g., the results can be available within days versus weeks or months. However, Leishmania PCR-based assays have varied in sensitivity, specificity, and utility for genus-or complexlevel versus species-level characterization (2, 3, 7, 10, 24-26, 28); some approaches have been highly sensitive for genuslevel detection, e.g., PCR methods that target minicircle kinetoplast DNA (kDNA), whereas others have been tailored for particular species/settings. We sought to develop a comprehensive molecular approach for characterizing Leishmania species; we focused on the rRNA internal transcribed spacer (ITS) region, which has been sequenced by other investigators and proposed as a target for molecular typing (12, 25). Here we describe our identification and validation of a diagnostic fragment that was amplified from the ITS2 with a PCR generic primer pair that we designed. Rather than conduct a formal evaluation of the m...