Cryptosporidium species and subtypes identified in human domestic cases through the national microbiological surveillance programme in Sweden from 2018 to 2022.

BACKGROUND: The intestinal protozoan parasite Cryptosporidium is an important cause of diarrheal disease worldwide. A national microbiological surveillance programme was implemented in Sweden in 2018 in order to increase knowledge of the molecular epidemiology of human cryptosporidiosis to better understand transmission patterns and potential zoonotic sources. This article summarises the results of the first five years of the surveillance programme. METHODS: Cryptosporidium-positive faecal and DNA samples from domestically acquired infections were collected from clinical microbiological laboratories in Sweden. Species and subtype determination was performed using 60 kDa glycoprotein and/or small subunit ribosomal RNA gene analysis. RESULTS: Between 2018 and 2022, 1654 samples were analysed and 11 different species were identified: C. parvum (n = 1412), C. mortiferum (n = 59), C. hominis (n = 56), C. erinacei (n = 11), C. cuniculus (n = 5), C. meleagridis (n = 3), C. equi (n = 2), C. ubiquitum (n = 2), and one each of C. canis, C. ditrichi and C. felis. Subtyping revealed seven subtype families of C. parvum (new subtype families IIy and IIz) and 69 different subtypes (11 new subtypes). The most common C. parvum subtypes were IIdA22G1c, IIdA24G1, IIdA15G2R1 and IIaA16G1R1b. For C. hominis, four different subtype families and nine different subtypes (two new subtypes) were identified. For additional species, two new subtype families (IIIk and VId) and nine new subtypes were identified. All successfully subtyped C. mortiferum cases were subtype XIVaA20G2T1, confirming previous findings in Sweden. Several outbreaks were identified of which the majority were foodborne and a few were due to direct contact with infected animals. CONCLUSION: Infection with C. parvum is the leading cause of human cryptosporidiosis acquired in Sweden, where more than 90% of domestic cases are caused by this zoonotic species and only a small proportion of cases are due to infection with other species. The rodent-associated C. mortiferum is considered an emerging zoonotic species in Sweden and the number of domestically acquired human cases has surpassed that of infection with C. hominis. A high diversity of species and subtypes, as well as diversity within the same subtype, was detected. Also, cryptosporidiosis appears to affect adults to a great extent in Sweden.

Protozoan infections are under-recognized in Swedish patients with gastrointestinal symptoms.

In acute gastroenteritis (GE), identification of the infectious agent is important for patient management and surveillance. The prevalence of GE caused by protozoa may be underestimated in Swedish patients. The purpose was to compare the prevalence of E. histolytica, Cryptosporidium spp., G. intestinalis, and C. cayetanensis in samples from patients where the clinician had requested testing for gastrointestinal parasites only (n = 758) to where testing for bacterial GE only (n = 803) or where both parasite and bacterial testing (n = 1259) was requested and a healthy control group (n = 197). This prospective cohort study was conducted in Region Jönköping County, Sweden (October 2018-March 2019). Fecal samples were analyzed with microscopy and real-time PCR. Cryptosporidium spp. was detected in 16 patients in the bacterial GE group and in 13 in the both bacterial and parasite group; no cases were detected in the group were only parasite infection was suspected. C. cayetanensis was detected in two patients in the bacterial GE group. One case of E. histolytica was detected in the bacterial group and one in the both bacterial and parasite group. G. intestinalis was detected in 14 patients in the parasite only group, 12 in the both parasite and bacterial group, three in the bacterial GE group, and one in the control group. Diarrhea caused by protozoa, especially Cryptosporidium was under-recognized by clinicians and is likely more common than hitherto estimated in Sweden. A more symptom-based diagnostic algorithm may increase detection and knowledge about protozoan infections.

Optimization of routine microscopic and molecular detection of parasitic protozoa in SAF-fixed faecal samples in Sweden.

Background: Nucleic acid-based methods are increasingly used for screening of gastrointestinal parasites. Microscopy is still used and Swedish routine protocol consists of formalin ethyl-acetate concentration and do not include screening for trophozoites or Cryptosporidium spp. This study aimed to compare detection with the Swedish routine microscopy method to an extended method that includes screening for trophozoites and Cryptosporidium. Furthermore, we also developed a method for DNA recovery from SAF-fixed faecal samples and compared the real-time PCR detection of Giardia intestinalis, Dientamoeba fragilis, Cryptosporidium spp., Entamoeba histolytica and Entamoeba dispar from SAF-fixed and unpreserved faecal samples. PCR results were then compared with microscopy results.Methods: SAF-fixed and unpreserved faecal samples from 1000 patients at the Clinical microbiology laboratory in Region Jönköping County, Sweden, were included. Samples were analysed with routine formalin ethyl-acetate concentration, wet mounts from both concentrated and unconcentrated samples, Ziehl-Neelsen staining on patients with certain symptoms and real-time PCR.Results: We found a significant higher detection rate of parasites with the extended microscopy method compared to the Swedish routine microscopy method when SAF-fixed samples were used. The detection rate with real-time PCR in SAF-fixed samples was equal to the detection rate in unpreserved samples. There was no significant difference in detection comparing extended microscopy and real-time PCR.Conclusion: In conclusion, this study showed that the extended microscopy method increased detection of intestinal protozoa with detection of both trophozoites and Cryptosporidium spp. We also showed that SAF-fixative can be used for detection of parasite-DNA with real-time PCR.

Prevalence of Dientamoeba fragilis, Giardia duodenalis, Entamoeba histolytica/dispar, and Cryptosporidium spp in Da Nang, Vietnam, detected by a multiplex real-time PCR.

We surveyed the prevalence of Dientamoeba fragilis, Giardia duodenalis, Entamoeba histolytica, Entamoeba dispar, and Cryptosporidium spp in individuals with and without gastrointestinal (GI) symptoms residing in and around Da Nang city, Vietnam. Fecal samples were collected from children (n = 100) and adults (n = 80) with GI symptoms and from healthy individuals (n = 88) reporting no GI symptoms. Parasite detection was performed by multiplex real-time PCR. Overall, except for G. duodenalis, we found a low prevalence (<5%) of D. fragilis and E. dispar and no detection of E. histolytica and C. spp in all participants with GI symptoms. Specifically for D. fragilis this contrasts with findings in European populations of children with GI symptoms showing prevalence up to 73%. Moreover, our results indicate that the prevalence of G. duodenalis is higher in patients with GI symptoms compared to asymptomatic individuals and this difference is most obvious in young patients.

Evaluation of the BD Max Enteric Parasite Panel for Clinical Diagnostics.

We compared the performance of the BD Max enteric parasite panel to routine microscopy and an in-house PCR for the detection of Giardia intestinalis, Entamoeba histolytica, and Cryptosporidium spp. The enteric parasite panel showed good specificity for all targets and good sensitivity for E. histolytica and Cryptosporidium spp. Sensitivity for G. intestinalis with the BD Max enteric parasite panel was equivalent to that with microscopy.

Dientamoeba fragilis DNA detection in Enterobius vermicularis eggs.

Dientamoeba fragilis is an intestinal protozoan suspected of causing gastrointestinal symptoms, and its mode of transmission is unknown, although first described almost a century ago. A hypothesis is that Enterobius vermicularis is a vector for D. fragilis, and recently, D. fragilis DNA was detected within surface-sterilized eggs of E. vermicularis. Using real-time PCR, we detected D. fragilis DNA in 18 (85%) of 21 samples of E. vermicularis eggs collected from patients harbouring D. fragilis in faeces. This finding supports the hypothesis that E. vermicularis may have an important role in the transmission of D. fragilis.