Identification and Evaluation of Cryptosporidium Species from New York City Cases of Cryptosporidiosis (2015 to 2018): a Watershed Perspective.

Watersheds that supply residents with drinking water have the potential for contamination with Cryptosporidium oocysts. To evaluate any potential similarities between Cryptosporidium species previously found in the New York City (NYC) watershed and those causing disease in NYC, the species were identified in stool specimens from residents with cryptosporidiosis. Genetic analysis was performed on 628 positive stool samples collected from NYC residents between 2015 and 2018 to determine the species present. A total of 547 samples yielded positive results by real-time PCR. Of these samples, 512 (93.6%) were identified to the species level, with 94.7% positive for either Cryptosporidium hominis or Cryptosporidium parvum (56.4% and 38.5%, respectively), including one coinfection. Less common Cryptosporidium species identified included C. felis, C. canis, C. ubiquitum, C. meleagridis, and a Cryptosporidium sp. chipmunk genotype. Results were evaluated and compared to species and genotypes of Cryptosporidium previously identified from stormwater collected within the NYC watershed. While there was overlap with some of the rare species found in case specimens, the prevalence and distribution of species did not suggest a connection between sources previously identified in the watershed and the species causing human cases of cryptosporidiosis in NYC residents. IMPORTANCE It is important to identify the species causing human cryptosporidiosis in a population in order to investigate possible sources or routes of contamination. Many species of Cryptosporidium are host-adapted and therefore have the potential to be tracked back to specific sources that can subsequently be managed. There has been no evidence to suggest that the water supply has ever been a source of cryptosporidiosis cases in NYC, and since 2013, the New York City Department of Environmental Protection has further reduced the risk of disease through the use of ultraviolet treatment to inactivate any Cryptosporidium present in the source water. However, as one of the largest unfiltered water supplies in the country, it is important to evaluate watershed sources for potential impacts to public health. In this unique study, species of Cryptosporidium causing disease in NYC residents were identified and compared with previously identified species from the watershed.

Assessing an Adaptation of the Universal Parasite Diagnostic Assay for Bloodborne Parasites in a US State Public Health Laboratory.

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.

Investigation of US Cyclospora cayetanensis outbreaks in 2019 and evaluation of an improved Cyclospora genotyping system against 2019 cyclosporiasis outbreak clusters.

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.

Evaluation of an ensemble-based distance statistic for clustering MLST datasets using epidemiologically defined clusters of cyclosporiasis.

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.

Detecting Cryptosporidium in Stool Samples Submitted to a Reference Laboratory.

When considering methods of detecting Cryptosporidium in patient samples, clinical and public health laboratories have historically relied primarily on microscopy. However, microscopy is time intensive and requires trained personnel to accurately identify pathogens that are present. Even with skilled analysts, the parasitemia level has the potential to fall below the level of detection. In addition, public health laboratories do not always receive specimens in fixatives that are compatible with the desired microscopic method. Antigen-based and molecular methods have proven to be effective at identifying Cryptosporidium at low levels and require less training and hands-on time. Here, we have developed and validated a real-time polymerase chain reaction (RT-PCR) laboratory-developed test (LDT) that identifies Cryptosporidium hominis and Cryptosporidium parvum, and also includes detection at the genus level to identify additional species that occasionally cause disease in humans. Results of the molecular test were compared with those obtained from modified acid-fast microscopy, immunofluorescent microscopy, an antigen-based detection rapid test, and a commercial gastrointestinal panel (GI panel). Of 40 positive samples, microscopy and antigen-based methods were able to detect Cryptosporidium in only 20 and 21 samples, respectively. The GI panel detected 33 of the 40 positive samples, even though not all specimens were received in the recommended preservative. The LDT detected Cryptosporidium in all 40 positive samples. When comparing each method for the detection of Cryptosporidium, our results indicate the LDT is an accurate, reliable, and cost-effective method for a clinical public health reference laboratory.

Lexis and Grammar of Mitochondrial RNA Processing in Trypanosomes.

Trypanosoma brucei spp. cause African human and animal trypanosomiasis, a burden on health and economy in Africa. These hemoflagellates are distinguished by a kinetoplast nucleoid containing mitochondrial DNAs of two kinds: maxicircles encoding ribosomal RNAs (rRNAs) and proteins and minicircles bearing guide RNAs (gRNAs) for mRNA editing. All RNAs are produced by a phage-type RNA polymerase as 3' extended precursors, which undergo exonucleolytic trimming. Most pre-mRNAs proceed through 3' adenylation, uridine insertion/deletion editing, and 3' A/U-tailing. The rRNAs and gRNAs are 3' uridylated. Historically, RNA editing has attracted major research effort, and recently essential pre- and postediting processing events have been discovered. Here, we classify the key players that transform primary transcripts into mature molecules and regulate their function and turnover.

Frequency and magnitude of seroreactivity to Babesia microti in 245 patients diagnosed by PCR in New York State.

Multiple methodologies have been used to detect antibodies to Babesia microti. Use of an indirect immunofluorescence assay (IFA) has been the most widely used approach, but IFAs have varied as to which antibody class or classes are being detected and in regard to cutoff titers. In this study, 245 different patients with polymerase chain reaction (PCR)-confirmed B. microti infection were tested by a polyvalent IFA using serum collected within 3 days of the date the blood sample for PCR testing was obtained. Of the 245 patients, 243 (99.2%) had a positive serologic test result (i.e., ≥1:64). Of the 243 patients who were seropositive, 242 (99.6%) had a titer of ≥1:256, 236 (97.1%) had a titer of ≥1:512, and 210 (86.4%) had a titer of ≥1:1024. In conclusion, high titer seropositivity based on a polyvalent IFA is to be expected at the time of PCR confirmation of active babesiosis in clinical practice.

Epidemiology of Cryptosporidiosis, New York City, New York, USA, 1995-20181.

Cryptosporidiosis is a parasitic diarrheal infection that is transmitted by the fecal-oral route. We assessed trends in incidence and demographic characteristics for the 3,984 cases diagnosed during 1995-2018 in New York City, New York, USA, and reported to the New York City Department of Health and Mental Hygiene. Reported cryptosporidiosis incidence decreased with HIV/AIDS treatment rollout in the mid-1990s, but the introduction of syndromic multiplex diagnostic panels in 2015 led to a major increase in incidence and to a shift in the demographic profile of reported patients. Incidence was highest among men 20-59 years of age, who consistently represented most (54%) reported patients. In addition, 30% of interviewed patients reported recent international travel. The burden of cryptosporidiosis in New York City is probably highest among men who have sex with men. Prevention messaging is warranted for men who have sex with men and their healthcare providers, as well as for international travelers.

Emerging Tick-Borne Diseases.

Increases in tick-borne disease prevalence and transmission are important public health issues. Efforts to control these emerging diseases are frustrated by the struggle to control tick populations and to detect and treat infections caused by the pathogens that they transmit. This review covers tick-borne infectious diseases of nonrickettsial bacterial, parasitic, and viral origins. While tick surveillance and tracking inform our understanding of the importance of the spread and ecology of ticks and help identify areas of risk for disease transmission, the vectors are not the focus of this document. Here, we emphasize the most significant pathogens that infect humans as well as the epidemiology, clinical features, diagnosis, and treatment of diseases that they cause. Although detection via molecular or immunological methods has improved, tick-borne diseases continue to remain underdiagnosed, making the scope of the problem difficult to assess. Our current understanding of the incidence of tick-borne diseases is discussed in this review. An awareness of the diseases that can be transmitted by ticks in specific locations is key to detection and selection of appropriate treatment. As tick-transmitted pathogens are discovered and emerge in new geographic regions, our ability to detect, describe, and understand the growing public health threat must also grow to meet the challenge.

Investigation of a case of suspected transfusion-transmitted malaria.

BACKGROUND: Transfusion-transmitted malaria (TTM) is a rare occurrence with serious consequences for the recipient. A case study is presented as an example of best practices for conducting a TTM investigation. CASE REPORT: A 15-year-old male with a history of sickle cell disease developed fever after a blood transfusion. He was diagnosed with Plasmodium falciparum malaria and was successfully treated. The American Red Cross, New York State Department of Health, and the Centers for Disease Control and Prevention investigated the eight donors who provided components to the transfusion. The investigation to identify a malaria-positive donor included trace back of donors, serologic methods to identify donor(s) with a history of malaria exposure, polymerase chain reaction (PCR) testing, microsatellite analysis to identify the parasite in a donor and match its genotype to the parasite in the recipient, and reinterview of all donors to clarify malaria risk factors. RESULTS: One donor had evidence of infection with P. falciparum by PCR, elevated antibody titers, and previously undisclosed malaria risk factors. Reinterview revealed that the donor immigrated to the United States from Togo just short of 3 years before the blood donation. The donor was treated for asymptomatic low parasitemia infection. CONCLUSION: This investigation used standard procedures for investigating TTM but also demonstrated the importance of applying sensitive laboratory techniques to identify the infected donor, especially a donor with asymptomatic infection with low parasitemia. Repeat interview of all donors identified as having contributed to the transfused component provides complementary epidemiologic information to confirm the infected donor.

Performance Evaluation of a Prototype Architect Antibody Assay for Babesia microti.

The tick-borne protozoan Babesia microti is responsible for more than 200 cases of transfusion-transmitted babesiosis (TTB) infection in the United States that have occurred over the last 30 years. Measures to mitigate the risk of TTB include nucleic acid testing (NAT) and B. microti antibody testing. A fully automated prototype B. microti antibody test was developed on the Architect instrument. The specificity was determined to be 99.98% in volunteer blood donors (n = 28,740) from areas considered to have low endemicity for B. microti The sensitivity of the prototype test was studied in experimentally infected macaques; a total of 128 samples were detected as positive whereas 125 were detected as positive with an indirect fluorescent antibody (IFA) test; additionally, 83 (89.2%) of the PCR-positive samples were detected in contrast to 81 (87.1%) using an IFA test. All PCR-positive samples that tested negative in the prototype antibody test were preseroconversion period samples. Following seroconversion, periods of intermittent parasitemia occurred; 17 PCR-negative samples drawn in between PCR-positive bleed dates tested positive both by the prototype test (robust reactivity) and IFA test (marginal reactivity) prior to the administration of therapeutic drugs, indicating that the PCR test failed to detect samples from persistently infected macaques. The prototype assay detected 56 of 58 (96.6%) human subjects diagnosed with clinical babesiosis by both PCR and IFA testing. Overall, the prototype anti-Babesia assay provides a highly sensitive and specific test for the diagnosis of B. microti infection. While PCR is preferred for detection of window-period parasitemia, antibody tests detect infected subjects during periods of low-level parasitemia.

Next-Generation Sequencing and Bioinformatics Protocol for Malaria Drug Resistance Marker Surveillance.

The recent advances in next-generation sequencing technologies provide a new and effective way of tracking malaria drug-resistant parasites. To take advantage of this technology, an end-to-end Illumina targeted amplicon deep sequencing (TADS) and bioinformatics pipeline for molecular surveillance of drug resistance in P. falciparum, called malaria resistance surveillance (MaRS), was developed. TADS relies on PCR enriching genomic regions, specifically target genes of interest, prior to deep sequencing. MaRS enables researchers to simultaneously collect data on allele frequencies of multiple full-length P. falciparum drug resistance genes (crt, mdr1, k13, dhfr, dhps, and the cytochrome b gene), as well as the mitochondrial genome. Information is captured at the individual patient level for both known and potential new single nucleotide polymorphisms associated with drug resistance. The MaRS pipeline was validated using 245 imported malaria cases that were reported to the Centers for Disease Control and Prevention (CDC). The chloroquine resistance crt CVIET genotype (mutations underlined) was observed in 42% of samples, the highly pyrimethamine-resistant dhpsIRN triple mutant in 92% of samples, and the sulfadoxine resistance dhps mutation SGEAA in 26% of samples. The mdr1 NFSND genotype was found in 40% of samples. With the exception of two cases imported from Cambodia, no artemisinin resistance k13 alleles were identified, and 99% of patients carried parasites susceptible to atovaquone-proguanil. Our goal is to implement MaRS at the CDC for routine surveillance of imported malaria cases in the United States and to aid in the adoption of this system at participating state public health laboratories, as well as by global partners.

Giardiasis Outbreak Associated with Asymptomatic Food Handlers in New York State, 2015.

Giardia duodenalis is a protozoan that causes a gastrointestinal illness called giardiasis. Giardiasis outbreaks in the United States are most commonly associated with waterborne transmission and are less commonly associated with food, person-to-person, and zoonotic transmission. During June to September 2015, an outbreak of 20 giardiasis cases occurred and were epidemiologically linked to a local grocery store chain on Long Island, New York. Further investigation revealed three asymptomatic food handlers were infected with G. duodenalis, and one food handler and one case were coinfected with Cryptosporidium spp. Although G. duodenalis was not detected in food samples, Cryptosporidium was identified in samples of spinach dip and potato salad. The G. duodenalis assemblage and subtype from one of the food handlers matched two outbreak cases for which genotyping could be performed. This outbreak highlights the potential role of asymptomatically infected food handlers in giardiasis outbreaks.

Community Laboratory Testing for Cryptosporidium: Multicenter Study Retesting Public Health Surveillance Stool Samples Positive for Cryptosporidium by Rapid Cartridge Assay with Direct Fluorescent Antibody Testing.

Cryptosporidium is a common cause of sporadic diarrheal disease and outbreaks in the United States. Increasingly, immunochromatography-based rapid cartridge assays (RCAs) are providing community laboratories with a quick cryptosporidiosis diagnostic method. In the current study, the Centers for Disease Control and Prevention (CDC), the Association of Public Health Laboratories (APHL), and four state health departments evaluated RCA-positive samples obtained during routine Cryptosporidium testing. All samples underwent "head to head" re-testing using both RCA and direct fluorescence assay (DFA). Community level results from three sites indicated that 54.4% (166/305) of Meridian ImmunoCard STAT! positives and 87.0% (67/77) of Remel Xpect positives were confirmed by DFA. When samples were retested by RCA at state laboratories and compared with DFA, 83.3% (155/186) of Meridian ImmunoCard STAT! positives and 95.2% (60/63) of Remel Xpect positives were confirmed. The percentage of confirmed community results varied by site: Minnesota, 39.0%; New York, 63.9%; and Wisconsin, 72.1%. The percentage of confirmed community results decreased with patient age; 12.5% of community positive tests could be confirmed by DFA for patients 60 years of age or older. The percentage of confirmed results did not differ significantly by sex, storage temperature, time between sample collection and testing, or season. Findings from this study demonstrate a lower confirmation rate of community RCA positives when compared to RCA positives identified at state laboratories. Elucidating the causes of decreased test performance in order to improve overall community laboratory performance of these tests is critical for understanding the epidemiology of cryptosporidiosis in the United States (US).

Determination of the ribosome structure to a resolution of 2.5 Å by single-particle cryo-EM.

With the advance of new instruments and algorithms, and the accumulation of experience over decades, single-particle cryo-EM has become a pivotal part of structural biology. Recently, we determined the structure of a eukaryotic ribosome at 2.5 Å for the large subunit. The ribosome was derived from Trypanosoma cruzi, the protozoan pathogen of Chagas disease. The high-resolution density map allowed us to discern a large number of unprecedented details including rRNA modifications, water molecules, and ions such as Mg2+ and Zn2+ . In this paper, we focus on the procedures for data collection, image processing, and modeling, with particular emphasis on factors that contributed to the attainment of high resolution. The methods described here are readily applicable to other macromolecules for high-resolution reconstruction by single-particle cryo-EM.

Structure and assembly model for the Trypanosoma cruzi 60S ribosomal subunit.

Ribosomes of trypanosomatids, a family of protozoan parasites causing debilitating human diseases, possess multiply fragmented rRNAs that together are analogous to 28S rRNA, unusually large rRNA expansion segments, and r-protein variations compared with other eukaryotic ribosomes. To investigate the architecture of the trypanosomatid ribosomes, we determined the 2.5-Å structure of the Trypanosoma cruzi ribosome large subunit by single-particle cryo-EM. Examination of this structure and comparative analysis of the yeast ribosomal assembly pathway allowed us to develop a stepwise assembly model for the eight pieces of the large subunit rRNAs and a number of ancillary "glue" proteins. This model can be applied to the characterization of Trypanosoma brucei and Leishmania spp. ribosomes as well. Together with other details, our atomic-level structure may provide a foundation for structure-based design of antitrypanosome drugs.

Neurocysticercosis in a Rhesus Macaque (Macaca mulatta).

An 8-y-old, intact, male rhesus macaque (Macaca mulatta) was sedated to undergo MRI in preparation for the implantation of cranial hardware. During imaging, 9 focal lesions were noted in the brain and musculature of the head. The lesions were hyperechoic with hypoechoic rims. The animal was deemed inappropriate for neuroscience research, and euthanasia was elected. Gross examination revealed multiple round, thick-walled, fluid-filled cysts (diameter, approximately 0.5 cm) in multiple tissues: one each in the left caudal lung lobe, left masseter muscle, and the dura overlying the brain and 8 throughout the gray and white matter of the brain parenchyma. Formalin-fixed sections of cyst-containing brain were stained with hematoxylin and eosin. Microscopic examination and molecular analysis of the COX1 (COI) gene recognized the causative organism as Taenia solium at 99.04% identity.

Healthcare-Associated Transmission of Plasmodium falciparum in New York City.

A patient with no risk factors for malaria was hospitalized in New York City with Plasmodium falciparum infection. After investigating all potential sources of infection, we concluded the patient had been exposed to malaria while hospitalized less than 3 weeks earlier. Molecular genotyping implicated patient-to-patient transmission in a hospital setting. Infect. Control Hosp. Epidemiol. 2015;37(1):113-115.

High-resolution cryo-electron microscopy structure of the Trypanosoma brucei ribosome.

Ribosomes, the protein factories of living cells, translate genetic information carried by messenger RNAs into proteins, and are thus involved in virtually all aspects of cellular development and maintenance. The few available structures of the eukaryotic ribosome reveal that it is more complex than its prokaryotic counterpart, owing mainly to the presence of eukaryote-specific ribosomal proteins and additional ribosomal RNA insertions, called expansion segments. The structures also differ among species, partly in the size and arrangement of these expansion segments. Such differences are extreme in kinetoplastids, unicellular eukaryotic parasites often infectious to humans. Here we present a high-resolution cryo-electron microscopy structure of the ribosome of Trypanosoma brucei, the parasite that is transmitted by the tsetse fly and that causes African sleeping sickness. The atomic model reveals the unique features of this ribosome, characterized mainly by the presence of unusually large expansion segments and ribosomal-protein extensions leading to the formation of four additional inter-subunit bridges. We also find additional rRNA insertions, including one large rRNA domain that is not found in other eukaryotes. Furthermore, the structure reveals the five cleavage sites of the kinetoplastid large ribosomal subunit (LSU) rRNA chain, which is known to be cleaved uniquely into six pieces, and suggests that the cleavage is important for the maintenance of the T. brucei ribosome in the observed structure. We discuss several possible implications of the large rRNA expansion segments for the translation-regulation process. The structure could serve as a basis for future experiments aimed at understanding the functional importance of these kinetoplastid-specific ribosomal features in protein-translation regulation, an essential step towards finding effective and safe kinetoplastid-specific drugs.