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Polymerase chain reaction (PCR) is increasingly used in the diagnosis of soil-transmitted helminth infections. Despite this, few studies have evaluated the impact of different fecal fixatives on the outcome of fecal helminth qPCR analysis, and none have evaluated the effect of commercial parasitology fixatives commonly used in diagnostic laboratories. We fixed dog feces containing Ancylostoma spp. hookworm eggs in zinc polyvinyl alcohol (Zn-PVA) and Total-Fix, and with 70% ethanol (EtOH) as a control. DNA was extracted at timepoints 11, 33, 64, and 94 days and subjected to Ancylostoma spp. quantitative PCR (qPCR). A linear regression model was created to assess the effect of preservative types on the temporal change of qPCR quantification cycle number (Cq) values, accounting for variances among individual animals. Fixation in 70% EtOH least affected Cq values over 94 days. Total-Fix preservation yielded a higher Cq overall, but there was no significant difference compared with 70% EtOH fixation. Fixation in Zn-PVA resulted in significantly (P < 0.001) higher Cq values than 70% EtOH after only 33 days and loss of amplification at 64 days. Consistent with other helminth fixation studies, 70% EtOH performed well in preserving hookworm DNA over 94 days. Total-Fix provided a comparable alternative for qPCR analysis for hookworm. Fixation in Zn-PVA resulted in loss of detectable hookworm DNA at 64 days, as determined by qPCR.

The apicomplexan parasite Cyclospora cayetanensis causes seasonal foodborne outbreaks of the gastrointestinal illness cyclosporiasis. Prior to the coronavirus disease-2019 pandemic, annually reported cases were increasing in the USA, leading the US Centers for Disease Control and Prevention to develop a genotyping tool to complement cyclosporiasis outbreak investigations. Thousands of US isolates and 1 from China (strain CHN_HEN01) were genotyped by Illumina amplicon sequencing, revealing 2 lineages (A and B). The allelic composition of isolates was examined at each locus. Two nuclear loci (CDS3 and 360i2) distinguished lineages A and B. CDS3 had 2 major alleles: 1 almost exclusive to lineage A and the other to lineage B. Six 360i2 alleles were observed – 2 exclusive to lineage A (alleles A1 and A2), 2 to lineage B (B1 and B2) and 1 (B4) was exclusive to CHN_HEN01 which shared allele B3 with lineage B. Examination of heterozygous genotypes revealed that mixtures of A- and B-type 360i2 alleles occurred rarely, suggesting a lack of gene flow between lineages. Phylogenetic analysis of loci from whole-genome shotgun sequences, mitochondrial and apicoplast genomes, revealed that CHN_HEN01 represents a distinct lineage (C). Retrospective examination of epidemiologic data revealed associations between lineage and the geographical distribution of US infections plus strong temporal associations. Given the multiple lines of evidence for speciation within human-infecting Cyclospora, we provide an updated taxonomic description of C. cayetanensis, and describe 2 novel species as aetiological agents of human cyclosporiasis: Cyclospora ashfordi sp. nov. and Cyclospora henanensis sp. nov. (Apicomplexa: Eimeriidae).

Background Acanthamoeba is a free-living ameba that can cause severe disease affecting the central nervous system, skin, sinuses, and other organs, particularly in immunocompromised individuals. These rare but severe infections are often fatal, yet incompletely described. Methods Cases included were either reported to the Centers for Disease Control and Prevention (CDC) Free-Living Ameba program or published in scientific literature. Characteristics of all patients in the United States with laboratory-confirmed non-keratitis Acanthamoeba infections were described using descriptive statistics, and associations with survival were determined using χ2 and Fisher exact tests. Results Of 173 patients identified, 71% were male and the median age was 44 years (range, 0–87 years). Of these, 26 (15%) survived. Most patients (88%) had at least 1 immunocompromising condition, most commonly human immunodeficiency virus (39%), cancer (28%), and solid organ or hematopoietic stem cell transplant (28%). Granulomatous amebic encephalitis (GAE) was the most common disease presentation (71%). Skin (46%), sinuses (29%), lungs (13%), and bone (6%) were also involved. Nearly half of patients (47%) had involvement of >1 organ system. Survival was less frequent among those with GAE (3%, P < .001) compared with cutaneous disease, rhinosinusitis, or multiorgan disease not including GAE. Of 7 who received the currently recommended treatment regimen, 5 (71%) survived. Conclusions Non-keratitis Acanthamoeba infections occur primarily in immunocompromised individuals and are usually fatal. Survival may be associated with disease presentation and treatment. Providers who care for at-risk patients should be aware of the various disease manifestations to improve early recognition and treatment.

A previously described universal parasite diagnostic (nUPDx) based on PCR amplification of the 18S rDNA and deep-amplicon sequencing, can detect human blood parasites with a sensitivity comparable to real-time PCR. To date, the efficacy of this assay has only been assessed on human blood. This study assessed the utility of nUPDx for the detection of parasitic infections in animals using blood, tissues, and other biological sample types from mammals, birds, and reptiles, known to be infected with helminth, apicomplexan, or pentastomid parasites (confirmed by microscopy or PCR), as well as negative samples. nUPDx confirmed apicomplexan and/or nematode infections in 24 of 32 parasite-positive mammals, while also identifying several undetected coinfections. nUPDx detected infections in 6 of 13 positive bird and 1 of 2 positive reptile samples. When applied to 10 whole parasite specimens (worms and arthropods), nUPDx identified all to the genus or family level, and detected one incorrect identification made by morphology.

Cyclosporiasis is a diarrheal illness caused by the foodborne parasite Cyclospora cayetanensis. Annually reported cases have been increasing in the United States prompting development of genotyping tools to aid cluster detection. A recently developed Cyclospora genotyping system based on 8 genetic markers was applied to clinical samples collected during the cyclosporiasis peak period of 2020, facilitating assessment of its epidemiologic utility. While the system performed well and helped inform epidemiologic investigations, inclusion of additional markers to improve cluster detection was supported. Consequently, investigations have commenced to identify additional markers to enhance performance.

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.

Cyclosporiasis is an illness characterised by watery diarrhoea caused by the food-borne parasite Cyclospora cayetanensis. The increase in annual US cyclosporiasis cases led public health agencies to develop genotyping tools that aid outbreak investigations. A team at the Centers for Disease Control and Prevention (CDC) developed a system based on deep amplicon sequencing and machine learning, for detecting genetically-related clusters of cyclosporiasis to aid epidemiologic investigations. An evaluation of this system during 2018 supported its robustness, indicating that it possessed sufficient utility to warrant further evaluation. However, the earliest version of CDC's system had some limitations from a bioinformatics standpoint. Namely, reliance on proprietary software, the inability to detect novel haplotypes and absence of a strategy to select an appropriate number of discrete genetic clusters would limit the system's future deployment potential. We recently introduced several improvements that address these limitations and the aim of this study was to reassess the system's performance to ensure that the changes introduced had no observable negative impacts. Comparison of epidemiologically-defined cyclosporiasis clusters from 2019 to analogous genetic clusters detected using CDC's improved system reaffirmed its excellent sensitivity (90%) and specificity (99%), and confirmed its high discriminatory power. This C. cayetanensis genotyping system is robust and with ongoing improvement will form the basis of a US-wide C. cayetanensis genotyping network for clinical specimens.

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.

Background Outbreaks of cyclosporiasis, a diarrheal illness caused by Cyclospora cayetanensis, have been a public health issue in the USA since the mid 1990’s. In 2018, 2299 domestically acquired cases of cyclosporiasis were reported in the USA as a result of multiple large outbreaks linked to different fresh produce commodities. Outbreak investigations are hindered by the absence of standardized molecular epidemiological tools for C. cayetanensis. For other apicomplexan coccidian parasites, multicopy organellar DNA such as mitochondrial genomes have been used for detection and molecular typing. Methods We developed a workflow to obtain complete mitochondrial genome sequences from cilantro samples and clinical samples for typing of C. cayetanensis isolates. The 6.3 kb long C. cayetanensis mitochondrial genome was amplified by PCR in four overlapping amplicons from genomic DNA extracted from cilantro, seeded with oocysts, and from stool samples positive for C. cayetanensis by diagnostic methods. DNA sequence libraries of pooled amplicons were prepared and sequenced via next-generation sequencing (NGS). Sequence reads were assembled using a custom bioinformatics pipeline. Results This approach allowed us to sequence complete mitochondrial genomes from the samples studied. Sequence alterations, such as single nucleotide polymorphism (SNP) profiles and insertion and deletions (InDels), in mitochondrial genomes of 24 stool samples from patients with cyclosporiasis diagnosed in 2014, exhibited discriminatory power. The cluster dendrogram that was created based on distance matrices of the complete mitochondrial genome sequences, indicated distinct strain-level diversity among the 2014 C. cayetanensis outbreak isolates analyzed in this study. Conclusions Our results suggest that genomic analyses of mitochondrial genome sequences may help to link outbreak cases to the source.

Outbreaks of cyclosporiasis, a food-borne illness caused by the coccidian parasite Cyclospora cayetanensis have increased in the USA in recent years, with approximately 2300 laboratory-confirmed cases reported in 2018. Genotyping tools are needed to inform epidemiological investigations, yet genotyping Cyclospora has proven challenging due to its sexual reproductive cycle which produces complex infections characterized by high genetic heterogeneity. We used targeted amplicon deep sequencing and a recently described ensemble-based distance statistic that accommodates heterogeneous (mixed) genotypes and specimens with partial genotyping data, to genotype and cluster 648 C. cayetanensis samples submitted to CDC in 2018. The performance of the ensemble was assessed by comparing ensemble-identified genetic clusters to analogous clusters identified independently based on common food exposures. Using these epidemiologic clusters as a gold standard, the ensemble facilitated genetic clustering with 93.8% sensitivity and 99.7% specificity. Hence, we anticipate that this procedure will greatly complement epidemiologic investigations of cyclosporiasis.

Background Cyclospora cayetanensis is a food-borne intestinal human parasite that causes outbreaks of diarrhea. There is a need for efficient laboratory methods for strain-level characterization to assist in outbreak investigations. By using next generation sequencing, genomic sequences can be obtained and compared to identify potential genotyping markers. However, there is no method available to propagate this parasite in the laboratory. Therefore, genomic DNA must be extracted from oocysts purified from human stool. The objective of this study was to apply optimized methods to purify C. cayetanensis oocysts and extract DNA in order to obtain high-quality whole genome sequences with minimum contamination of DNA from other organisms. Results Oocysts from 21 human stool specimens were separated from other stool components using discontinuous density gradient centrifugation and purified further by flow cytometry. Genomic DNA was used to construct Ovation Ultralow libraries for Illumina sequencing. MiSeq sequencing reads were taxonomically profiled for contamination, de novo assembled, and mapped to a draft genome available in GenBank to assess the quality of the resulting genomic sequences. Following all purification steps, the majority (81–99%) of sequencing reads were from C. cayetanensis. They could be assembled into draft genomes of around 45 MB in length with GC-content of 52%. Conclusions Density gradients performed in the presence of a detergent followed by flow cytometry sorting of oocysts yielded sufficient genomic DNA largely free from contamination and suitable for whole genome sequencing of C. cayetanensis. The methods described here will facilitate the accumulation of genomic sequences from various samples, which is a prerequisite for the development of typing tools to aid in outbreak investigations.

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.

Because the lack of typing tools for Cyclospora cayetanensis has hampered outbreak investigations, we sequenced its genome and developed a genotyping tool. We observed 2 to 10 geographically segregated sequence types at each of 5 selected loci. This new tool could be useful for case linkage and infection/contamination source tracking.

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.

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.

Cyclospora cayetanensis is an intestinal parasite responsible for the diarrheal illness, cyclosporiasis. Molecular genotyping, using targeted amplicon sequencing, provides a complementary tool for outbreak investigations, especially when epidemiological data are insufficient for linking cases and identifying clusters. The goal of this study was to identify candidate genotyping markers using a novel workflow for detection of segregating single nucleotide polymorphisms (SNPs) in C. cayetanensis genomes. Four whole C. cayetanensis genomes were compared using this workflow and four candidate markers were selected for evaluation of their genotyping utility by PCR and Sanger sequencing. These four markers covered 13 SNPs and resolved parasites from 57 stool specimens, differentiating C. cayetanensis into 19 new unique genotypes.Développement d’un flux de travail pour l’identification de marqueurs de génotypage nucléaire pour Cyclospora cayetanensis.Cyclospora cayetanensis est un parasite intestinal responsable de la cyclosporose, maladie diarrhéique. Le génotypage moléculaire, utilisant le séquençage ciblé des amplicons, fournit un outil complémentaire pour les enquêtes sur les épidémies, en particulier lorsque les données épidémiologiques sont insuffisantes pour relier les cas et identifier les grappes. Le but de cette étude était d’identifier des marqueurs candidats de génotypage à l’aide d’un nouveau flux de travail pour la détection des polymorphismes d’un seul nucléotide (SNP) différentiateurs dans les génomes de C. cayetanensis. Quatre génomes entiers de C. cayetanensis ont été comparés à l’aide de ce flux de travail et quatre marqueurs candidats ont été sélectionnés pour l’évaluation de leur utilité de génotypage par PCR et séquençage Sanger. Ces quatre marqueurs couvraient 13 SNP et ont résolu les parasites provenant de 57 spécimens de selles, différenciant C. cayetanensis en 19 nouveaux génotypes uniques.

Background Protozoan parasites such as Giardia duodenalis, Cryptosporidium spp., Cyclospora cayetanensis, Toxoplasma gondii and Entamoeba histolytica represent a great challenge to the systems producing water for human consumption because their cystic forms are persistent in the environment and resist to the disinfection methods conventionally used for their control. In this study, we investigated the presence of these protozoan pathogens in both raw and treated water samples used for the production of drinking water in Nariño Department, southwest Colombia. We collected 110 water samples (10 lof each sample) and analyzed them with real-time PCR (qPCR). qPCR-positive samples were genotyped with PCR and DNA sequencing. Results Giardia duodenalis was detected in 35/110 (31.8%) of the samples and Cryptosporidium spp. in 9/110 (8.2%) of the samples; no sample was positive for T. gondii, E. histolytica or C. cayetanensis. Giardia duodenalis was detected in samples of both raw water (Drinking Water Treatment Plants (DWTP): 47.83%;Drinking Water Rural Plants (DWRP): 18.42%) and water collected either after conventional physicochemical treatment (26.09%) or after disinfection by chlorine (50%), whereas Cryptosporidium spp. were only detected in raw waters (DWTP: 17.39%; DWRP: 13.16%). The two pathogens were detected in both types of treatment plants supplying water to urban areas and to rural zones. Analysis of gdh and tpi markers identified assemblages AI, AII and H of G. duodenalis, while analysis of the small subunit rRNA and gp60 markers of Cryptosporidium-positive samples identified C. parvum (Subtype IIcA5G3c), C. galli, C. molnari, Cryptosporidium sp. genotype II of bats and Cryptosporidium sp. genotype VIII of birds. Conclusions The results obtained demonstrate the presence of protozoan parasites in the water of the study region, and the need to improve the surveillance systems for these pathogens and identify the corresponding sources of contamination. Electronic supplementary material The online version of this article (10.1186/s13071-018-3147-3) contains supplementary material, which is available to authorized users.

Cyclospora cayetanensisis a coccidian parasite associated with diarrheal illness. In the USA, foodborne outbreaks of cyclosporiasis have been documented almost every year since the mid-1990s. The typical approach used to identify this parasite in human stools is an examination of acid-fast-stained smears under bright-field microscopy. UV fluorescence microscopy of wet mounts is more sensitive and specific than acid-fast staining but requires a fluorescence microscope with a special filter not commonly available in diagnostic laboratories. In this study, we evaluated a new DNA extraction method based on the Universal Nucleic Acid Extraction (UNEX) buffer and compared the performances of four published real-time polymerase chain reaction (PCR) assays for the specific detection ofC. cayetanensisin stool. The UNEX-based method had an improved capability to recover DNA from oocysts compared with the FastDNA stool extraction method. The best-performing real-time PCR assay was aC. cayetanensis-specific TaqMan PCR that targets the 18S ribosomal RNA gene. This new testing algorithm should be useful for detection ofC. cayetanensisin human stool samples.

Cyclospora cayetanensis is a human parasite transmitted via ingestion of contaminated food or water. Cases of C. cayetanensis infection acquired in the United States often go unexplained, partly because of the difficulties associated with epidemiologic investigations of such cases and the lack of genotyping methods. A Multilocus Sequence Typing (MLST) method for C. cayetanensis based on five microsatellite loci amplified by nested PCR was described in 2016. The MLST loci had high variability, but many specimens could not be assigned a type because of poor DNA sequencing quality at one or more loci. We analyzed Cyclospora-positive stool specimens collected during 1997–2016 from 54 patients, including 51 from the United States. We noted limited inter-specimen variability for one locus (CYC15) and the frequent occurrence of unreadable DNA sequences for two loci (CYC3 and CYC13). Overall, using the remaining two loci (CYC21 and CYC22), we detected 17 different concatenated sequence types. For four of five clusters of epidemiologically linked cases for which we had specimens from >1 case-patient, the specimens associated with the same cluster had the same type. However, we also noted the same type for specimens that were geographically and temporally unrelated, indicating poor discriminatory power. Furthermore, many specimens had what appeared to be a mixture of sequence types at locus CYC22. We conclude that it may be difficult to substantially improve the performance of the MLST method because of the nucleotide repeat features of the markers, along with the frequent occurrence of mixed genotypes in Cyclospora infections.

Background Cyclospora cayetanensis is an emerging coccidian parasite that causes endemic and epidemic diarrheal disease called cyclosporiasis, and this infection is associated with consumption of contaminated produce or water in developed and developing regions. Food-borne outbreaks of cyclosporiasis have occurred almost every year in the USA since the 1990s. Investigations of these outbreaks are currently hampered due to lack of molecular epidemiological tools for trace back analysis. The apicoplast of C. cayetanensis, a relict non-photosynthetic plastid with an independent genome, provides an attractive target to discover sequence polymorphisms useful as genetic markers for detection and trace back analysis of the parasite. Distinct differences in the apicoplast genomes of C. cayetanensis could be useful in designing advanced molecular methods for rapid detection and, subtyping and geographical source attribution, which would aid outbreak investigations and surveillance studies. Methods To obtain the genome sequence of the C. cayetanensis apicoplast, we sequenced the C. cayetanensis genomic DNA extracted from clinical stool samples, assembled and annotated a 34,146 bp-long circular sequence, and used this sequence as a reference genome in this study. We compared the genome and the predicted proteome to the data available from other apicomplexan parasites. To initialize the search for genetic markers, we mapped the raw sequence reads from an additional 11 distinct clinical stool samples originating from Nepal, New York, Texas, and Indonesia to the apicoplast reference genome. Results We identified several high quality single nucleotide polymorphisms (SNPs) and small insertion/deletions spanning the apicoplast genome supported by extensive sequencing reads data, and a 30 bp sequence repeat at the terminal spacer region in a Nepalese sample. The predicted proteome consists of 29 core apicomplexan peptides found in most of the apicomplexans. Cluster analysis of these C. cayetanensis apicoplast genomes revealed a familiar pattern of tight grouping with Eimeria and Toxoplasma, separated from distant species such as Plasmodium and Babesia. Conclusions SNPs and sequence repeats identified in this study may be useful as genetic markers for identification and differentiation of C. cayetanensis isolates found and could facilitate outbreak investigations. Electronic supplementary material The online version of this article (doi:10.1186/s13071-016-1896-4) contains supplementary material, which is available to authorized users.

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.

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 near complete mitochondrial genome for Cyclospora cayetanensis is 6184 bp in length with three protein-coding genes (Cox1, Cox3, CytB) and numerous lsrDNA and ssrDNA fragments. Gene arrangements were conserved with other coccidia in the Eimeriidae, but the C. cayetanensis mitochondrial genome is not circular-mapping. Terminal transferase tailing and nested PCR completed the 5′-terminus of the genome starting with a 21 bp A/T-only region that forms a potential stem-loop. Regions homologous to the C. cayetanensis mitochondrial genome 5′-terminus are found in all eimeriid mitochondrial genomes available and suggest this may be the ancestral start of eimeriid mitochondrial genomes.

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 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 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.

Background A century after its discovery, Chagas disease still represents a major neglected tropical threat. Accurate diagnostics tools as well as surrogate markers of parasitological response to treatment are research priorities in the field. The purpose of this study was to evaluate the performance of PCR methods in detection of Trypanosoma cruzi DNA by an external quality evaluation. Methodology/Findings An international collaborative study was launched by expert PCR laboratories from 16 countries. Currently used strategies were challenged against serial dilutions of purified DNA from stocks representing T. cruzi discrete typing units (DTU) I, IV and VI (set A), human blood spiked with parasite cells (set B) and Guanidine Hidrochloride-EDTA blood samples from 32 seropositive and 10 seronegative patients from Southern Cone countries (set C). Forty eight PCR tests were reported for set A and 44 for sets B and C; 28 targeted minicircle DNA (kDNA), 13 satellite DNA (Sat-DNA) and the remainder low copy number sequences. In set A, commercial master mixes and Sat-DNA Real Time PCR showed better specificity, but kDNA-PCR was more sensitive to detect DTU I DNA. In set B, commercial DNA extraction kits presented better specificity than solvent extraction protocols. Sat-DNA PCR tests had higher specificity, with sensitivities of 0.05–0.5 parasites/mL whereas specific kDNA tests detected 5.10−3 par/mL. Sixteen specific and coherent methods had a Good Performance in both sets A and B (10 fg/µl of DNA from all stocks, 5 par/mL spiked blood). The median values of sensitivities, specificities and accuracies obtained in testing the Set C samples with the 16 tests determined to be good performing by analyzing Sets A and B samples varied considerably. Out of them, four methods depicted the best performing parameters in all three sets of samples, detecting at least 10 fg/µl for each DNA stock, 0.5 par/mL and a sensitivity between 83.3–94.4%, specificity of 85–95%, accuracy of 86.8–89.5% and kappa index of 0.7–0.8 compared to consensus PCR reports of the 16 good performing tests and 63–69%, 100%, 71.4–76.2% and 0.4–0.5, respectively compared to serodiagnosis. Method LbD2 used solvent extraction followed by Sybr-Green based Real time PCR targeted to Sat-DNA; method LbD3 used solvent DNA extraction followed by conventional PCR targeted to Sat-DNA. The third method (LbF1) used glass fiber column based DNA extraction followed by TaqMan Real Time PCR targeted to Sat-DNA (cruzi 1/cruzi 2 and cruzi 3 TaqMan probe) and the fourth method (LbQ) used solvent DNA extraction followed by conventional hot-start PCR targeted to kDNA (primer pairs 121/122). These four methods were further evaluated at the coordinating laboratory in a subset of human blood samples, confirming the performance obtained by the participating laboratories. Conclusion/Significance This study represents a first crucial step towards international validation of PCR procedures for detection of T. cruzi in human blood samples., Author Summary A century after its discovery, Chagas disease, caused by the parasite Trypanosoma cruzi, still represents a major neglected tropical threat. Accurate diagnostics tools as well as surrogate markers of parasitological response to treatment are research priorities in the field. The polymerase chain reaction (PCR) has been proposed as a sensitive laboratory tool for detection of T. cruzi infection and monitoring of parasitological treatment outcome. However, high variation in accuracy and lack of international quality controls has precluded reliable applications in the clinical practice and comparisons of data among cohorts and geographical regions. In an effort towards harmonization of PCR strategies, 26 expert laboratories from 16 countries evaluated their current PCR procedures against sets of control samples, composed by serial dilutions of T.cruzi DNA from culture stocks belonging to different lineages, human blood spiked with parasite cells and blood samples from Chagas disease patients. A high variability in sensitivities and specificities was found among the 48 reported PCR tests. Out of them, four tests with best performance were selected and further evaluated. This study represents a crucial first step towards device of a standardized operative procedure for T. cruzi PCR.

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.

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.

In a concluding session of the workshop, the participants developed a list of 115 research and outreach needs, outlining the top 5-7 needs in each of 8 areas (Table). For complete information, including presenter details and abstracts, visit the workshop website at www.hawaii.edu/cowielab/Angio%20website%20home.htm.