Diagnostic evaluation of nested PCR and microscopy for Cryptosporidiosis in goats: can COWP gene based qRT-PCR be useful in assessment of active Cryptosporidial infections?

The present research delved into the transmission patterns, diagnostic methods, molecular traits, and phylogenetic analysis of Cryptosporidium species. The research was undertaken to enhance comprehension of the epidemiology and the potential for zoonotic transmission. A total of 80 goat-kid samples were tested, 7 were confirmed positive by mZN microscopy and 12 by nested-PCR. By PCR, 18SSUrRNA, HSP70, and GP60 amplicons were tested for Cryptosporidium. The restriction enzymes viz., SspI, VspI and MboII were used to genotype 12 Cryptosporidium positive samples by which C. parvum and C. bovis mixed infections were detected. Quantitative reverse transcription real-time PCR was used to transcriptionally screen the COWP-subunit genes to assess the severity of the infection in goat-kids, which showed upregulation of COWP6 and COWP4, while COWP9 and COWP3 genes were downregulated. A silent mutation was found at the codon CCA→CCC, which is being reported for the first time in goat field isolates. Phylogenetic and sequencing analyses confirmed the presence of the anthropozoonotic IIe subtype.

Assessment of a Multiplex PCR for the Simultaneous Diagnosis of Intestinal Cryptosporidiosis and Microsporidiosis: Epidemiologic Report from a French Prospective Study.

Microsporidiosis and cryptosporidiosis are associated with chronic diarrhea in immunocompromised patients. The objectives of this study were to: i) assess a multiplex quantitative PCR assay targeting Cryptosporidium spp and the microsporidian Enterocytozoon bieneusi and Encephalitozoon spp, and ii) provide an update on the epidemiology of these pathogens. A prospective study was conducted from January 2017 to January 2019. Performance of the assay was assessed, and all cryptosporidia and microsporidia isolates were genotyped. The sensitivity of the multiplex PCR method reached 1 copy/µL for each targeted pathogen. The sensitivity of co-proantigen testing in the diagnosis of cryptosporidiosis was 73%. The sensitivity of microscopy in the diagnosis of cryptosporidiosis was 64%, and microsporidiosis, 50%. Among the 456 patients included, 14 were positive for Cryptosporidium spp (4 different species); 5, for E. bieneusi; and 2, for Encephalitozoon intestinalis. The overall prevalence of cryptosporidia was 3.1%, and of microsporidia, 1.5%; in kidney transplant recipients (n = 82), corresponding values were 7.3% and 2.4% (6 and 2 patients), respectively. Two cases of E. intestinalis infection were diagnosed in children who had traveled to the tropics. This study is the first to assess a multiplex quantitative PCR method for the simultaneous diagnosis of intestinal microsporidiosis and cryptosporidiosis. The highest prevalences of both pathogens were observed in kidney transplant recipients.

Hydrodynamic cavitation for the rapid separation and electrochemical detection of Cryptosporidium parvum and Escherichia coli O157:H7 in ground beef.

Foodborne illnesses are a major contributor to misery and health challenges in both rich and poor nations. Illnesses from pathogens such as Escherichia coli and Cryptosporidium parvum oocysts account for most of the cases of diarrhea in the world. Many standard methods exist for detecting these pathogens in water. However, these standard methods do not readily translate to the detection of the same pathogens in food. Detection techniques for pathogens in food are often inadequate, due to their inability to completely separate pathogens from food matrices. In this paper, we present a technique to separate and detect both Escherichia coli cells and Cryptosporidium parvum oocysts that have been embedded in ground meat. We achieve this objective by combining enzymatic digestion of the meat, hydrodynamic cavitation to disassemble pathogens from the meat, immunomagnetic separation to purify meat samples and indirect electrochemical detection of the target pathogens. Our use of hydrodynamic cavitation to separate pathogens is compared against an industry standard separation technique. Results indicate that the use of hydrodynamic cavitation amplifies the detection capabilities of our sensing technique and is overall comparable to or better than conventional stomacher sample preparation.

First detection of Cryptosporidium parvum in falcons (Falconiformes): Diagnosis, molecular sequencing, therapeutic trial and epidemiological assessment of a possible emerging disease in captive falcons.

Cryptosporidiosis in raptors and falcons is well-known to be caused by Cryptosporidium baileyi and associated mainly with respiratory pathology. This report presents the diagnosis of an atypical cryptosporidiosis event caused by Cryptosporidium parvum, that to the authors' knowledge, is a case observed for the first time in falcons. Two falcons (Gyrfalcon x Peregrine hybrids) were presented for annual check without any clinical signs. Hematology, biochemistry, fecal and crop parasitology, radiographic and endoscopic examinations were performed. Endoscopy revealed microcystic formation of the caudal lung field in the two falcons, adhesions and air sac alterations. Sampling and subsequent cytology revealed fungal spores and acid fast stain organisms (identified as Cryptosporidium spp.). Feces and affected lung tissue was further send for Cryptosporidium spp.-DNA detection. Fecal samples and lung tissue tested positive for Cryptosporidium spp. gp60 gene by PCR. By sequence analysis of the gp60 gene locus, diagnosis of C. parvum was confirmed with 100% homology. Despite the fact that falcons didn't recover after 1 month of therapy, eight months after the initial examination they were clinically healthy and had satisfactory flying performance. No other falcons were observed with C. parvum infections in the facility so far. The possible source, infection route and implications are discussed.

Sensitivity, specificity and comparison of three commercially available immunological tests in the diagnosis of Cryptosporidium species in animals

ABSTRACT The study was conducted to compare the specificity of immunological diagnostic methods used for the diagnosis of Cryptosporidium species capable of causing life-threatening infection in both immunosuppressed and immunocompetent patients. For the detection of Cryptosporidium species in 79 animals with diarrhoea, we used three Copro-antigen tests: RIDASCREEN® Cryptosporidium test, Cryptosporidium 2nd Generation (ELISA) and RIDA®QUICK Cryptosporidium. For immunoassays we used positive and negative samples detected by means of polymerase chain reaction and validated by sequencing and nested polymerase chain reaction to confirm the presence six different species of Cryptosporidium species. Prevalence of cryptosporidiosis in the entire group determined by enzyme immunoassay, enzyme linked immunosorbent assay, immuno-chromatographic test and polymerase chain reaction was 34.17%, 27.84%, 6.33% and 27.84%, respectively. Sensitivity of animal samples with enzyme immunoassay, enzyme linked immunosorbent assay, and immuno-chromatographic test was 63.6%, 40.9% and 22.7%, resp., when questionable samples were considered positive, whereas specificity of enzyme immunoassay, enzyme linked immunosorbent assay and immuno-chromatographic test was 75.9%, 78.9% and 100%, respectively. Positive predictive values and negative predictive values were different for all the tests. These differences results are controversial and therefore reliability and reproducibility of immunoassays as the only diagnostic method is questionable. The use of various Cryptosporidium species in diagnosis based on immunological testing and different results obtained by individual tests indicate potential differences in Copro-antigens produced by individual Cryptosporidium species.

Sensitivity, specificity and comparison of three commercially available immunological tests in the diagnosis of Cryptosporidium species in animals.

The study was conducted to compare the specificity of immunological diagnostic methods used for the diagnosis of Cryptosporidium species capable of causing life-threatening infection in both immunosuppressed and immunocompetent patients. For the detection of Cryptosporidium species in 79 animals with diarrhoea, we used three Copro-antigen tests: RIDASCREEN®Cryptosporidium test, Cryptosporidium 2nd Generation (ELISA) and RIDA®QUICK Cryptosporidium. For immunoassays we used positive and negative samples detected by means of polymerase chain reaction and validated by sequencing and nested polymerase chain reaction to confirm the presence six different species of Cryptosporidium species. Prevalence of cryptosporidiosis in the entire group determined by enzyme immunoassay, enzyme linked immunosorbent assay, immuno-chromatographic test and polymerase chain reaction was 34.17%, 27.84%, 6.33% and 27.84%, respectively. Sensitivity of animal samples with enzyme immunoassay, enzyme linked immunosorbent assay, and immuno-chromatographic test was 63.6%, 40.9% and 22.7%, resp., when questionable samples were considered positive, whereas specificity of enzyme immunoassay, enzyme linked immunosorbent assay and immuno-chromatographic test was 75.9%, 78.9% and 100%, respectively. Positive predictive values and negative predictive values were different for all the tests. These differences results are controversial and therefore reliability and reproducibility of immunoassays as the only diagnostic method is questionable. The use of various Cryptosporidium species in diagnosis based on immunological testing and different results obtained by individual tests indicate potential differences in Copro-antigens produced by individual Cryptosporidium species.

Economic Assessment of Waterborne Outbreak of Cryptosporidiosis.

In 2007, a waterborne outbreak of Cryptosporidium hominis infection occurred in western Ireland, resulting in 242 laboratory-confirmed cases and an uncertain number of unconfirmed cases. A boil water notice was in place for 158 days that affected 120,432 persons residing in the area, businesses, visitors, and commuters. This outbreak represented the largest outbreak of cryptosporidiosis in Ireland. The purpose of this study was to evaluate the cost of this outbreak. We adopted a societal perspective in estimating costs associated with the outbreak. Economic cost estimated was based on totaling direct and indirect costs incurred by public and private agencies. The cost of the outbreak was estimated based on 2007 figures. We estimate that the cost of the outbreak was >€19 million (≈€120,000/day of the outbreak). The US dollar equivalent based on today's exchange rates would be $22.44 million (≈$142,000/day of the outbreak). This study highlights the economic need for a safe drinking water supply.

Assessment of microscopic and molecular tools for the diagnosis and follow-up of cryptosporidiosis in patients at risk.

Cryptosporidiosis is an important though underreported public health concern. Molecular tools might be helpful in improving its diagnosis. In this study, ZR Fecal DNA MiniPrep™ Kit (ZR) and NucliSens® easyMAG® (EM) were compared using four Cryptosporidium-seeded feces and 29 Cryptosporidium-positive stools. Thereafter, ZR was selected for prospective evaluation of Cryptosporidium detection by 18S rDNA and LAXER quantitative PCR (qPCR) in 69 stools from 56 patients after Cryptosporidium detection by glycerin, modified Ziehl-Neelsen (ZN) and auramine-phenol (AP) stainings. The combination of any of the two extraction methods with 18S qPCR yielded adequate detection of Cryptosporidium in seeded stools, but the ZR kit showed the best performance. All 29 Cryptosporidium-positive samples were positive with 18S qPCR, after both ZR and EM extraction. However, false-negative results were found with LAXER qPCR or nested PCR. Cryptosporidiosis was diagnosed in 7/56 patients. All the microscopic methods enabled the initial diagnosis, but Cryptosporidium was detected in 12, 13, and 14 samples from these seven patients after glycerin, ZN, and AP staining respectively. Among these samples, 14 and 12 were positive with 18S and LAXER qPCR respectively. In two patients, Cryptosporidium DNA loads were found to be correlated with clinical evolution. Although little known, glycerin is a sensitive method for the initial detection of Cryptosporidium. When combined with 18S qPCR, ZR extraction, which had not been evaluated so far for Cryptosporidium, was an accurate tool for detecting Cryptosporidium and estimating the oocyst shedding in the course of infection.

Approaches to the detection of very small, common, and easily missed outbreaks that together contribute substantially to human Cryptosporidium infection.

Water supply-associated cryptosporidiosis outbreaks have decreased in England since the application of risk reduction measures to public water supplies. We hypothesized that smaller outbreaks were occurring which could be better detected by enhanced surveillance. Rolling analysis of detailed questionnaire data was applied prospectively in a population of 2·2 million in the south of England in 2009 and 2010. Detection of spatiotemporal clusters using SaTScan was later undertaken retrospectively. Together these approaches identified eight outbreaks, compared to an expectation of less than one based on national surveillance data. These outbreaks were small and associated with swimming pool use or, less commonly, direct (e.g. petting-farm) contact with animals. These findings suggest that frequent small-scale transmission in swimming pools is an important contributor to disease burden. Identification of swimming pool-level risk factors may inform preventative measures. These findings and the approaches described may be applicable to many other populations and to some other diseases.

Nucleic acid test to diagnose cryptosporidiosis: lab assessment in animal and patient specimens.

Diarrheal diseases cause more morbidity and mortality around the world than human immunodeficiency virus (HIV), malaria, or tuberculosis. Given that effective treatment of persistent diarrheal illness requires knowledge of the causative organism, diagnostic tests are of paramount importance. The protozoan parasites of the genus Cryptosporidium are increasingly recognized to be responsible for a significant portion of diarrhea morbidity. We present a novel nucleic acid test to detect the presence of Cryptosporidium species in DNA extracted from stool samples. The assay uses the isothermal amplification technique recombinase polymerase amplification (RPA) to amplify trace amounts of pathogen DNA extracted from stool to detectable levels in 30 min; products are then detected visually on simple lateral flow strips. The RPA-based Cryptosporidium assay (RPAC assay) was developed and optimized using DNA from human stool samples spiked with pathogen. It was then tested using DNA extracted from the stool of infected mice where it correctly identified the presence or absence of 27 out of 28 stool samples. It was finally tested using DNA extracted from the stool of infected patients where it correctly identified the presence or absence of 21 out of 21 stool samples. The assay was integrated into a foldable, paper and plastic device that enables DNA amplification with only the use of pipets, pipet tips, and a heater. The performance of the integrated assay is comparable to or better than polymerase chain reaction (PCR), without requiring the use of thermal cycling equipment. This platform can easily be adapted to detect DNA from multiple pathogens.

Burden of disease from cryptosporidiosis.

PURPOSE OF REVIEW: The global significance of cryptosporidiosis is widespread and far-reaching. In this review, we present recent data about strain diversity and the burden of disease, along with developments in therapeutic and preventive strategies. RECENT FINDINGS: Cryptosporidium is an emerging pathogen that disproportionately affects children in developing countries and immunocompromised individuals. Without a diagnostic tool amenable for use in developing countries, the burden of infection and its relationship to growth faltering, malnutrition, and diarrheal mortality remain underappreciated. Disease incidence is also increasing in industrialized countries largely as a result of outbreaks in recreational water facilities. Advances in molecular methods, including subtyping analysis, have yielded new insights into the epidemiology of cryptosporidiosis. However, without practical point-of-care diagnostics, an effective treatment for immunocompromised patients, and a promising vaccine candidate, the ability to reduce the burden of disease in the near future is limited. This is compounded by inadequate coverage with antiretroviral therapy in developing countries, the only current means of managing HIV-infected patients with cryptosporidiosis. SUMMARY: Cryptosporidiosis is one of the most important diarrheal pathogens affecting people worldwide. Effective methods to control and treat cryptosporidiosis among high-risk groups present an ongoing problem in need of attention.

[Assessment of two commercially available immunochromatographic assays for a rapid diagnosis of Giardia duodenalis and Cryptosporidium spp. in human fecal specimens]. / Evaluación de dos métodos inmunocromatográficos comerciales para el diagnóstico rápido de Giardia duodenalis y Cryptosporidium spp. en muestras de heces.

INTRODUCTION: To assess and compare the performance of two immunochromatographic tests for the simultaneous detection of Giardia duodenalis and Cryptosporidium spp. in faeces. MATERIALS AND METHODS: In this study 254 faeces samples were tested using the two immunochromatography strips Cryto-Giardia (CerTest Biotec) and Stick Crypto-Giardia (Operon). RESULTS: In the diagnosis of G. duodenalis, the sensitivity and specificity of the kits were 97% and 100%, respectively for the CerTest; and 97% and 95% for Operon. In the diagnosis of Cryptosporidium spp. Certest strip rendering a sensitivity of 100%, compared to with a sensitivity of 92% using Operon. There were no false positives using either technique. CONCLUSIONS: Both methods yielded good sensitivity and specificity values and are thus useful tools for a rapid diagnosis of G. duodenalis and Cryptosporidium spp. The benefits of immunochromatography methods are that there is no requirement for expert microscopists or special equipment.

Assessment of cryptodiag for diagnosis of cryptosporidiosis and genotyping Cryptosporidium species.

The performance of a new commercial PCR-enzyme-linked immunosorbent assay (ELISA) (Cryptodiag; Bio Advance, France) for the diagnosis of cryptosporidiosis and the identification of Cryptosporidium hominis and C. parvum from stool samples was examined. This test is based on PCR amplification of Cryptosporidium DNA extracted from stools, followed by an ELISA based on hybridization with Cryptosporidium sp.-, C. hominis-, or C. parvum-specific probes. In spiking experiments, approximately five oocysts were detected either in water or in stool suspensions while assessing for the efficient removal of stool PCR inhibitors. No cross-reactivity was observed in the detection of C. parvum and C. hominis using the respective specific probes. Thirty-three fecal samples from patients with microscopically proven cryptosporidiosis and 118 from patients with or without other digestive protozoan infections were tested by Cryptodiag, blinded to the results of microscopy. Compared to microscopy, the sensitivity of Cryptodiag was 97.0% (32/33) and 100% (33/33), including the gray zone, and specificity was 98.3% (116/118) and 96.6% (114/118), including the gray zone. Among 34 positive results, Cryptodiag identified 19 due to C. hominis, 8 due to C. parvum, and 7 due to Cryptosporidium spp. Genotyping by Cryptodiag agreed with reference typing methods in 85% of cases of C. parvum or C. hominis infections. Cryptodiag proved to be reliable and sensitive for the diagnosis of cryptosporidiosis. The use of specific probes allowed the identification of C. hominis and C. parvum, i.e., the two main species responsible for human cryptosporidiosis, and rapidly provided information on the possible source of infection.

Multiattribute evaluation of two simple tests for the detection of Cryptosporidium parvum in calf faeces.

There is a need for simple and inexpensive diagnostic and screening tests for the detection of Cryptosporidium parvum infection in calves. A sucrose wet mount test and a lateral immunochromatography test were evaluated for epidemiological sensitivity and specificity, cost per test, simplicity, test time and ease of batching. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) of the Cryptosporidium oocyst wall protein (COWP) gene locus, with gel electrophoresis, was used as a gold standard. Cohen's kappa statistic of agreement (kappa) between the Ontario Veterinary College (OVC) sucrose wet mount test and COWP PCR-RFLP was 0.82, and the sensitivity and specificity of the OVC sucrose wet mount test were 88.6% and 93.8%, respectively. The sensitivity and specificity of the lateral immunochromatography test were 78.3% and 93.3%, respectively, and agreement between this test and PCR-RFLP was good (kappa=0.73). There was substantial agreement between the OVC sucrose wet mount test and the lateral immunochromatography test (kappa=0.84). Both tests were inexpensive and easy to use; however, the lateral immunochromatography test was faster and simpler to perform than the sucrose wet mount test, and was generally more user-friendly. These tests provide practitioners and researchers with cheap, quick and accurate methods of detecting C. parvum infection in young calves.

Evaluation of the Triage Micro Parasite Panel for detection of Giardia lamblia, Entamoeba histolytica/Entamoeba dispar, and Cryptosporidium parvum in patient stool specimens.

A study comparing the Triage Micro Parasite Panel (Biosite Diagnostics, Inc., San Diego, Calif.) to conventional O&P examination (O&P) was performed using patient fecal specimens. Five hundred twenty-three stool samples were compared. Nineteen specimens were found to be positive by Triage, and 29 were found to be positive by O&P. Seven specimens were positive for Giardia lamblia, four were positive for Entamoeba histolytica/E. dispar, and three were positive for Cryptosporidium parvum as determined by both methods. There was one false positive by Triage (C. parvum) and four false negatives by O&P (two G. lamblia, one E. histolytica/E. dispar, and one C. parvum). The Triage test accurately detected all 18 specimens that contained one of the three organisms that it was designed to detect. The Triage test is a rapid, easy-to-use enzyme immunoassay for the detection of G. lamblia, E. histolytica/E. dispar, and C. parvum in fresh or fresh-frozen fecal specimens. These data suggest that the Triage test can be used as a screen for the immediate testing of stool specimens for these three pathogenic parasites. If Triage test results are negative, O&P can be performed if parasitic infections other than G. lamblia, E. histolytica/E. dispar, or C. parvum are suspected.

Disease reporting from an automated laboratory-based reporting system to a state health department via local county health departments.

OBJECTIVE: The authors assessed the completeness of disease reporting from a managed care organization's automated laboratory-based reporting system to the California Department of Health Services (CDHS) via local public health departments. METHODS: The authors identified all positive laboratory tests for 1997 from the computerized database of Kaiser Permanente Northern California for seven infections for which there are statutory reporting requirements: Campylobacter jejuni, Chlamydia trachomatis, Cryptosporidium parvum, hepatitis A, Neisseria meningitidis, Neisseria gonorrhoeae, and Salmonella (N = 7,331 reports). Cases were then matched by computer query to records of cases reported to CDHS. To determine why cases were not found in CDHS records, a sample of un-matched cases was searched at two county health departments. RESULTS: Overall, 84.5% (95% CI 83.4, 85.6) of the laboratory reports submitted with accompanying demographic information were successfully matched with cases in the CDHS disease surveillance database. Frequency of matching for specific diseases ranged from 79.4% (95% CI 75.6, 83.3) for N. gonorrhoeae to 88.4% (95% CI 85.3, 91.6) for C. jejuni. Reports were more likely to be matched when the county of residence was the same as the county of the health care facility. At the county level, reasons for failure of cases to be forwarded to CDHS included: errors due to manual data entry, failure to forward information from the county of diagnosis to the county of residence, and incorrect disease coding. CONCLUSION: Automated laboratory-based reporting is highly effective, but some data are lost with off-line transfer of information. To optimize surveillance accuracy and completeness, reporting at all levels should be done via direct electronic data transfer.

Malaria, intestinal parasites, and schistosomiasis among Barawan Somali refugees resettling to the United States: a strategy to reduce morbidity and decrease the risk of imported infections.

In 1997, enhanced health assessments were performed for 390 (10%) of approximately 4,000 Barawan refugees resettling to the United States. Of the refugees who received enhanced assessments, 26 (7%) had malaria parasitemia and 128 (38%) had intestinal parasites, while only 2 (2%) had Schistosoma haematobium eggs in the urine. Mass therapy for malaria (a single oral dose of 25 mg/kg of sulfadoxine-pyrimethamine) was given to all Barawan refugees 1-2 days before resettlement. Refugees >2 years of age and nonpregnant women received a single oral dose of 600 mg albendazole for intestinal parasite therapy. If mass therapy had not been provided, upon arrival in the United States an estimated 280 (7%) refugees would have had malaria infections and 1,500 (38%) would have had intestinal parasites. We conclude that enhanced health assessments provided rapid on-site assessment of parasite prevalence and helped decrease morbidity among Barawan refugees, as well as, the risk of imported infections.

Assessment of maternal immunity to Cryptosporidium baileyi in chickens.

The degree of protection to Cryptosporidium baileyi in the progeny of infected chickens was studied. Hens at the beginning of their laying period were given orally three consecutive, large doses of C. baileyi oocysts at weekly intervals. The infection became patent after 6 days and lasted for another 6 days. Increasing serum IgG, and serum, bile, lachrymal and salivary IgA were demonstrated from their samples. These immunoglobulins were transferred to the eggs, since high levels of maternally derived IgG and lower amount of IgA were present in their yolks. Hatchlings of infected hens were divided into uninfected (UY) and infected (IY) groups, the birds in the latter receiving an oral inoculum of C. baileyi oocysts on the first day of their life. Two other groups, progeny of uninfected hens served as controls (uninfected UC, and infected IC). Maternal IgG was detected in serum samples of UY hatchlings which was eliminated by the third week. The total oocyst shedding of IY chickens was 54.3% lower than that of the controls (IC), however, the prepatent and patent periods did not show significant difference. In spite of the partial protection observed in IY birds, their humoral immune response to C. baileyi was significantly lower when compared to IC. A dot-ELISA was developed to evaluate seroconversion of infected chickens which was 100% in both infected groups. The findings of the present study suggest that infection of hens with C. baileyi results in partial protection of their progeny to this parasite, and factors other than immunoglobulins may also be transferred via the eggs.