• Acquired Immunodeficiency Syndrome:highly active antiretroviral therapy
• Acute kidney injury
• Cryptosporidium
• Human immunodeficiency virus
• Hypokalemia;

• Adult
• Humans
• Male
• Acquired Immunodeficiency Syndrome/complications
• Acquired Immunodeficiency Syndrome/immunology
• Acute Kidney Injury/immunology
• Acute Kidney Injury/microbiology
• Cryptosporidiosis/complications
• Cryptosporidiosis/diagnosis
• Cryptosporidiosis/immunology
• Cryptosporidium/isolation & purification

• Cryptosporidium
• DNA extraction
• lysis
• nanoparticles

• Calf
• Cryptosporidium bovis
• Cryptosporidium parvum
• Portugal
• Subtype

• Humans
• Animals
• Cattle
• Cryptosporidium/genetics
• Portugal/epidemiology
• Cryptosporidiosis/epidemiology
• Cryptosporidium parvum/genetics
• Environment
• Oocysts
• Cattle Diseases/epidemiology

• Cryptosporidium ssp.
• Egypt
• Horse
• Prevalence
• Working equid

• Animals
• Cryptosporidiosis/epidemiology
• Cryptosporidium
• Egypt/epidemiology
• Prevalence
• Feces/parasitology

• Zagazig University

• dead and live oocysts
• duplex real-time PCR assay
• oocyst wall protein

Cryptosporidium 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.

Of 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).

For 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.

The online version contains supplementary material available at 10.1186/s12917-022-03435-w.

• Cattle
• Cryptosporidium spp.
• ELISA
• Immunochromatography
• Kuwait
• PCR
• mZN

• Biosensor
• Cryptosporidium detection
• Lab-on-chip
• Label-free detection
• Microfluidics
• Nanotechnology

• Cryptosporidiosis
• Cryptosporidium/isolation & purification
• Humans
• Water Quality
• Water Supply

• engineering
• mechanical engineering
• dna nanotechnology
• nanoscale devices
• nanoscale materials
• other nanotechnology
• techniques and instrumentation

• Biosensing Techniques/instrumentation
• Colorimetry/instrumentation
• Cryptosporidiosis/parasitology
• Cryptosporidium/genetics
• Cryptosporidium/isolation & purification
• Equipment Design
• Gold/chemistry
• Humans
• Lab-On-A-Chip Devices
• Limit of Detection
• Metal Nanoparticles/chemistry
• Nucleic Acid Hybridization
• Oligonucleotides/chemistry
• Oligonucleotides/genetics
• RNA, Protozoan/analysis
• RNA, Protozoan/genetics
• Smartphone/instrumentation

As qualified microscopy of enteric parasitoses as defined by high diagnostic accuracy is difficult to maintain in non-endemic areas due to scarce opportunities for practicing with positive sample materials, molecular diagnostic options provide less investigator-dependent alternatives. Here, we compared three molecular targets for the real-time PCR-based detection of Cryptosporidium spp. From a population of 1000 individuals comprising both Ghanaian HIV (human immunodeficiency virus) patients and military returnees after deployment in the tropics, stool samples were assessed for Cryptosporidium spp. by real-time PCR targeting the small subunit ribosomal RNA (SSU rRNA) gene, the Cryptosporidium oocyst wall (COWP) gene, and the DnaJ-like protein gene (DnaJ), respectively. In declining order, sensitivity of 100% for the SSU rRNA gene PCR, 90.0% for the COWP PCR and 88.8% for the DnaJ PCR, respectively, as well as specificity of 99.6% for the COWP PCR and 96.9% for both the SSU rRNA gene PCR and the DnaJ PCR, respectively, were recorded. Substantial agreement (kappa value 0.663) between the three assays was observed. Further, an accuracy-adjusted Cryptosporidium spp. prevalence of 6.0% was calculated for the study population. In conclusion, none of the assessed real-time PCR assays were associated with perfect test accuracy. However, a combination of highly sensitive SSU rRNA gene PCR for screening purposes and more specific COWP PCR for confirmatory testing should allow reliable diagnosis of Cryptosporidium spp. in stool samples even in low prevalence settings.

• Cryptosporidium spp.
• diagnostic accuracy
• latent class analysis
• real-time PCR
• sensitivity
• specificity
• test comparison

Due to the delicate nature of berries and the reduced shelf-life once washed, producers usually do not wash berries. Therefore, consumers are expected to wash the berries prior to consumption, and this might be a more effective way of infection prevention. However, the efficacy of consumer berry-washing procedures in removing the parasite contaminants from the berries surface has not been investigated. The aim of the present study was, therefore, to compare the efficacy of three different washing techniques in removing parasite contaminants. Three alternatives to washing berries before consumption were compared on berries artificially contaminated with oo/cysts of Cyclospora cayetanensis, Cryptosporidium parvum, and Giardia duodenalis. The results show that simple washing of berries under the cold tap for 1 min could remove on average at least 80% of the parasites, except for C. cayetanensis, which seems to be stickier than both G. duodenalis and C. parvum. The percent removal was slightly lower for raspberries as compared to blueberries. Although the differences are expected, a relevant result of the study is that washing contaminated berries prior to consumption by the consumer removes a considerable proportion of parasites and thereby lowers the risk of ingesting parasites’ transmission stages.

• Cryptosporidium
• Cyclospora
• Giardia
• berries
• contamination
• foodborne parasite
• removal
• washing

• Cryptosporidium
• France
• Lamb
• Sheep
• Zoonosis

• Density gradients
• Dispersion solution
• Soil contamination
• Sucrose flotation
• Toxoplasma gondii
• qPCR detection

Cryptosporidium is an obligate intracellular protist parasite infecting a wide range of vertebrate hosts and causes significant intestinal disease in both animals and humans, as some species are zoonotic. Cattle and especially calves have been identified as one of the most common reservoirs of this protist. However, little is known about the genetics of Cryptosporidium in calves in some regions of France. The aim of this study was to detect and isolate Cryptosporidium spp. in faecal samples from naturally infected pre-weaned calves (≤45 days-old) in France. A total of 35 diarrhoeic pre-weaned calf faecal samples were collected from 26 dairy cattle farms in six departments (French administrative provinces). Cryptosporidium presence was established by microscopically screening samples for oocystes with an immunofluorescent (DFA) staining method. DFA-positive samples were then analysed by PCR-RFLP and 18S rRNA gene sequencing to determine species. Cryptosporidium parvum-positive samples were subtyped via nested PCR analysis of a partial fragment of the 60 kDa glycoprotein (gp60) gene product. Data were then integrated into phylogenetic tree analysis. DFA revealed the presence of Cryptosporidium oocysts in 31 out of 35 (88%) samples. Combined with 18S rRNA gene analysis results, C. parvum was detected in 30 samples. Subtyping analysis in 27/30 samples (90%) of the C. parvum isolates revealed two zoonotic subtype families, IIa (24/27) and IId (3/27). Four subtypes were recognised within the subtype family IIa, including the hypertransmissible IIaA15G2R1 subtype that is the most frequently reported worldwide (21/27), IIaA17G3R1 (1/27), IIaA17G1R1 (1/27), and IIaA19G1R1 (1/27). Two subtypes were recognised within the IId subtype family including IIdA22G1 (2/27) and IIdA27G1 (1/27). These findings illustrate the high occurrence of Cryptosporidium in calves in dairy herds and increase the diversity of molecularly characterised C. parvum isolates with the first description of IIaA17G3R1, IIaA19G1R1, and IId subtypes in France. The presence of zoonotic C. parvum subtype families (IIa, IId) in this study suggests that pre-weaned calves are likely to be a significant reservoir of zoonotic C. parvum, and highlights the importance of animal to human cryptosporidiosis transmission risk. Further molecular studies in calves and small ruminants from other French regions are required to better understand the epidemiology of cryptosporidiosis in France.

•

Faecal samples from pre-weaned diarrheal calves were analysed

• 18S rRNA gene
• Calves
• Cryptosporidium parvum
• France
• PCR-RFLP
• Zoonotic parasite
• gp60 subtype

• Antigenic
• Cryptosporidium
• Detection
• Microscopy
• Molecular
• Preservation

• Cryptosporidium
• diagnosis
• prophylaxis
• risk management
• treatment
• vaccines

• ELISA
• PCR-based diagnostics
• clinical intestinal disorders
• diarrhea
• intestinal protozoa
• microbial pathogenesis
• microscopy
• point of care
• polymerase chain reaction

Diarrhea is a common complication in solid organ transplant (SOT) recipients and may be attributed to immunosuppressive drugs or infectious organisms such as bacteria, viruses or parasites. Cryptosporidium usually causes self-limited diarrhea in immunocompetent hosts. Although it is estimated that cryptosporidium is involved in about 12% of cases of infectious diarrhea in developing countries and causes approximately 748000 cases each year in the United States, it is still an under recognized and important cause of infectious diarrhea in SOT recipients. It may run a protracted course with severe diarrhea, fluid and electrolyte depletion and potential for organ failure. Although diagnostic methodologies have improved significantly, allowing for fast and accurate identification of the parasite, treatment of the disease is difficult because antiparasitic drugs have modest activity at best. Current management includes fluid and electrolyte replacement, reduction of immunosuppression and single therapy with Nitazoxanide or combination therapy with Nitazoxanide and other drugs. Future drug and vaccine development may add to the currently poor armamentarium to manage the disease. The current review highlights key epidemiological, diagnostic and management issues in the SOT population.

• Cryptosporidium
• Solid organ transplantation
• Diarrhea
• Nitazoxanide
• Antiparasitic drugs

• Cryptosporidium avian genotype V
• Cryptosporidium avium
• Molecular analyses
• Morphology
• Transmission studies

PCR detection of intestinal protozoa is often restrained by a poor DNA recovery or by inhibitors present in feces. The need for an extraction protocol that can overcome these obstacles is therefore clear. QIAamp® DNA Stool Mini Kit (Qiagen) was evaluated for its ability to recover DNA from oocysts/cysts directly from feces. Twenty-five Giardia-positive, 15 Cryptosporidium-positive, 15 Entamoeba histolytica-positive, and 45 protozoa-free samples were processed as control by microscopy and immunoassay tests. DNA extracts were amplified using 3 sets of published primers. Following the manufacturer's protocol, the kit showed sensitivity and specificity of 100% towards Giardia and Entamoeba. However, for Cryptosporidium, the sensitivity and specificity were 60% (9/15) and 100%, respectively. A series of optimization experiments involving various steps of the kit's protocol were conducted using Cryptosporidium-positive samples. The best DNA recoveries were gained by raising the lysis temperature to the boiling point for 10 min and the incubation time of the InhibitEX tablet to 5 min. Also, using a pre-cooled ethanol for nucleic acid precipitation and small elution volume (50-100 µl) were valuable. The sensitivity of the amended protocol to Cryptosporidium was raised to 100%. Cryptosporidium DNA was successfully amplified by either the first or the second primer set. When applied on parasite-free feces spiked with variable oocysts/cysts counts, ≈ 2 oocysts/cysts were theoretically enough for detection by PCR. To conclude, the Qiagen kit with the amended protocol was proved to be suitable for protozoan DNA extraction directly from feces and support PCR diagnosis.

• Cryptosporidium
• DNA extraction
• Entamoeba histolytica
• Giardia
• diagnostic PCR
• feces

• cryptosporidiosis
• cryptosporidium
• detection
• diagnosis
• typing
• subtyping

• Animals
• Animals, Suckling
• Child
• Cryptosporidiosis/diagnosis
• Cryptosporidiosis/parasitology
• Cryptosporidium/classification
• Cryptosporidium/genetics
• Cryptosporidium/isolation & purification
• DNA, Protozoan/genetics
• Environmental Microbiology
• Europe
• Humans
• Immunocompromised Host
• Life Cycle Stages
• Molecular Typing
• Public Health
• Species Specificity
• Time Factors
• Zoonoses