Cryptosporidium-positive Samples Research Articles

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.

Source tracking of fecal contamination in Asian green mussels (Perna viridis) harvested in Manila Bay, Philippines by molecular detection and genotyping of Cryptosporidium spp.

Manila Bay, a multipurpose body of water located around Metro Manila, Philippines, is progressively deteriorating because of massive pollution. Reports have shown that the bay and its aquatic resources (i.e., seafood) are contaminated with fecal matter and enteric pathogens, posing a threat to public health and industry. This problem raises the need for a microbial source tracking methodology as a part of the rehabilitation efforts in the bay. Bivalve mollusks cultivated in water can serve as sentinel species to detect fecal pollution and can complement water monitoring. With the use of polymerase chain reaction and DNA sequence analysis, this study detected Cryptosporidium spp. in Asian green mussels (Perna viridis) cultivated and harvested in Manila Bay and sold in Bulungan Seafood Market, Parañaque, Philippines, from 2019 to 2021 with an overall occurrence of 8.77% (n = 57). The analysis of the 18S rDNA segment revealed three genotypes from Cryptosporidium-positive samples, namely, Cryptosporidium sp. rat genotype IV (60%), C. galli (20%), and C. meleagridis (20%). These findings suggest fecal pollution in bivalve cultivation sites coming from sewage, nonpoint, and agricultural sources. The presence of C. meleagridis, the third most common cause of human cryptosporidiosis, in mussels poses a threat to human health. Thus, there is a need to establish routine detection and source tracking of Cryptosporidium spp. in Manila Bay and to educate seafood consumers on food safety.

An Epidemiological Assessment of Cryptosporidium and Giardia spp. Infection in Pet Animals from Taiwan

Cryptosporidium spp. and Giardia duodenalis are enteric protozoan pathogens in humans. and animals. Companion animals infected with zoonotic species/assemblages are a matter of major public concern around the world. The objectives of the present study were to determine the prevalences of Cryptosporidium spp. and G. duodenalis infections and their co-infection statuses in dogs and cats living in Taiwan and to identify the species and assemblages. Fecal samples were collected from local animal shelters (n = 285) and a veterinary hospital (n = 108). Nested polymerase chain reaction (PCR) was performed using the SSU-rRNA, β-giardin, and glutamate dehydrogenase genes for Cryptosporidium spp. and G. duodenalis, respectively. Results showed that the overall prevalences of Cryptosporidium and G. duodenalis were 7.38% (29/393) and 10.69% (42/393). In addition, co-infection was detected in 1.02% (4/393) of all samples. Sample source, clinical sign, and breed may be risk factors that influence the infection rate. In Cryptosporidium-positive samples, C. canis and C. felis were detected most frequently. Although the canine-specific assemblages C and D (37/42) were dominant, the zoonotic human-specific assemblage A (1/42) was also found in Giardia-positive samples. Phylogenetic analysis revealed that most positive samples belonged to host-specific subtypes/assemblages, while some Cryptosporidium or Giardia-positive samples could be zoonotic. The findings suggested that pet animals could be a cause of zoonotic transmission, causing human cryptosporidiosis and giardiasis in Taiwan.

Molecular evaluation of Cryptosporidium spp. in sheep in southern Xinjiang, China.

Cryptosporidium spp. are diarrheagenic intestinal parasites with multiple hosts worldwide. A total of 1252 fresh fecal samples of sheep were collected from 10 large-scale farms in southern Xinjiang. Based on the small subunit ribosomal (SSU rRNA) gene of Cryptosporidium, 100 Cryptosporidium-positive samples (8.0%, 100/1252) were detected by PCR. Nine out of 10 farms were positive for Cryptosporidium, with the highest infection rate being 18.4% (23/125) on farm 9 in Qira. The infection rates of Cryptosporidium in pre-weaned lambs, weaned lambs, fattening sheep, and adult sheep were 20.3% (61/301), 10.3% (34/329), 0.9% (3/327), and 0.7% (2/295), respectively. Three Cryptosporidium species were identified, namely, C. xiaoi (n=61), C. parvum (n=22), and C. ubiquitum (n=17). Of them, C. xiaoi was detected on all positive farms and in different age groups of sheep. The subtypes of C. parvum and C. ubiquitum were identified by PCR at the 60 kDa glycoprotein (gp60) gene. Two C. parvum subtypes were identified: IIdA19G1 (n = 21) and IIdA15G1 (n = 1). One C. ubiquitum subtype was identified with XIIa (n = 17). These results indicated the common transmission and genetic diversity of Cryptosporidium in sheep in southern Xinjiang, and further investigations are needed on the zoonotic potential of C. parvum and C. ubiquitum in this region.

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

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. 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. 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.

Molecular evaluation of Cryptosporidium spp. among breeding calves of Lorestan province Western Iran.

Cryptosporidium spp. are opportunistic intestinal protozoans with global distribution and are of great importance as zoonotic protozoans are common to humans and domestic animals, including cattle and calves. Identification and detection of parasite species using precise methods including molecular methods can be an effective step in treating and controlling parasites. This study aimed to investigate the prevalence of Cryptosporidium among breeding calves of Khorramabad city, Lorestan province, Western Iran, using PCR. The faecal samples were taken from 181 healthy and diarrhoeal calves and after the Ziehl Neelsen Acid-fast staining and microscopic evaluation, the genomic DNA was extracted for molecular evaluations. To detect Cryptosporidium species, specific primers targeting the SAM-1 gene of Cryptosporidium and a commercial master mix were used for PCR. Out of 181 faecal samples of breeding calves in Khorramabad city, 9 samples (5%) were positive for Cryptosporidium spp. using the PCR method. Statistical analysis of the data showed that there was no significant statistical relationship between Cryptosporidium infection of the calves and variables of age, breed, type of water consumption, clinical signs of diarrhoea, and sampling location, while parasite infection had a significant relationship with calf gender so that all Cryptosporidium positive samples were from male calves (p ≤ 0.05). Considering the presence of Cryptosporidium infection, the region's traditional grazing system, and the close relationship between livestock and humans, there is a possibility of human infection in the region. So preventive measures such as periodic animal testing with sensitive and accurate diagnostic techniques including PCR, pharmacological treatment of livestock, water hygiene and the use of industrial grazing instead of traditional grazing to improve the hygiene of food consumed by livestock are recommended.

Subtyping of Cryptosporidium parvum Obtained from Humans and Calves in Van, Turkey.

We aimed to investigate the prevalence of Cryptosporidium species detected in humans and calves in the Van region of Turkey. A total of 150 patients, comprising 60 who were immunosup-pressed, 50 who were immunosuppressed and had diarrhea, and 40 who had only diarrhea, were enrolled in this study in the Department of Medical Parasitology, Van Yuzuncu Yıl University Faculty of Medicine, Turkey. Stool samples were taken from the rectums of a total of 50 calves that had 30 diarrhea and 20 that did not have diarrhea, from the stables and farms of 10 central villages of Van, Turkey. All samples were analyzed using modified acid-fast staining, immunochromatographic test, and PCR. Cryptosporidium positive samples were also subtyped. Only C. parvum subtypes were detected in all positive samples. C. parvum was detected in 30 (20%) of the 150 human stool samples, while it was detected in 5 (10%) of the 50 samples from the calves. The GP60 gene region was amplified and sent for sequence analysis to identify the C. parvum subtypes. As a result, C. parvum is found to be an active species that caused cryptosporidiosis is in the Van region. IIdA24G1 subtype of C. parvum were found in both human and calf. Therefore, due to the zoonotic feature of the C. parvum IIdA24G1 subtype, it has been shown that the calves in the region are a significant risk for humans.

Performance of diagnostic assays used to detect Cryptosporidium oocysts in faecal samples of cattle in Kuwait and genotyping of Cryptosporidium species

BackgroudCryptosporidium species are zoonotic protozoan parasites responsible for gastroenteritis in various animals and humans. The diagnosis of Cryptosporidium presents many challenges. This research attempted to match the diagnostic efficiency of the modified Ziehl–Neelsen technique (mZN), immunochromatographic assays (IC), and enzyme-linked immunosorbent assay (ELISA) for the detection of Cryptosporidium in faecal samples of cattle in Kuwait. In addition, polymerase chain reaction (PCR) was utilised to determine the predominant species infecting cattle in Kuwait and correlating the detected species with the results of different diagnostic tests used, the presence or absence of clinical signs, and the age group of the infected cattle.ResultsOf 400 analysed faecal samples, Cryptosporidium positive samples were 23%, 15.25%, and 14% using IC, ELISA, and mZN. IC had the highest sensitivity (74.07%), and mZN had the highest specificity (98.29%) using a composite reference standard (CRS) as a gold standard. The rapid IC test results in high false-positive results of cryptosporidiosis, whereas using mZN alone is insufficient to declare a negative faecal sample. Only 74.5% (35/47) of Cryptosporidium-positive samples by the three assays could be amplified by PCR. This study was the first to genotype Cryptosporidium in Kuwait. Cryptosporidium parvum (n = 26) was the dominant species detected from cattle samples, followed by C. andersoni (n = 6), C. bovis (n = 2), and C. raynae (n = 1). The findings showed a statistically relevant relationship between diarrhoea and the detection of Cryptosporidium spp. in faecal samples of cattle (p-value = 0.0003). Pre-weaned calves were the most vulnerable age group to Cryptosporidium spp. infection (p-value = 0.0007).ConclusionFor screening of Cryptosporidium infection in faecal samples, antigen detection or PCR methods combined with one of the microscopy techniques should be used. Cryptosporidium parvum was the prepoderant Cryptosporidium spp. recovered from cattle samples in Kuwait followed by C. andersoni. Cryptosporidium parvum is a significant risk factor for diarrhoea in pre-weaned calves. However, further study is needed as many other causes of diarrhoea in calves must be ruled out before a diagnosis of Cryptosporidium diarrhoea can be made.

Genotyping and subtyping of Cryptosporidium spp. and Giardia duodenalis isolates from two wild rodent species in Gansu Province, China

Cryptosporidium spp. and Giardia duodenalis are commonly detected intestinal protozoa species in humans and animals, contributing to global gastroenteritis spread. The present study examined the prevalence and zoonotic potential of Cryptosporidium spp. and G. duodenalis in Himalayan marmots and Alashan ground squirrels in China's Qinghai-Tibetan Plateau area (QTPA) for the first time. Four hundred ninety-eight intestinal content samples were collected from five counties of QTPA of Gansu province, China.All samples were examined for Cryptosporidium spp. and G. duodenalis by PCR amplification. The resultant data were statistically analyzed by chi-square, Fisher's test and Bonferroni correction using SPSS software 25. 0. Cryptosporidium positive samples were further subtyped through analysis of the 60-kDa glycoprotein (gp60) gene sequence. A total of 11 and 8 samples were positive for Cryptosporidium spp. and G. duodenalis, respectively. Prevalence of Cryptosporidium spp. and G. duodenalis were 2.5% (10/399) and 1.5% (6/399) in Himalayan marmots, 1.0% (1/99) and 2.0% (2/99) in Alashan ground squirrels, respectively. Sequence analysis confirmed the presence of C. rubeyi (n = 2), ground squirrel genotype II (n = 7), chipmunk genotype V (n = 1) and horse genotype (n = 1). The horse genotype was further subtyped as novel subtype VIbA10. G. duodenalis zoonotic assemblages A (n = 1), B (n = 6), E (n = 1) were identified in the present study. This is the first study to identify Cryptosporidium spp. and G. duodenalis in Himalayan marmots and Alashan ground squirrels, suggesting the potential zoonotic transmission of the two pathogens in QTPA.

Cross-sectional household transmission study of Cryptosporidium shows that C. hominis infections are a key risk factor for spread

BackgroundInfection with the Cryptosporidium parasite causes over 4000 cases of diagnosed illness (cryptosporidiosis) in England and Wales each year. The incidence of sporadic disease has not been sufficiently established, and how frequently this arises from contact with other infected people is not well documented. This project aimed to explore potential transmission in the home and attempt to identify asymptomatic infections, which might play a role in transmission. Risk factors and characteristics associated with spread of infection in the home were described including any differences between Cryptosporidium species.MethodsThe study identified cryptosporidiosis cases from North West England and Wales over a year and invited them and their household to take part. Each household was sent a study pack containing study information and a questionnaire, and stool sample kits to provide samples from consenting household members. Cryptosporidium-positive stool samples, identified by immunofluorescence microscopy, were characterised using molecular methods to help describe any patterns of transmission. Characteristics of households with and without additional cases were described, and compared using odds ratios (OR) and a multivariable logistic regression identified independent risk factors for household transmission. Data collection ran for one year, beginning in September 2018 with an initial pilot phase.ResultsWe enrolled 128 index cases and their households. Additional illness occurred in over a quarter of homes, each reporting an average of two additional cases. The majority of these were undiagnosed and unreported to surveillance. This burden was even greater in households where the index case was infected with C. hominis versus C. parvum, or the index case was under five years old, with mums and siblings most at risk of secondary infection. Only having an index case of C. hominis was independently associated with transmission in the multivariable model (OR 4.46; p = 0.01).ConclusionsCryptosporidium was a considerable burden in the home. At-risk homes were those where the index was less than five years old and/or infected with C. hominis. Of particular risk were female caregivers and siblings. Hygiene advice should be specifically directed here.This work provides evidence for humans as sources of C. hominis infection and that person-person is a key pathway. We recommend that all stools submitted for the investigation of gastrointestinal pathogens are tested for Cryptosporidium to better capture cases, inclusion of speciation data in routine surveillance, and the consideration of specific clinical advice on prevention for high-risk homes.

A survey of Cryptosporidium prevalence among birds in two zoos in China.

BackgroundCryptosporidiosis is an important zoonotic protozoan disease worldwide, but few studies on this disease have been performed in wild birds; thus, our knowledge of this disease is insufficient, even in zoo birds. Animals in zoos are possible zoonotic disease reservoirs, potentially resulting in zoonotic agent spillover to humans; accordingly, our understanding of such phenomena should be improved.MethodsA total of 263 fresh fecal samples from 43 avian species were randomly collected from the Beijing Zoo and Harbin North Forest Zoo and screened for the prevalence of Cryptosporidium by 18S rRNA gene sequencing. Cryptosporidium species were distinguished based on the combined results of phylogenetic tree and genetic distance analyses conducted with the inclusion of seven avian Cryptosporidium species and 13 avian Cryptosporidium genotypes. The genetic diversity of Cryptosporidium parvum among different hosts, including humans, cattle, dogs, and birds, and the genetic diversity of avian C. parvum among avian hosts in China, Iraq and Brazil were determined based on C. parvum 18S rRNA haplotypes.ResultsThe results of PCR targeting the 18S rRNA gene revealed that 1.9% (5/263) of the samples were Cryptosporidium-positive. Four of the five Cryptosporidium-positive samples originated from white cranes (Grus leucogeranus), and one originated from a flamingo (Phoenicopteridae). Avian C. parvum isolates, including the isolates examined in the present study, showed gene flow with other isolates from different types of hosts, including humans, cattle and dogs, indicating that zoo birds potentially pose zoonotic and pathogenic risks to humans and animals. Additionally, gene flow between avian C. parvum isolates from China and Brazil was detected.ConclusionsTo the best of our knowledge, our results demonstrate C. parvum infection in a flamingo (Phoenicopteridae) and white cranes (Grus leucogeranus) for the first time. The results of our study provide an important reference for understanding the host range, biological characteristics, and molecular epidemiology of C. parvum.

Molecular characterization of Cryptosporidium spp. obtained from fecal samples of immunosuppressed patients from Brazil

Background: Cryptosporidium spp. are pathogenic protozoans that play an important role in developing diseases in the elderly, children, and immunosuppressed individuals. Methods: The objective of this study was to detect and genetically characterize Cryptosporidium spp. in kidney transplanted patients (n = 97 samples; group 1) and immunosuppressed individuals from an outpatient clinic suspected of having Cryptosporidium infection (n = 53 samples; group 2). All fecal samples were analyzed by parasitological stool examination, immunochromatographic test, and real-time polymerase chain reaction (real-time PCR). Cryptosporidium-positive samples were tested using nested PCR for the gp60 gene, followed by sequencing for subtype determination. Results: Parasitological examination was negative in all Group 1, and positive in four Group 2 samples. Real-time PCR revealed Cryptosporidium in 13 samples: four in Group 1 (three C. hominis and one C. parvum) and nine in Group 2 (seven C. hominis, one C. parvum, and one mixed C. hominis/C. parvum). The immunochromatographic test was reactive in 11 samples (four in Group 1 and seven in Group 2). All 11 C. hominis isolates were identified as subtype IbA10G2 and one C. parvum as subtype IIbA15G2R1. All C. hominis belonged to subtype IbA10G2, which is recognized as the most prevalent and pathogenic subtype. Conclusions: This study showed, for the first time, that the presence of Cryptosporidium subtypes is considered more virulent in Brazilian transplanted kidney patients.

Genetic diversity within the 18S rRNA and actin locus of Cryptosporidium scrofarum (Apicomplexa: Cryptosporidiidae) infecting domestic pigs (Sus scrofa domesticus) of India

The genetic diversity was studied of Cryptosporidium scrofarum (syn Cryptosporidium pig genotype II) of domestic pigs (Sus scrofa domesticus) from Punjab, India. Nested PCR amplification targeting the 18S rRNA and actin gene loci from Cryptosporidium positive samples was carried out, and the amplicons were sequenced. Phylogenetic comparison of a partial 18S rRNA gene revealed that they were genetically most similar to C. scrofarum isolated from other parts of the world. However, comparison of sequences representing a fragment of the genomic actin locus identified a new genotype conserved within the isolates sampled from India but distinct from other published sequences, suggesting the presence of a different Indian genotype.

Megasphaera in the Stool Microbiota Is Negatively Associated With Diarrheal Cryptosporidiosis.

BackgroundThe protozoan parasites in the Cryptosporidium genus cause both acute diarrheal disease and subclinical (ie, nondiarrheal) disease. It is unclear if the microbiota can influence the manifestation of diarrhea during a Cryptosporidium infection.MethodsTo characterize the role of the gut microbiota in diarrheal cryptosporidiosis, the microbiome composition of both diarrheal and surveillance Cryptosporidium-positive fecal samples from 72 infants was evaluated using 16S ribosomal RNA gene sequencing. Additionally, the microbiome composition prior to infection was examined to test whether a preexisting microbiome profile could influence the Cryptosporidium infection phenotype.ResultsFecal microbiome composition was associated with diarrheal symptoms at 2 timepoints. Megasphaera was significantly less abundant in diarrheal samples compared with subclinical samples at the time of Cryptosporidium detection (log2 [fold change] = –4.3; P = 10–10) and prior to infection (log2 [fold change] = –2.0; P = 10–4); this assigned sequence variant was detected in 8 children who had diarrhea and 30 children without diarrhea. Random forest classification also identified Megasphaera abundance in the pre- and postexposure microbiota as predictive of a subclinical infection.ConclusionsMicrobiome composition broadly, and specifically low Megasphaera abundance, was associated with diarrheal symptoms prior to and at the time of Cryptosporidium detection. This observation suggests that the gut microenvironment may play a role in determining the severity of a Cryptosporidium infection.Clinical Trials Registration. NCT02764918.

High Diversity of Cryptosporidium Species and Subtypes Identified in Cryptosporidiosis Acquired in Sweden and Abroad.

The intestinal protozoan parasite Cryptosporidium is an important cause of diarrheal disease worldwide. The aim of this study was to expand the knowledge on the molecular epidemiology of human cryptosporidiosis in Sweden to better understand transmission patterns and potential zoonotic sources. Cryptosporidium-positive fecal samples were collected between January 2013 and December 2014 from 12 regional clinical microbiology laboratories in Sweden. Species and subtype determination was achieved using small subunit ribosomal RNA and 60 kDa glycoprotein gene analysis. Samples were available for 398 patients, of whom 250 (63%) and 138 (35%) had acquired the infection in Sweden and abroad, respectively. Species identification was successful for 95% (379/398) of the samples, revealing 12 species/genotypes: Cryptosporidium parvum (n = 299), C. hominis (n = 49), C. meleagridis (n = 8), C. cuniculus (n = 5), Cryptosporidium chipmunk genotype I (n = 5), C. felis (n = 4), C. erinacei (n = 2), C. ubiquitum (n = 2), and one each of C. suis, C. viatorum, C. ditrichi, and Cryptosporidium horse genotype. One patient was co-infected with C. parvum and C. hominis. Subtyping was successful for all species/genotypes, except for C. ditrichi, and revealed large diversity, with 29 subtype families (including 4 novel ones: C. parvum IIr, IIs, IIt, and Cryptosporidium horse genotype VIc) and 81 different subtypes. The most common subtype families were IIa (n = 164) and IId (n = 118) for C. parvum and Ib (n = 26) and Ia (n = 12) for C. hominis. Infections caused by the zoonotic C. parvum subtype families IIa and IId dominated both in patients infected in Sweden and abroad, while most C. hominis cases were travel-related. Infections caused by non-hominis and non-parvum species were quite common (8%) and equally represented in cases infected in Sweden and abroad.

Molecular Characterisation of Cryptosporidium spp. in Mozambican Children Younger than 5 Years Enrolled in a Matched Case-Control Study on the Aetiology of Diarrhoeal Disease.

Cryptosporidium is a leading cause of childhood diarrhoea and associated physical and cognitive impairment in low-resource settings. Cryptosporidium-positive faecal samples (n = 190) from children aged ≤ 5 years enrolled in the Global Enteric Multicenter Study (GEMS) in Mozambique detected by ELISA (11.5%, 430/3754) were successfully PCR-amplified and sequenced at the gp60 or ssu rRNA loci for species determination and genotyping. Three Cryptosporidium species including C. hominis (72.6%, 138/190), C. parvum (22.6%, 43/190), and C. meleagridis (4.2%, 8/190) were detected. Children ≤ 23 months were more exposed to Cryptosporidium spp. infections than older children. Both C. hominis and C. parvum were more prevalent among children with diarrhoeal disease compared to those children without it (47.6% vs. 33.3%, p = 0.007 and 23.7% vs. 11.8%, p = 0.014, respectively). A high intra-species genetic variability was observed within C. hominis (subtype families Ia, Ib, Id, Ie, and If) and C. parvum (subtype families IIb, IIc, IIe, and IIi) but not within C. meleagridis (subtype family IIIb). No association between Cryptosporidium species/genotypes and child’s age was demonstrated. The predominance of C. hominis and C. parvum IIc suggests that most of the Cryptosporidium infections were anthroponotically transmitted, although zoonotic transmission events also occurred at an unknown rate. The role of livestock, poultry, and other domestic animal species as sources of environmental contamination and human cryptosporidiosis should be investigated in further molecular epidemiological studies in Mozambique.

Veterinary Students Have a Higher Risk of Contracting Cryptosporidiosis when Calves with High Fecal Cryptosporidium Loads Are Used for Fetotomy Exercises.

An outbreak of cryptosporidiosis among veterinary students performing fetotomy exercises on euthanized calves took place in September 2018 in Denmark. A prospective cohort investigation was performed to identify risk factors and provide guidance for preventing outbreaks of cryptosporidiosis in this setting. Ninety-seven students attended the fetotomy exercises and completed a questionnaire about symptoms and potential risk behavior. Real-time PCR was used to detect Cryptosporidium spp. in stool samples from students and to quantify the fecal parasite load in the calves used for the exercises. gp60 subtyping was carried out for the Cryptosporidium-positive samples. Our case definition was based on participation in a fetotomy exercise, reported symptoms, and laboratory results. Eleven laboratory-confirmed or probable cases (11%) were identified in two outbreaks during the prospective study period, with attack rates of 4/10 (40%) and 7/9 (78%), respectively. The risk factors for cryptosporidiosis we identified were performing the exercise on a diarrheic calf, reporting visible fecal contamination on the personal protective equipment (PPE), and reporting problems with PPE during the exercise. Cryptosporidium parvum IIaA15G2R1 was detected in both cases and calves. A significantly higher proportion of the calves aged 7 days old and above were positive compared with younger calves. Furthermore, a high fecal Cryptosporidium load in a calf was associated with a higher probability of an outbreak among the students. Based on our results, using noninfected calves for the exercises, appropriate use of PPE, and thorough hand hygiene are recommended to reduce the risk of contracting cryptosporidiosis in connection with fetotomy exercises.IMPORTANCECryptosporidium spp. can cause severe diarrhea in infected individuals. Cryptosporidium parvum is zoonotic, and cattle are the main reservoir. In several countries, outbreaks of cryptosporidiosis have occurred in veterinary students after handling calves. We carried out a 1-year-long prospective study to investigate the occurrence of these recurrent cryptosporidiosis outbreaks in Denmark. Our investigation used a One Health approach and combined comprehensive epidemiological approaches and laboratory methods applied to both students and calves in the setting of the fetotomy exercises. Two outbreaks took place during the study period; additionally, we retrospectively identified two more suspected outbreaks prior to the study period. The results illustrated a high risk of contracting cryptosporidiosis among veterinary students in the setting of the fetotomy exercises, especially when using calves with high fecal Cryptosporidium loads. Our data can be used to inform future efforts to prevent transmission of Cryptosporidium parvum to students during fetotomy exercises.

The red fox (Vulpes vulpes) as a potential natural reservoir of human cryptosporidiosis by Cryptosporidium hominis in Northwest Spain.

Giardia duodenalis and Cryptosporidium spp. are ubiquitous intestinal protozoa that parasitize domestic and wild animals, as well as human beings. Due to their zoonotic potential, the objective of the present study was to determine the presence of these pathogens in the fox population (Vulpes vulpes) located in Northwest Spain. A total of 197 faecal samples from legally hunted foxes were collected in the autonomous region of Galicia. The presence of G. duodenalis and Cryptosporidium spp. was investigated by PCR-based methods amplifying the small subunit ribosomal RNA (ssu rRNA) gene of the parasites. Attempts to genotype obtained positive samples were subsequently conducted at the glutamate dehydrogenase (gdh) and β-giardin (bg) genes of G. duodenalis, and the 60kDa glycoprotein (gp60) gene of Cryptosporidium. Giardia duodenalis and Cryptosporidium spp. were identified in 19 (9.6%) and 12 (6.1%) of the investigated samples, respectively. However, five Cryptosporidium species were detected at the ssu rRNA locus: C. hominis (33.4%, 4/12), C. canis (25.0%, 3/12), C. parvum (16.7%, 2/12), C. ubiquitum (8.3%, 1/12) and C. suis (8.3%, 1/12). An additional Cryptosporidium-positive sample was identified at the genus level only. Typing and subtyping of Giardia- and Cryptosporidium-positive samples were unsuccessful. The detection of C. hominis in wild foxes indicates the probable overlapping of sylvatic and domestic cycles of this parasite in rural settings. Besides, this finding raises the question of whether red foxes may act as natural reservoirs of C. hominis. The detection of C. parvum and C. suis is suggestive of active transmission events between farm and wild animals, opening up the possibility of transmission to human beings.

Identifying human enteric parasitic infections in Greece, with focus on Giardia and Cryptosporidium

A study was conducted in two different areas in Greece to investigate the presence of intestinal human parasitic infections (targeting healthy and individuals with diarrhoea). In total, 876 stool samples were collected from 822 adults and 54 children. Both sedimentation (acid/ether) and concentration/flotation techniques were performed in all samples to detect intestinal parasites. Additionally, a quantitative direct immunofluorescence assay was used specifically for the detection of Giardia and Cryptosporidium. PCR followed by sequencing was applied to genotype Giardia and Cryptosporidium positive samples. Thirty-five (4%) of the individuals examined harboured at least one species of intestinal parasite, the majority of which were protozoa (3.8%). The species found were Blastocystis hominis (1.8%), Giardia duodenalis (1.3%), Cryptosporidium spp. (0.6%), Entamoeba coli (0.2%) and E. histolytica/E. dispar (0.1%). Two persons were positive for Enterobius vermicularis. Genotyping results revealed the presence of G. duodenalis sub-assemblage AII, whereas sequencing was not successful for Cryptosporidium positive samples. A novel multi-locus genotype of G. duodenalis was identified, which has not been described in humans or animals previously. Overall, in the studied population, infection rates with intestinal parasites were low and similar to previous published data. As infection levels were low, no associations could be made between infection status and clinical relevance, risk factors or indication of potential sources of infection, apart from the fact that infections with Giardia were positively correlated to diarrhoea. Based on the parasite species and genotypes detected, there was no indication that animals were an important source of infection. Thus, it is suggested that Giardia infections were more likely to be acquired via human-to-human transmission, either involving indirect pathways such as contaminated food or water, or via direct contact.

Performance of three rapid diagnostic tests for the detection of Cryptosporidium spp. and Giardia duodenalis in children with severe acute malnutrition and diarrhoea

BackgroundThere is significant need for accurate diagnostic tools for Cryptosporidium spp. and Giardia duodenalis infections in resource limited countries where diarrhoeal disease caused by these parasites is often prevalent. The present study assessed the diagnostic performance of three commercially available rapid diagnostic tests (RDTs) based on faecal-antigen detection for Cryptosporidium spp. and/or G. duodenalis infections in stool samples of children admitted with severe acute malnutrition (SAM) and diarrhoea. An established multiplex PCR was used as reference test.MethodsStool samples from children with SAM and diarrhoea enrolled in a randomized controlled trial (registered at clinicaltrials.gov/ct2/show/NCT02246296) in Malawi (n = 175) and Kenya (n = 120) between December 2014 and December 2015 were analysed by a multiplex PCR for the presence of Cryptosporidium spp., G. duodenalis or Entamoeba histolytica parasite DNA. Cryptosporidium-positive samples were species typed using restriction fragment length polymorphism analysis. A sub-sample of the stool specimens (n = 236) was used for testing with three different RDTs. Diagnostic accuracy of the tests under evaluation was assessed using the results of PCR as reference standard using MedCalc software. Pearson Chi-square test and Fisher’s exact test were used to determine (significant) difference between the number of cryptosporidiosis or giardiasis cases found by PCR in Malawi and Kenya. The overall diagnostic accuracy of each RDT was calculated by plotting a receiver operating characteristic (ROC) curve for each test and to determine the area under the curve (AUC) using SPSS8 software.ResultsPrevalence of Cryptosporidium spp. by PCR was 20.0 and 21.7% in Malawi and Kenya respectively, mostly C. hominis. G. duodenalis prevalence was 23.4 and 5.8% in Malawi and Kenya respectively. E. histolytica was not detected by PCR. RDT testing followed the same pattern of prevalence. RDT sensitivities ranged for cryptosporidiosis from 42.9 to 76.9% and for G. duodenalis from 48.2 to 85.7%. RDT specificities ranged from 88.4 to 100% for Cryptosporidium spp. and from 91.2 to 99.2% for G. duodenalis infections. Based on the estimated area under the curve (AUC) values, all tests under evaluation had an acceptable overall diagnostic accuracy (> 0.7), with the exception of one RDT for Cryptosporidium spp. in Malawi.ConclusionsAll three RDTs for Cryptosporidium spp. and Giardia duodenalis evaluated in this study have a moderate sensitivity, but sufficient specificity. The main value of the RDTs is within their rapidness and their usefulness as screening assays in surveys for diarrhoea.