Dead-End Ultrafiltration and DNA-Based Methods for Detection of Cyclospora cayetanensis in Agricultural Water.

Cyclospora cayetanensis is a protozoan parasite that causes foodborne and waterborne diarrheal illness outbreaks worldwide. Most of these outbreaks are associated with the consumption of fresh produce. Sensitive and specific methods to detect C. cayetanensis in agricultural water are needed to identify the parasite in agricultural water used to irrigate crops that have been implicated in outbreaks. In this study, a method to detect C. cayetanensis in water by combining dead-end ultrafiltration (DEUF) with sensitive and specific molecular detection was developed and evaluated. Triplicates of 10-liter agricultural water samples were seeded with 200, 100, 25, 12, and 6 C. cayetanensis oocysts. Surface water samples were also collected in the Mid-Atlantic region. All water samples were processed by DEUF and backflushed from the ultrafilters. DNA was extracted from concentrated samples and analyzed by quantitative PCR (qPCR) targeting the C. cayetanensis 18S rRNA gene. All water samples seeded with 12, 25, 100, and 200 oocysts were positive, and all unseeded samples were negative. Samples seeded with 6 oocysts had a detection rate of 66.6% (8/12). The method was also able to detect C. cayetanensis isolates in surface water samples from different locations of the Chesapeake and Ohio Canal (C&O Canal) in Maryland. This approach could consistently detect C. cayetanensis DNA in 10-liter agricultural water samples contaminated with low levels of oocysts, equivalent to the levels that may be found in naturally incurred environmental water sources. Our data demonstrate the robustness of the method as a useful tool to detect C. cayetanensis from environmental sources.IMPORTANCECyclospora cayetanensis is a protozoan parasite that causes foodborne and waterborne outbreaks of diarrheal illness worldwide. These foodborne outbreaks associated with the consumption of fresh produce and agricultural water could play a role in the contamination process. In this study, a method to detect C. cayetanensis in agricultural water by combining a robust filtration system with sensitive and specific molecular detection was developed and validated by the FDA. The results showed that this approach could consistently detect low levels of C. cayetanensis contamination in 10 liters of agricultural water, corresponding to the levels that may be found in naturally occurring environmental water sources. The method was also able to detect C. cayetanensis in surface water samples from a specific location in the Mid-Atlantic region. Our data demonstrate the robustness of the method to detect C. cayetanensis in agricultural water samples, which could be very useful to identify environmental sources of contamination.

Peptidyl-prolyl cis-trans isomerase A - A novel biomarker of multi-episodic (recurrent) ocular toxoplasmosis.

Ocular toxoplasmosis (OT) is the most common etiology of posterior uveitis. The high incidence of macular scarring associated with OT is a leading cause of visual morbidity. Serum biomarkers of the disease would aid in its diagnosis. This study sought, for the first time, to elucidate serum biomarkers for OT by mass spectrometry. Blood samples were collected from four groups of nine patients each; toxoplasmosis IgG-with no history of uveitis, non-toxoplasmosis uveitis, first episode OT, and symptomatic recurrent OT. Serum was isolated and subjected to proteomics analysis using 2-dimensional gel electrophoresis (2D-GE) and surface-enhanced laser desorption ionization mass spectrometry (SELDI-MS). Selected proteins were further separated by SDS-PAGE and sequenced using tandem MS. Results were cross-validated with a T. gondii outbreak biomarker database that occurred in Brazil. Fifty markers of OT and 46 markers of recurrent disease were discovered by SELDI-MS of which 30 and 15, respectively, were cross-validated. 2D-GE analysis yielded 57 bands, selected based on the intensity of the bands, leading to the identification of 20 proteins. Eleven of those identified candidates were also found by SELDI-MS. Four candidates were chosen for immunoblotting. One serum protein, peptidyl-prolyl cis-trans isomerase A (PPIA), was confirmed as a biomarker of multi-episodic OT by immunoblotting in patients. PPIA can identify the patient with active recurrent OT from acute OT, other forms of uveitis and other parasitic infections. A validated PPIA assay may have a role in the diagnosis of the atypical OT patient before more invasive anterior chamber or vitreous tap is performed for PCR analysis or for Goldmann-Witner coefficient calculations. Base-line PPIA levels need to be studied to understand its possible use when deciding for prophylactic antibiotic use in the immunosuppressed sero-positive patient.

Molecular detection of Cyclospora cayetanensis in human stool specimens using UNEX-based DNA extraction and real-time PCR.

Cyclospora cayetanensis is 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 of C. cayetanensis in 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 a C. cayetanensis-specific TaqMan PCR that targets the 18S ribosomal RNA gene. This new testing algorithm should be useful for detection of C. cayetanensis in human stool samples.

Evaluation of the U.S. Food and Drug Administration validated method for detection of Cyclospora cayetanensis in high-risk fresh produce matrices and a method modification for a prepared dish.

The performance of the U.S. Food and Drug Administration (FDA) validated method for regulatory detection of Cyclospora cayetanensis in leafy greens and berries was evaluated in additional high-risk fresh produce items and in a dish prepared with these produce commodities. The method was robust and reproducible in basil, parsley, shredded carrots, shredded cabbage and carrot mix, and could detect as few as 5 oocysts in 25 g samples. Some differences in C. cayetanensis detection were found among the fresh produce analyzed. Significantly lower target gene copy numbers per reaction were obtained with shredded carrots, and shredded cabbage and carrot mix compared to leafy greens, which highlights the importance of evaluating the performance characteristics of validated methods in different food matrices. In the prepared dish, coleslaw with dressing, the method was optimized to detect 5 oocysts in a 25 g sample by using 1.0% Alconox® in the washing solution instead of 0.1% as originally described. These data are important to assess the prevalence of C. cayetanensis in different produce items and to support outbreak investigations.

Interlaboratory validation of an improved method for detection of Cyclospora cayetanensis in produce using a real-time PCR assay.

A collaborative validation study was performed to evaluate the performance of a new U.S. Food and Drug Administration method developed for detection of the protozoan parasite, Cyclospora cayetanensis, on cilantro and raspberries. The method includes a sample preparation step in which oocysts are recovered from produce using an enhanced produce washing solution containing 0.1% Alconox and a commercially available method to disrupt the C. cayetanensis oocysts and extract DNA. A real-time PCR assay targeting the C. cayetanensis 18S rDNA gene with an internal amplification control to monitor PCR inhibition provides species-specific identification. Five laboratories blindly analyzed a total of 319 samples consisting of 25 g of cilantro or 50 g of raspberries which were either uninoculated or artificially contaminated with C. cayetanensis oocysts. Detection rates for cilantro inoculated with 200, 10, and 5 oocysts, were 100%, 80%, and 31%, respectively. For raspberries, the detection rates for samples inoculated with 200, 10, and 5 oocysts were 100%, 90% and 50%, respectively. All uninoculated samples, DNA blank extracts, and no-template PCR controls were negative. Reproducibility between laboratories and analysts was high and the method was shown to be an effective analytical tool for detection of C. cayetanensis in produce.

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

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

Detection and Differentiation of Leishmania spp. in Clinical Specimens by Use of a SYBR Green-Based Real-Time PCR Assay.

Leishmaniasis in humans is caused by Leishmania spp. in the subgenera Leishmania and Viannia Species identification often has clinical relevance. Until recently, our laboratory relied on conventional PCR amplification of the internal transcribed spacer 2 (ITS2) region (ITS2-PCR) followed by sequencing analysis of the PCR product to differentiate Leishmania spp. Here we describe a novel real-time quantitative PCR (qPCR) approach based on the SYBR green technology (LSG-qPCR), which uses genus-specific primers that target the ITS1 region and amplify DNA from at least 10 Leishmania spp., followed by analysis of the melting temperature (Tm) of the amplicons on qPCR platforms (the Mx3000P qPCR system [Stratagene-Agilent] and the 7500 real-time PCR system [ABI Life Technologies]). We initially evaluated the assay by testing reference Leishmania isolates and comparing the results with those from the conventional ITS2-PCR approach. Then we compared the results from the real-time and conventional molecular approaches for clinical specimens from 1,051 patients submitted to the reference laboratory of the Centers for Disease Control and Prevention for Leishmania diagnostic testing. Specimens from 477 patients tested positive for Leishmania spp. with the LSG-qPCR assay, specimens from 465 of these 477 patients also tested positive with the conventional ITS2-PCR approach, and specimens from 10 of these 465 patients had positive results because of retesting prompted by LSG-qPCR positivity. On the basis of the Tm values of the LSG-qPCR amplicons from reference and clinical specimens, we were able to differentiate four groups of Leishmania parasites: the Viannia subgenus in aggregate; the Leishmania (Leishmania) donovani complex in aggregate; the species L (L) tropica; and the species L (L) mexicana, L (L) amazonensis, L (L) major, and L (L) aethiopica in aggregate.

Cross-reactivity of the 31 kDa antigen of Angiostrongylus cantonensis - Dealing with the immunodiagnosis of meningoencephalitis.

The primary causative agent of eosinophilic meningoencephalitis (EoM) in endemic regions is the nematode Angiostrongylus cantonensis. The occurrence of EoM was previously restricted to countries in Southeast Asia and the Pacific Islands; however, more recently, it has been reported from other regions, including Brazil. The commonly used diagnosis is detection of specific antibody reactivity to the 31 kDa antigen, which is derived from female worm somatic extracts. Here we report the occurrence of cross-reactivity to this antigen in sera from other parasitic infections, especially those that may cause EoM, such as gnathostomiasis, toxocariasis, hydatidosis and strongyloidiasis. We also demonstrated that the cross-reactivity, in part, is dependent of the concentration of antigen used in Western blot assays. We discuss the importance of these findings on the interpretation of this test.

Transmission of Balamuthia mandrillaris by Organ Transplantation.

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

The first association of a primary amebic meningoencephalitis death with culturable Naegleria fowleri in tap water from a US treated public drinking water system.

BACKGROUND: 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. METHODS: Clinical and environmental testing and a case investigation were initiated to determine the cause of death and to identify potential exposures. RESULTS: 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. CONCLUSIONS: 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.

Performance of a real time PCR for leishmaniasis diagnosis using a L. (L.) infantum hypothetical protein as target in canine samples.

Visceral leishmaniasis represents an important public health issue in different parts of the world, requiring that measures be put in place to control the spread of the disease worldwide. The canine leishmaniasis diagnosis is not easy based on clinical signs, since dogs may not develop the infection with recognizable signs. Thus, the laboratorial diagnosis is essential to ascertain the incidence and prevalence of canine leishmaniasis especially in areas with major control efforts. Although, the diagnosis can be performed by the use of different approaches, the molecular methods such as PCR have become an indispensable tool for leishmaniases diagnosis. A TaqMan assay for real-time PCR (Linj31-qPCR) was developed to determine the parasite occurrence in clinical cases of leishmaniasis. The assay targets an L. (L.) infantum hypothetical protein region. The specificity of the assay was verified by using Leishmania World Health Organization reference strains including parasites belonging to subgenus L. (Leishmania), subgenus L. (Viannia), other Leishmania species and Trypanosoma cruzi. The sensitivity was verified by using isolates of L. (L.) amazonensis and L. (L.) infantum. The usefulness of the assay for diagnosis was ascertained by testing 277 samples from dogs in regions endemic for visceral and/or cutaneous leishmaniasis and from regions in which leishmaniasis was not endemic in São Paulo State, Brazil. Diagnosis of canine visceral leishmaniasis (CVL) was determined on these animals by conventional PCR and three serological tests. The dog samples were divided into four groups. I, dogs with CVL (n = 101); II, dogs with other diseases and without CVL (n = 97); III, dogs with American cutaneous leishmaniasis (n = 7), and, IV, dogs without CVL (n = 72) from areas where leishmaniasis was not endemic as control group. Results indicated that Linj31-qPCR was able to identify parasites belonging to subgenus L. (Leishmania) with no cross-amplification with other parasite subgenera. The Linj31-qPCR detected Leishmania parasites DNA in 98% of samples from Group I. In conclusion this methodology can be used as routine diagnostic tools to detect parasites from subgenus Leishmania.

A Diagnostic Algorithm for Detection of Leishmania spp. in Human Fresh and Fixed Tissue Samples.

Leishmaniasis is an important travel-related parasitic infection in the United States. Treatment regimens vary by Leishmania species and require an accurate diagnosis. The sensitivity and specificity of diagnostic methods depend on the type and condition of specimen analyzed. To identify the best algorithm for detection of parasites in fresh and fixed tissue samples, we evaluated parasite cultures, two PCR methods, and Leishmania immunohistochemistry (IHC) in samples received by the CDC from 2012 through 2019. The sensitivity and specificity of IHC assays were evaluated in fresh specimens tested. Diagnostic accuracy for formalin-fixed tissue was evaluated by using PCR-based methods and IHC. Of 100 suspected cases with fresh tissue available, Leishmania spp. infection was identified by PCR in 56% (56/100) of specimens; from these, 80% (45/56) were positive by parasite culture and 59% (33/56) by IHC. Of 420 possible cases where only fixed specimens were available, 58% (244/420) were positive by IHC and/or PCR. Of these, 96% (235/420) were positive by IHC and 84% (204/420) by PCR-based methods. Overall parasite detection using all methodologies was similar for fresh and formalin-fixed tissue specimens (56% versus 58%, respectively). Although PCR-based methods were superior for diagnosis of leishmaniasis and species identification in fresh samples, IHC in combination with PCR increased the accuracy for Leishmania spp. detection in fixed samples. In conclusion, PCR is the most effective method for detecting Leishmania infection in fresh tissue samples, whereas for formalin-fixed samples, IHC and PCR-based methods should be used in combination.

High throughput sequencing of the Angiostrongylus cantonensis genome: a parasite spreading worldwide.

Angiostrongylus cantonensis is a parasitic nematode of rodents and a leading aetiological agent of eosinophilic meningitis in humans. Definitive diagnosis is difficult, often relying on immunodiagnostic methods which utilize crude antigens. New immunodiagnostic methods based on recombinant proteins are being developed, and ideally these methods would be made available worldwide. Identification of diagnostic targets, as well as studies on the biology of the parasite, are limited by a lack of molecular information on Angiostrongylus spp. available in databases. In this study we present data collected from DNA random high-throughput sequencing together with proteomic analyses and a cDNA walking methodology to identify and obtain the nucleotide or amino acid sequences of unknown immunoreactive proteins. 28 080 putative ORFs were obtained, of which 3371 had homology to other deposited protein sequences. Using the A. cantonensis genomic sequences, 156 putative ORFs, matching peptide sequences obtained from previous proteomic studies, were considered novel, with no homology to existing sequences. Full-length coding sequences of eight antigenic target proteins were obtained. In this study we generated not only the complete nucleotide sequences of the antigenic protein targets but also a large amount of genomic data which may help facilitate future genomic, proteomic, transcriptomic or metabolomic studies on Angiostrongylus.

[Molecular characterization of Cryptosporidium spp. isolated in humans in two different locations in Spain]. / Caracterización molecular de aislados humanos de Cryptosporidium spp. procedentes de 2 diferentes localizaciones de España.

Molecular PCR based diagnostic techniques have enabled us to distinguish between the different, morphologically identical, Cryptosporidium species that can infect humans. Of the 23 recognized species in the genus, at least 9 are able to infect humans. As the intensity of the clinical manifestations, pathogenicity, excretion of oocysts, and incidence, are different between this species, molecular studies are crucial for a better understanding of the epidemiology of human cryptosporidiosis. Samples form two independent studies are analyzed in this publication. One included 23 samples from Madrid, and the other, 72 samples from La Coruña. All of them positive for Cryptosporidium spp. by microscopic methods and belonging to isolated cases of human cryptosporidiosis. For the identification of the species responsible for the infection, the 18S rDNA diagnostic region and the COWP gene diagnostic regions were used. Out of the 95 samples tested, in 77 cases we were able to extract and amplify DNA. In those cases the species responsible for the infection were: C. parvum (40 cases, 2 Madrid and 38 La Coruña), C. hominis (30 cases, 10 Madrid and 20 La Coruña) and C. meleagridis (2 cases, 1 Madrid and 1 La Coruña). In 5 samples it was impossible to detect the species responsible for the infection, but their positivity was confirmed by PCR (4 Madrid and 1 La Coruña). The genotypes of the isolates from patients correlated well with animals from the same regions.

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

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

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

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

Workshop on research priorities for management and treatment of angiostrongyliasis(1).

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.

Characterization of Angiostrongylus cantonensis excretory-secretory proteins as potential diagnostic targets.

Angiostrongyliasis results from infections with intra-arterial nematodes that accidentally infect humans. Specifically, infections with Angiostrongylus cantonensis cause eosinophilic meningitis and Angiostrongylus costaricensis infections result in eosinophilic enteritis. Immunological tests are the primary means of diagnosing infections with either pathogen since these parasites are usually not recoverable in fecal or cerebrospinal fluid. However, well-defined, purified antigens are not currently available in sufficient quantities from either pathogen for use in routine immunodiagnostic assays. Since A. costaricensis and A. cantonensis share common antigens, sera from infected persons will recognize antigens from either species. In addition to their potential use in angiostrongyliasis diagnosis, characterization of these proteins that establish the host-parasite interphase would improve our understanding of the biology of these parasites. The main objective of the present work was to characterize A. cantonensis excretory-secretory (ES) products by analyzing ES preparations by two-dimensional gel electrophoresis coupled with immunoblotting using pools of positive sera (PS) and sera from healthy individuals (SC). Protein spots recognized by PS were excised and analyzed by electrospray ionization (ESI) mass spectrometry. MASCOT analysis of mass spectrometry data identified 17 proteins: aldolase; CBR-PYP-1 protein; beta-amylase; heat shock protein 70; proteosome subunit beta type-1; actin A3; peroxiredoxin; serine carboxypeptidase; protein disulfide isomerase 1; fructose-bisphosphate aldolase 2; aspartyl protease inhibitor; lectin-5; hypothetical protein F01F1.12; cathepsin B-like cysteine proteinase 1; hemoglobinase-type cysteine proteinase; putative ferritin protein 2; and a hypothetical protein. Molecular cloning of these respective targets will next be carried out to develop a panel of Angiostrongylus antigens that can be used for diagnostic purposes and to further study host-Angiostrongylus interactions.

Development of a Molecular Marker Based on the Mitochondrial Genome for Detection of Cyclospora cayetanensis in Food and Water Samples.

Cyclospora cayetanensis is a coccidian parasite that causes diarrheal illness outbreaks worldwide. The development of new laboratory methods for detection of C. cayetanensis is of critical importance because of the high potential for environmental samples to be contaminated with a myriad of microorganisms, adversely impacting the specificity when testing samples from various sources using a single molecular assay. In this study, a new sequencing-based method was designed targeting a specific fragment of C. cayetanensis cytochrome oxidase gene and developed as a complementary method to the TaqMan qPCR present in the U.S. FDA BAM Chapter 19b and Chapter 19c. The comparative results between the new PCR protocol and the qPCR for detection of C. cayetanensis in food and water samples provided similar results in both matrices with the same seeding level. The target region and primers in the protocol discussed in this study contain sufficient Cyclospora-specific sequence fidelity as observed by sequence comparison with other Eimeriidae species. The sequence of the PCR product appears to represent a robust target for identifying C. cayetanensis on samples from different sources. Such a sensitive method for detection of C. cayetanensis would add to the target repertoire of qPCR-based screening strategies for food and water samples.

Identification of Leishmania spp. by molecular amplification and DNA sequencing analysis of a fragment of rRNA internal transcribed spacer 2.

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.