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.

[Prevalence and molecular detection of human Cryptosporidium infections: a review].

Cryptosporidium is an important intestinal parasite that is mainly transmitted through the fecal-oral route. Human infection may occur following ingestion of water and food contaminated by Cryptosporidium oocysts, and children and immunocompromised individuals are at a high risk of infections. The main symptoms of Cryptosporidium infections include diarrhea, vomiting, malnutrition, and even death. Because of high sensitivity and rapid procedures, molecular tests are helpful for the diagnosis of cryptosporidiosis and may reduce the public health risk of cryptosporidiosis. This review summarizes the advances in the latest prevalence and molecular detection of human Cryptosporidium infections during recent years.

Development of a "Rapid-Crypto Colorimetric LAMP Test" to Detect Cryptosporidiosis in Feces of Newborns Calves.

BACKGROUND: Cryptosporidiosis is a disease that causes major intestinal damage in humans and animals. The causative agents of the disease are Cryptosporidium species. In newborn calves, diarrhea can lead to death, resulting in significant economic losses for the farms. Therefore, accurate, rapid, and cost-effective diagnosis of the disease is very important. MATERIAL AND METHODS: In this study, a novel colorimetric loop-mediated isothermal amplification (LAMP) test named "Rapid-Crypto Colorimetric LAMP test" targeting Cryptosporidium spp. 18S rRNA gene was developed to detect cryptosporidiosis in the feces of newborn calves. The analytical sensitivity of the test was determined by plasmid controls. Clinical sensitivity was determined using the feces of 127 calves collected from farms in Izmir and Manisa provinces. All of the samples were also investigated with Real-Time PCR targeting the Cryptosporidium spp. COWP gene. Cross-reactivity was tested using the DNA of other parasites and bacteria. RESULTS: According to the results, the analytical sensitivity of the "Rapid-Crypto Colorimetric LAMP test" was found as 1 copy plasmid/reaction. When the results were compared with the Real-Time PCR test, the sensitivity of the "Rapid-Crypto Colorimetric LAMP test" was 100% and the specificity was 97.4%. The test did not cross-react with other parasites and bacteria.   CONCLUSION: The "Rapid-Crypto Colorimetric LAMP test" developed in this study provides an advantage in the diagnosis of Cryptosporidium spp. in calf stool samples since it can be applied in basic laboratories or in the field, does not require experienced personnel, and has high sensitivity. Moreover, diagnosis can be made with the naked eye without using any device.

Increased number of cryptosporidiosis cases with travel history to Croatia might be related to swimming pools, Germany, 2023.

In August and September 2023, an unusually high number of cryptosporidiosis cases identified by routine German surveillance had travelled to Croatia (n = 23). Nine cases had stayed in the same camping resort and seven further cases had stayed at other camping sites within 15 km. Based on our standardised questionnaires, the most likely source of infection was swimming pools (93%). Further environmental investigations on site might reveal potential common sources of contamination that could be targeted by control measures.

Diagnosis of gastrointestinal infections: comparison between traditional microbiology and a commercial syndromic molecular-based panel.

Traditional diagnosis of infectious gastroenteritis is based on culture, microscopy and antigen detection. The development of gastrointestinal syndromic panels based on molecular techniques have allowed rapid and simultaneous identification of multiple pathogens. The objective was to evaluate the implementation of Allplex™ Gastrointestinal Panel Assays (AGPA): Allplex™ GI-Virus, Allplex™ GI-Bacteria (I) and Allplex™ GI-Parasite by comparing with traditional diagnosis. A retrospective comparative study was conducted at Hospital Universitario La Paz, between the first year of implementation of the AGPA (April 1, 2018 to March 31, 2019) and the results obtained during the previous year with traditional methods (April 1, 2017 to March 31, 2018). With the implementation of AGPA we obtained an increase in the detection of rotavirus and adenovirus, being statistically significant for rotavirus ([CI95%:3.60-6.79]; P < 0.05) and an increase in the positivity rates of all the bacteria tested, with the exception of Salmonella spp. ([CI95%:3.60-6.79]; P < 0.05). Comparing the bacteria recovered by culture, we obtained an increase in the case of Shigella spp. cultivation during the AGPA period. Regarding protozoa, we achieved a significant increase in the positivity rates for Cryptosporidium spp. ([CI95%:1.98-3.01] P < 0.05), Giardia intestinalis ([CI95%:3.94-5.25]; P < 0.05) and Blastocystis spp. ([CI95%:9.44-11.36]; P < 0.05). There was an improvement in report turnaround time when comparing molecular diagnosis to bacterial culture and concentration plus microscopy for parasites; but not compared with antigen detection. The molecular diagnosis approach with AGPA were more sensitive and had a faster turnaround time for some targets, and in our setting, enabled an increased diagnostic capacity for viruses and protozoa.

Common Intestinal Parasites.

Parasites are a source of significant illness worldwide. In the United States, giardiasis, cryptosporidiosis, cyclosporiasis, and trichinellosis are nationally notifiable conditions. Pinworm, the most common intestinal parasite in children, is not a locally notifiable infection. Intestinal parasites have a wide range of acute and chronic symptoms but should be suspected in those who present with diarrhea lasting more than seven days. Infections most often occur through a fecal-oral route. Symptoms tend to be worse for children, older adults, or immunocompromised individuals. To diagnose Giardia infection, stool microscopy with direct fluorescent antibody testing is recommended; metronidazole, nitazoxanide, or tinidazole is used for treatment. Microscopy with immunofluorescence is sensitive and specific for diagnosing Cryptosporidium infection. This infection is often self-resolving, but treatment with nitazoxanide is effective for symptoms lasting more than two weeks. Microscopy or polymerase chain reaction assays are recommended to diagnose Cyclospora infections, and sulfamethoxazole/trimethoprim may be used to treat patients with persistent diarrhea. Trichinella infection is diagnosed by serum antibody testing, and severe symptoms are treated with albendazole in patients older than one year. Pinworm infections are diagnosed visually or by a tape test or paddle test; albendazole and pyrantel pamoate are both effective treatments.

Preliminary investigation of a significant national Cryptosporidium exceedance in the United Kingdom, August 2023 and ongoing.

Routine laboratory surveillance has identified an unprecedented and ongoing exceedance of Cryptosporidium spp. across the United Kingdom, notably driven by C. hominis transmission, since 14 August 2023. Information from 477 reported cases in England and Wales, followed up with a standardised exposure questionnaire as of 25 September 2023, identified foreign travel in 250 (54%) of 463 respondents and swimming in 234 (66%) of 353 cases. A significant, common exposure has not yet been identified in first analyses.

Examination of ELISA against PCR for assessing treatment efficacy against Cryptosporidium in a clinical trial context.

BACKGROUND: Cryptosporidium is a gastrointestinal pathogen that presents a serious opportunistic infection in immunocompromised individuals including those living with human immunodeficiency syndrome. The CRYPTOFAZ trial, previously published, was conducted in Malawi to evaluate the efficacy of clofazimine in response to an unmet need for drugs to treat cryptosporidiosis in HIV populations. A combination of rapid diagnostic tests, ELISA, qPCR, and conventional sequencing were employed to detect Cryptosporidium in 586 individuals during pre-screening and monitor oocyst shedding and identify enteric co-pathogens in 22 enrolled/randomized participants during the in-patient period and follow-up visits. METHODOLOGY: Oocyst shedding as measured by qPCR was used to determine primary trial outcomes, however pathogen was detected even at trial days 41-55 in individuals randomized to either clofazimine or placebo arms of the study. Therefore, in this work we re-examine the trial outcomes and conclusions in light of data from the other diagnostics, particularly ELISA. ELISA data was normalized between experiments prior to comparison to qPCR. The amount of all identified enteric pathogens was examined to determine if co-pathogens other than Cryptosporidium were major causative agents to a participant's diarrhea. CONCLUSION: ELISA had higher sample-to-sample variability and proved to be equally or less sensitive than qPCR in detecting Cryptosporidium positive samples. Compared to qPCR, ELISA had equal or greater specificity in detecting Cryptosporidium negative samples. Sequencing identified several Cryptosporidium species including viatorum which has never been identified in Malawi and Southern Africa. In addition to Cryptosporidium, enterotoxigenic E. coli was also identified as a pathogen in diarrheagenic amounts in 4 out of 22 participants.

Investigating Cryptosporidium spp. Using Genomic, Proteomic and Transcriptomic Techniques: Current Progress and Future Directions.

Cryptosporidiosis is a widespread disease caused by the parasitic protozoan Cryptosporidium spp., which infects various vertebrate species, including humans. Once unknown as a gastroenteritis-causing agent, Cryptosporidium spp. is now recognized as a pathogen causing life-threatening disease, especially in immunocompromised individuals such as AIDS patients. Advances in diagnostic methods and increased awareness have led to a significant shift in the perception of Cryptosporidium spp. as a pathogen. Currently, genomic and proteomic studies play a main role in understanding the molecular biology of this complex-life-cycle parasite. Genomics has enabled the identification of numerous genes involved in the parasite's development and interaction with hosts. Proteomics has allowed for the identification of protein interactions, their function, structure, and cellular activity. The combination of these two approaches has significantly contributed to the development of new diagnostic tools, vaccines, and drugs for cryptosporidiosis. This review presents an overview of the significant achievements in Cryptosporidium research by utilizing genomics, proteomics, and transcriptomics approaches.

Respiratory cryptosporidiosis detected by commercial multiplex-PCR in immunosuppressed pediatric patients.

We report the off-label use of a commercial gastrointestinal protozoa multiplex-PCR panel for bronchoalveolar lavage samples, detecting respiratory cryptosporidiosis in 2 immunocompromised pediatric patients. We suggest that implying this readily available assay in cases in which systemic cryptosporidiosis is suspected, may widen our understanding regarding this rarely reported syndrome.

Biosensors as early warning detection systems for waterborne Cryptosporidium.

Waterborne disease is a global health threat contributing to a high burden of diarrhoeal disease, and growing evidence indicates a prospective increase in incidence coinciding with the profound effects of climate change. A major causative agent of gastrointestinal disease is Cryptosporidium, a protozoan waterborne parasite identified in over 70 countries. Cryptosporidium is a cause of high disease morbidity in children and the immunocompromised with limited treatment options for patients at risk of severe illness. The hardy nature of the organism leads to its persistence in various water sources, with certain water treatment procedures proving inefficient for its complete removal. While diagnostic methods for Cryptosporidium are well-defined in the clinical sphere, detection of Cryptosporidium in water sources remains suboptimal due to low dispersion of organisms in large sample volumes, lengthy processing times and high costs of equipment and reagents. A need for improvement exists to identify the organism as an emerging threat in domestic water systems, and the technological advantages that biosensors offer over current analytical methods may provide a preventative approach to outbreaks of Cryptosporidium. Biosensors are innovative, versatile and adaptable analytical tools that could provide highly sensitive, rapid, on-site analysis needed for Cryptosporidium detection in low-resource settings.

Treatment of Urticaria caused by severe cryptosporidiosis in a 17-month-old child - a case report.

BACKGROUND: Cryptosporidium is an intracellular protozoan that causes gastrointestinal symptoms in humans and animals. In immunocompromised patients and children under 5 years of age, the infection is severe and can be life-threatening due to severe diarrhea. CASE PRESENTATION: We report a case of urticaria associated with Cryptosporidium in a 17-month-old female Iranian child. The patient had moderate diarrhea (> 3 loose, watery stools but not more than 10 diarrhea stools in a day), weight loss, and acute urticarial (rash clears completely within 6 weeks). Since the child's father worked in livestock farming, the parasite may have been transferred from the cow or calve to the house and the child. Several Cryptosporidium oocysts were detected in the modified acid-fast staining of the child's stool sample. The patient was successfully treated with nitazoxanide (100 mg twice daily) and became negative for parasites three days after treatment and one week after discharge from the hospital. The child was observed to produce < 3 loose stools in the previous 24 h after 1-week post-treatment and after 6 months of follow-up. CONCLUSION: A number of parasites are associated with urticaria, but to our knowledge, there is no information on Cryptosporidium-induced urticaria. Therefore, our result may be evidence for the role of this parasite in the development of urticaria if other causes such as food allergies, autoimmune diseases and etc. don't role in urticaria.

Epidemiology of gastrointestinal infections: lessons learned from syndromic testing, Region Zealand, Denmark.

The aim of this study was to investigate the value of syndromic diagnostic testing for a better understanding of the epidemiology of gastrointestinal infections in Denmark. Here we evaluated the QIAstat-Dx® Gastrointestinal (GI) Panel 1 assay on 18,610 fecal samples requested for analysis for enteric pathogens in Region Zealand, Denmark, in 1 year (October 1, 2021, to September 30, 2022). In total, 6905 (37%) samples were detected positive for one or more diarrhoeal bacteria, viruses, and protozoa. The most common bacterial, viral, and parasitic pathogens detected with the QIAstat-Dx® Gastrointestinal Panel 1 were EPEC (in patients ≥ 2 years of age) (n = 1420 (20.6%)), rotavirus (n = 948 (13.7%)), and Cryptosporidium spp. (n = 196 (2.84%)). We identified a large diversity in infections likely reflecting substantial differences in the epidemiology including origin of infections, mode of transmission, seasonality, age-dependent susceptibility to disease, severity, and travel history. All pathogens were detected as both single and coinfections. Viral infections peaked in March with a positive rate of 31.6%, and bacterial infections peaked in August with a positive rate of 35.3%. ETEC, Shigella/EIEC, EAEC, and P. shigelloides were most related to travel activity, and coinfections were frequent. The distribution of Ct values varied significantly between the pathogens, with the lowest Ct values (median 17-18) observed in astrovirus, adenovirus, and rotavirus. Our results highlight the value of providing extensive diagnostic testing on fecal samples for sufficient detection of relevant diarrhoeal pathogens for optimal clinical care.

Cryptosporidium infection and associated risk factors among children from rural areas in Peru: a nested case-control study.

This study aimed to detect the frequency of Cryptosporidium infection and associated risk factors among children from rural areas in Peru. A case-control study was conducted, nested in a cohort in two rural communities that included children between 6 and 13 months who were followed for 6 months. Cases were children whose fecal samples tested positive for Cryptosporidium infection using an immunochromatography test. Logistic regression was used to calculate odds ratios (ORs) and 95% confidence intervals (CIs) to analyze risk factors associated with Cryptosporidium infection. Among 72 children, 13 (18%) were cases. Cryptosporidium infection was associated with below secondary education of the mother (OR 7.62, 95% CI 1.50-36.72) and with having more siblings living at home (OR 1.71, 95% CI 1.04-2.82). An important frequency of Cryptosporidium infection among children from rural areas in Peru was reported, more research is needed to understand its true burden and risk factors among children in Peru.

Successful management of life-threatening post-COVID-19 cryptosporidiosis in a renal transplant patient: a case report.

Worldwide, Cryptosporidium spp. is a common parasite that affects domestic and wild animals, including humans, and causes diarrhea in both immunocompetent and immunocompromised hosts. The fecal-oral pathway accounts for the majority of its transfer. Although C. parvum and C. hominis are the most common zoonotic species in humans, other zoonotic species can also infect immunocompetent and immunocompromised people. Patients undergoing renal transplants are more likely to contract cryptosporidiosis, which can cause severe and potentially fatal diarrhea. A 41-year-old male patient who presented to the emergency department complained of a sudden onset, severe and continuous fatigue, and a feverish sensation of two-day duration. Two days prior to the current admission, the patient started to complain of weakness affecting his whole body, as well as a fever of 39°C and continuous yellowish diarrhea occurring 4-5 times daily without blood. Stool analysis revealed a cryptosporidium infection. The patient underwent surgery for kidney transplantation. The donated kidney was the left one from his brother and was attached to the patient´s right groin. As illustrated by our example, cryptosporidiosis should be considered a significant cause of acute, persistent, watery diarrhea in immunocompromised kidney transplant recipients. Patients undergoing renal transplants should be instructed to wash their hands frequently, stay away from young animals, sick people, and swimming pools in order to lower their risk of infection.

An end-point multiplex PCR/reverse transcription-PCR for detection of five agents of bovine neonatal diarrhea.

Neonatal calf diarrhea (NCD) is frequently associated with single or mixed viral, bacterial and/or protozoal infections. Consequently, laboratory diagnostic of NCD usually requires specific tests for each potential agent; a time-consuming, laborious and expensive process. Herein, we describe an end-point multiplex PCR/reverse transcription-PCR (RT-PCR) for detection of five major NCD agents: bovine rotavirus (BRV), bovine coronavirus (BCoV), Escherichia coli K99 (E. coli K99), Salmonella enterica (S. enterica) and Cryptosporidium parvum (C. parvum). Initially, we selected and/or designed high-coverage primers. Subsequently, we optimized multiplex PCR/RT-PCR conditions. Next, we evaluated the analytical sensitivity of the assay and assessed the performance of the reaction by testing 95 samples of diarrheic calf feces. The analytical specificity was evaluated against bovine viral diarrhea virus (BVDV), E. coli heat-stable enterotoxin (STa) and Eimeria spp. The detection limit of our assay was about 10 infectious units of BRV, 10-2 dilution of a BCoV positive sample pool, about 5 × 10-4 CFU for S. enterica, 5 × 10-6 CFU for E. coli K99 and 50 oocysts for C. parvum. No non-specific amplification of other bovine diarrhea agents was detected. Out of 95 samples analyzed, 50 were positive for at least one target, being 35 single and 15 mixed infections. BRV was the most frequent agent detected in single infections (16/35), followed by Cryptosporidium spp. (11/35), which was the most frequent in mixed infections (11/15). Positive and negative multiplex results were confirmed in individual reactions. In conclusion, we described an end-point multiplex PCR/RT-PCR for faster and easier NCD diagnosis, which may be useful for routine diagnosis and surveillance studies.

Giardia duodenalis in Ugandan Children Aged 9-36 Months in Kampala, Uganda: Prevalence and Associated Factors.

Giardia duodenalis is a common gastrointestinal pathogen globally that has been associated with growth failure in children. Most of the studies have been done in school-age children, and there is a paucity of data in pre-school children. We determined the prevalence and factors associated with G. duodenalis infection in children aged 9-36 months presenting to Mulago Hospital with diarrhea or cough. Demographic and socio-economic characteristics, animal ownership, medical history, and physical examination findings were recorded. Stool was tested for G. duodenalis using real-time quantitative polymerase chain reaction (qPCR), and additional tests included stool microscopy and qPCR for Cryptosporidium. The overall prevalence of G. duodenalis infection was 6.7% (214/3,173). In children with diarrhea the prevalence was 6.9% (133/1,923), whereas it was 6.5% (81/1,250) in those with cough as the main symptom. Of 214 children with G. duodenalis infection, 19 (8.9%) were co-infected with Cryptosporidium. Older children (25-36 months) were more likely to have G. duodenalis infection (adjusted odds ratio [aOR]: 2.93, 95% CI: 1.93-4.43). Use of an unimproved toilet (aOR: 1.38, 95% CI: 1.04-1.83) and the wet season (aOR: 1.33, 95% CI: 1.00-1.77) were associated with increased infection. Other factors associated with infection were recurrent diarrhea (aOR: 2.46, 95% CI: 1.64-3.70) and passing of mucoid stool (aOR: 2.25, 95% CI: 1.08-4.66). Having a ruminant at the homestead was also associated with infection (aOR: 1.83, 95% CI: 1.20-2.79). Giardia duodenalis infection occurred in 1 of 15 children aged 9-36 months with diarrhea or cough in Kampala, Uganda. Further studies are needed to clarify the zoonotic significance of G. duodenalis infection in this setting.

Persisting symptoms after Cryptosporidium hominis outbreak: a 10-year follow-up from Östersund, Sweden.

In late 2010, an outbreak of Cryptosporidium hominis affected 27,000 inhabitants (45%) of Östersund, Sweden. Previous research shows that abdomen and joint symptoms commonly persist up to 5 years post-infection. It is unknown whether Cryptosporidium is associated with sequelae for a longer duration, how persisting symptoms present over time, and whether sequelae are associated with prolonged infection. In this prospective cohort study, a randomly selected cohort in Östersund was surveyed about cryptosporidiosis symptoms in 2011 (response rate 69.2%). A case was defined as a respondent reporting new diarrhoea episodes during the outbreak. Follow-up questionnaires were sent after 5 and 10 years. Logistic regressions were used to examine associations between case status and symptoms reported after 10 years, with results presented as adjusted odds ratios (aOR) with 95% confidence intervals. Consistency of symptoms and associations with case status and number of days with symptoms during outbreak were analysed using X2 and Mann-Whitney U tests. The response rate after 10 years was 74% (n = 538). Case status was associated with reporting symptoms, with aOR of ~3 for abdominal symptoms and ~2 for joint symptoms. Cases were more likely to report consistent symptoms. Cases with consistent abdominal symptoms at follow-up reported 9.2 days with symptoms during the outbreak (SD 8.1), compared to 6.6 days (SD 6.1) for cases reporting varying or no symptoms (p = 0.003). We conclude that cryptosporidiosis was associated with an up to threefold risk for reporting symptoms 10 years post-infection. Consistent symptoms were associated with prolonged infection.

Aptamer-Based Electrochemical Microfluidic Biosensor for the Detection of Cryptosporidium parvum.

Cryptosporidium parvum is a high-risk and opportunistic waterborne parasitic pathogen with highly infectious oocysts that can survive harsh environmental conditions for long periods. Current state-of-the-art methods are limited to lengthy imaging and antibody-based detection techniques that are slow, labor-intensive, and demand trained personnel. Therefore, the development of new sensing platforms for rapid and accurate identification at the point-of-care (POC) is essential to improve public health. Herein, we propose a novel electrochemical microfluidic aptasensor based on hierarchical 3D gold nano-/microislands (NMIs), functionalized with aptamers specific to C. parvum. We used aptamers as robust synthetic biorecognition elements with a remarkable ability to bind and discriminate among molecules to develop a highly selective biosensor. Also, the 3D gold NMIs feature a large active surface area that provides high sensitivity and a low limit of detection (LOD), especially when they are combined with aptamers,. The performance of the NMI aptasensor was assessed by testing the biosensor's ability to detect different concentrations of C. parvum oocysts spiked in different sample matrices, i.e., buffer, tap water, and stool, within 40 min detection time. The electrochemical measurements showed an acceptable LOD of 5 oocysts mL-1 in buffer medium, as well as 10 oocysts mL-1 in stool and tap water media, over a wide linear range of 10-100,000 oocysts mL-1. Moreover, the NMI aptasensor recognized C. parvum oocysts with high selectivity while exhibiting no significant cross-reactivity to other related coccidian parasites. The specific feasibility of the aptasensor was further demonstrated by the detection of the target C. parvum in patient stool samples. Our assay showed coherent results with microscopy and real-time quantitative polymerase chain reaction, achieving high sensitivity and specificity with a significant signal difference (p < 0.001). Therefore, the proposed microfluidic electrochemical biosensor platform could be a stepping stone for the development of rapid and accurate detection of parasites at the POC.

Cryptosporidiosis in India and the World: A Review.

Cryptosporidiosis is caused by infection with a coccidian parasite belonging to the genus Cryptosporidium. Initially, human cryptosporidiosis was believed to be caused only by one species, but since the advent of molecular studies, 15 more species have been discovered to cause this infection. Among them, Cryptosporidium hominis and Cryptosporidium parvum are the most common species involved. This mainly affects children and causes diarrhea in most cases. It is mainly diagnosed by microscopy, especially in low-middle-income countries. This review covers the epidemiology, life cycle, risk factors, clinical manifestations, different diagnostic methods and treatment of this disease.