The epidemiology of sporadic human infections with unusual cryptosporidia detected during routine typing in England and Wales, 2000-2008

Routine typing of 14 469 isolates from human cryptosporidiosis cases between 2000 and 2008 revealed that 7439 (51·4%) were Cryptosporidium (C.) hominis, 6372 (44·0%) C. parvum, 51 (0·4%) both C. hominis and C. parvum, 443 (3·1%) were not typable and 164 (1·1%) were other Cryptosporidium species or genotypes. Of the latter, 109 were C. meleagridis, 38 C. felis, 11 C. ubiquitum, one C. canis, two horse, two novel and one skunk genotype. C. hominis monkey genotype and C. cuniculus were identified in a separate study. Patients with unusual infections were older than those with C. hominis (P<0·01) or C. parvum (P<0·01) and were more likely to be immunocompromised (Fisher's exact P<0·01). Forty-one percent of unusual cases had travelled abroad, mainly to the Indian subcontinent. Significant risk factors in those with unusual species were travel abroad (C. meleagridis, P<0·01), being immunocompromised (C. felis, Fisher's exact P=0·02), and contact with cats (C. felis, Fisher's exact P=0·02).     

Multilocus sequence typing and genetic structure of Cryptosporidium hominis from children in Kolkata, India.

Endemicity of cryptosporidiosis in India has been documented with little genetic characterization of the parasites. Fifty Cryptosporidium-positive specimens collected between 2001 and 2004 from pediatric patients in Kolkata, India were analyzed for parasite genetic structure using multilocus sequence typing (MLST). Genotype analyses showed the presence of Cryptosporidium hominis, Cryptosporidium meleagridis and Cryptosporidium felis in 49, 2 and 1 patients, respectively (two patients had mixed infections of C. hominis and C. meleagridis). To assess the extent of genetic heterogeneity of C. hominis, minisatellites, microsatellites and polymorphic markers in three different chromosomes were sequenced, including genes encoding the 60kDa glycoprotein (GP60), a 47kDa protein (CP47), a mucin-like protein (Mucin1), a serine repeat antigen (MSC6-7), and a 56kDa trans-membrane protein (CP56) in chromosome 6, the 70kDa heat shock protein (HSP70) in chromosome 2, and a T-rich gene fragment (Chrom3T) in chromosome 3. Population sub-structure of C. hominis based on multilocus gene sequences showed that there were 25 multilocus subtypes defined by combined sequence length and nucleotide polymorphism, which formed four distinct groups in this population. Significant intra- and inter-genic linkage disequilibria were observed with minimum recombination or expansion of limited subtypes, all indicative of a mostly clonal population structure. The results highlight the importance of high resolution MLST in studying Cryptosporidium population sub-structure especially when length polymorphism may be inadequate in identifying unique subtypes. The significance of the diverse MLST within C. hominis in relation to geographical and temporal factors and clinical manifestations of disease warrants further investigations.     

Cryptosporidium species and subtypes and clinical manifestations in children, Peru

To determine whether clinical manifestations are associated with genotypes or subtypes of Cryptosporidium spp., we studied a 4-year longitudinal birth cohort of 533 children in Peru. A total of 156 infection episodes were found in 109 children. Data from first infections showed that C. hominis was associated with diarrhea, nausea, vomiting, general malaise, and increased oocyst shedding intensity and duration. In contrast, C. parvum, C. meleagridis, C. canis, and C. felis were associated with diarrhea only. C. hominis subtype families were identified (Ia, Ib, Id, and Ie); all were associated with diarrhea. Ib was also associated with nausea, vomiting, and general malaise. All C. parvum specimens belonged to subtype family IIc. Analysis of risk factors did not show associations with specific Cryptosporidium spp. genotypes or subtypes. These findings strongly suggest that Cryptosporidium spp. and subtypes are linked to different clinical manifestations in children.     

Prevalence of Mycoplasma genitalium, Mycoplasma hominis and Chlamydia trachomatis among Danish patients requesting abortion

The aim of the study was to determine lower genital tract carriage rate of Mycoplasma genitalium (M. genitalium) and to compare it to the carriage rates of Mycoplasma hominis (M. hominis ) and Chlamydia trachomatis (C. trachomatis) among 102 women requesting termination of pregnancy at the Horsens Hospital in Denmark. Real-Time PCR was used for the detection of bacterial DNA, and the presence of antibodies to the three microorganisms was determined by ELISA and immunoblotting. Real-Time PCR detected M. genitalium in one swab sample (0.98%) only, while the prevalence of C. trachomatis was high (15.69%) and M. hominis colonization (18.63%) was similar to colonization observed among sexually experienced adults. There was a significant difference in prevalence of M. hominis infection in the different age groups. C. trachomatis load in the cervical samples was significantly higher among young patients. There was no correlation between the presence of genital infection with C. trachomatis and genital mycoplasmas and no correlation between the presence of antibodies to these bacteria. In conclusion, in Danish patients it is not necessary to test for M. genitalium before abortion since less than 1% were found positive. The prevalence of genital C. trachomatis infections was high among the abortion-seeking patients.     

河南省三株人源隐孢子虫分离株鉴定

目的隐孢子虫病是一种人兽共患寄生虫病,在艾滋病病人和儿童中的感染率可分别高达48％和17．5％,可导致严重的水样腹泻甚至危及生命,免疫功能正常者也能感染出现急性自限性腹泻。本文通过不同软件对三株人源隐孢子虫分离株进行种类鉴定。方法参照Xiao L H 1999年发表的资料合成隐孢子虫18S rDNA特异性引物(上游引物:5’-AACCTGGTTGATCCTGCCAGTAGTC -3’,下游引物:5’-TGAGCCTTCTGCAGGTTCACCTACG-3’,由大连宝生物工程技术公司合成。),FCR 扩增,产物测序、序列分别用Paup4．0、rhylip3．6、DNAstar4．0等软件分析并与12株隐孢子虫相应序列进行同源性分析,绘制了系统发育进化树,分析鉴定。1、2号株采自非免疫抑制病人,3号株采自免疫抑制病人。结果①三株隐孢子虫均能扩增出1700bp左右的目的片段。②构建系统发生树;用DNAstar4．0软件进行分析:1与2号株的同源性为99．8％,1和2号株与 AF108864 C．parvum-cattle的同源性分别为99．5％和99．3％,与AF112569 C．hominis进化关系较远。3号株与AF112574 C． meleagridis(火鸡源 C．meleagridis)的同源性为99．8％,3号株与C．suis(AF108861)的同源性为98．9％,而与AF108864 C．parvum -cattle、AF112569 C．hominis、AF108865 C．parvum(Homo sapiens)、AY642591 C．muris、AB210854 C．canis、AF108862 C． felis等均不处于同一进化枝,亲缘关系较远。利用PAUP4．0、Phylip3.6的方法与DNAstar4．0 软件分析的结果基本一致。结论 1和2号分离株均为C．parvum牛基因型,3号分离株是C．meleagridis(火鸡隐孢子虫)。本文结果为深入研究人隐孢子虫病的分子流行病学打下基础。     

Antibody induction after combined application of an adjuvanted recombinant FeLV vaccine and a multivalent modified live virus vaccine with a chlamydial component

The compatibility, safety and interaction on antibody induction of a combined vaccine application were assessed. Specific pathogen-free cats were vaccinated with either a modified live virus vaccine containing feline calici- (FCV), herpes- (FHV-1), parvovirus (FPV) and Chlamydophila felis (C. felis), an adjuvanted recombinant feline leukaemia virus (FeLV) vaccine or both vaccines in one syringe. After combined application, FeLV ELISA antibody titres were unaltered, However antibody production based on indirect immunofluorescence assay was remarkably enhanced for FCV and was at selected time points also enhanced for FHV-1 and C. felis but diminished for FPV. The use of these vaccines in combination was safe and will simplify vaccination schedules in veterinary practice.     

Extended outbreak of cryptosporidiosis in a pediatric hospital, China

Four Cryptosporidium spp. and 6 C. hominis subtypes were isolated from 102 of 6,284 patients in 3 pediatric hospitals in People's Republic of China. A cryptosporidiosis outbreak was identified retrospectively. The outbreak lasted >1 year and affected 51.4% of patients in 1 hospital ward, where 2 C. hominis subtypes with different virulence were found.     

Human infection with Cryptosporidium felis: case report and literature review.

An infection with Cryptosporidium felis in an HIV-positive man from Italy was successfully treated with paromomycin, despite the patient's having a CD4+ cell count of 31/mL. Fourteen cases of human infection with C. felis have been described, all in the past 3 years, emphasizing the public health importance of Cryptosporidium parasites other than C. parvum.     

Differential evolution of repetitive sequences in Cryptosporidium parvum and Cryptosporidium hominis.

Cryptosporidium parvum and Cryptosporidium hominis are two morphologically identical species of Apicomplexan protozoa infecting humans. Although the genomes of these species are 97% identical, their host range is strikingly different. C. parvum infects humans and animals and is primarily a zoonotic infection, whereas C. hominis is typically not detected in animals. The extent of genetic polymorphism in both species has been surveyed locally, but not on a larger geographical scale. Herein, a collection of unrelated C. parvum and C. hominis isolates was genotyped using multiple, randomly distributed micro- and minisatellites. In average, minisatellites, consisting of tandemly repeated sequence motifs of 6-24 basepair, were more polymorphic than microsatellites. When the average number of micro- and minisatellite alleles per locus was used as a measure of heterogeneity, no difference between C. parvum and C. hominis was found. However, the frequency distribution of alleles in both species was significantly different and in 6 of the 14 loci the size of the C. parvum and C. hominis repeats did not overlap. Assuming that C. parvum and C. hominis evolved from a common ancestor, these observations suggest a differential evolution of repeat length at these loci.     

Incidence of cryptosporidiosis species in paediatric patients in Malawi

We determined the incidence of cryptosporidiosis in children aged <5 years presenting with diarrhoea in an urban and rural hospital-based setting in Malawi. Stools were collected over a 22-month period during both rainy and dry seasons. A range of microscopic methods were used to determine the presence of Cryptosporidium spp. oocysts. Species determination was by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) of oocyst-extracted DNA using 18S rRNA and COWP gene loci. Cryptosporidium spp. oocysts were seen in 5.9% (50/848) of samples, of which 43 amplified by PCR-RFLP indicated the following species: C. hominis, C. parvum, C. hominis/C. parvum, C. meleagridis and C. andersoni. Seven samples could not be amplified by PCR. Wider species diversity was found in the rural setting, and may be a result of increased malnutrition and zoonotic exposure in this area. Improvements in water, sanitation, household hygiene and animal control are required to reduce the incidence of infection in this population.     

Human Rickettsia felis infection, Canary Islands, Spain

We report the first cases of human infection by Rickettsia felis in the Canary Islands. Antibodies against R. felis were found in 5 adsorbed serum samples from 44 patients with clinically suspected rickettsiosis by Western blot serology. Fleas from 1 patient's dog were positive for R. felis by polymerase chain reaction.     

Prevalence and clinical relevance of Blastocystis hominis in diverse patient cohorts

The pathogenicity of Blastocystis hominis is extensively debated in the medical literature. Therefore, we did a prevalence study to investigate the association between the presence of several intestinal parasites and gastrointestinal symptoms in diverse patient cohorts. The study population consisted of 1216 adults, including immunocompromised patients, institutionalized psychiatric or elder subjects, immigrants from developing countries, travellers to developing tropical countries and controls. Several variables for each risk group were considered. Stools specimens, collected in triplicate, were processed by the same technicians. Clinical data about each subject were provided by standardized questionnaires. The presence of gastrointestinal symptoms were related to the presence of any parasite. In addition, on the basis of microbiological results, five subgroups of subjects were evaluated. The results showed a high prevalence of parasites in all the risk groups. Immunocompromised status, recent arrival from developing countries and the presence of behavioural aberrations were significantly related to presence of parasites. B. hominis was the parasite most frequently detected in each studied group. B. hominis showed a significant correlation with gastrointestinal symptoms only when detected in the group including subjects with a severe immunodepression. Immunodepression seems to be a factor of primary importance of the pathogenic role of B. hominis.     

Comparative genome analysis of two Cryptosporidium parvum isolates with different host range

Parasites of the genus Cryptosporidium infect the intestinal and gastric epithelium of different vertebrate species. Some of the many Cryptosporidium species described to date differ with respect to host range; whereas some species' host range appears to be narrow, others have been isolated from taxonomically unrelated vertebrates. To begin to investigate the genetic basis of Cryptosporidium host specificity, the genome of a Cryptosporidium parvum isolate belonging to a sub-specific group found exclusively in humans was sequenced and compared to the reference C. parvum genome representative of the zoonotic group. Over 12,000 single-nucleotide polymorphisms (SNPs), or 1.4 SNP per kilobase, were identified. The genome distribution of SNPs was highly heterogeneous, but non-synonymous and silent SNPs were similarly distributed. On many chromosomes, the most highly divergent regions were located near the ends. Genes in the most diverged regions were almost twice as large as the genome-wide average. Transporters, and ABC transporters in particular, were over-represented among these genes, as were proteins with predicted signal peptide. Possibly reflecting the presence of regulatory sequences, the distribution of intergenic SNPs differed according to the function of the downstream open reading frame. A 3-way comparison of the newly sequenced anthroponotic C. parvum, the reference zoonotic C. parvum and the human parasite Cryptosporidium hominis identified genetic loci where the anthroponotic C. parvum sequence is more similar to C. hominis than to the zoonotic C. parvum reference. Because C. hominis and anthroponotic C. parvum share a similar host range, this unexpected observation suggests that proteins encoded by these genes may influence the host range.     

Genetic analysis of Giardia and Cryptosporidium from people in Northern Australia using PCR-based tools

To date, there has been limited genetic study of the gastrointestinal pathogens Giardia and Cryptosporidium in northern parts of Australia. Here, PCR-based methods were used for the genetic characterization of Giardia and Cryptosporidium from 695 people with histories of gastrointestinal disorders from the tropical North of Australia. Genomic DNAs from fecal samples were subjected to PCR-based analyses of regions from the triose phosphate isomerase (tpi), small subunit (SSU) of the nuclear ribosomal RNA and/or the glycoprotein (gp60) genes. Giardia and Cryptosporidium were detected in 13 and four of the 695 samples, respectively. Giardia duodenalis assemblages A and B were found in 4 (31%) and 9 (69%) of the 13 samples in persons of < 9 years of age. Cryptosporidium hominis (subgenotype IdA18), Cryptosporidium mink genotype (subgenotype IIA16R1) and C. felis were also identified in single patients of 11–21 years of age. Future studies might focus on a comparative study of these and other protists in rural communities in Northern Australia.     

Mixed Cryptosporidium infections and HIV

Mixed Cryptosporidium infections were detected in 7 of 21 patients with a diagnosis of rare Cryptosporidium canis or C. felis infections; 6 patients were infected with 2 Cryptosporidium spp. and 1 patient with 3 species. Mixed infections may occur more frequently than previously believed and should be considered when assessing cryptosporidiosis.     

New cryptosporidium genotypes in HIV-infected persons.

Using DNA sequencing and phylogenetic analysis, we identified four distinct Cryptosporidium genotypes in HIV-infected patients: genotype 1 (human), genotype 2 (bovine) Cryptosporidium parvum, a genotype identical to C. felis, and one identical to a Cryptosporidium sp. isolate from a dog. This is the first identification of human infection with the latter two genotypes.     

Heavy cryptosporidial infections in children in northeast Brazil: comparison of Cryptosporidium hominis and Cryptosporidium parvum

Cryptosporidium is an important cause of infectious diarrhoea worldwide, but little is known about the course of illness when infected with different species. Over a period of 5 years, Cryptosporidium was identified in the stools of 58 of 157 children prospectively followed from birth in an urban slum (favela) in northeast Brazil. Forty isolates were available for quantification and 42 for speciation (24 Cryptosporidium hominis and 18 C. parvum). Children with C. hominis shed significantly more oocysts/ml of stool (3.5 x 10(6) vs. 1.7 x 10(6)perml; P=0.001), and oocyst counts were higher among symptomatic children (P=0.002). Heavier C. parvum shedding was significantly associated with symptoms (P=0.004), and symptomatic C. parvum-infected children were significantly more likely than asymptomatic children to be lactoferrin-positive (P=0.004). Height-for-age (HAZ) Z-scores showed significant declines within 3 months of infection for children infected with either C. hominis (P=0.028) or C. parvum (P=0.001). However, in the 3-6 month period following infection, only C. hominis-infected children continued to demonstrate declining HAZ score and asymptomatic children showed even greater decline (P=0.01). Cryptosporidium hominis is more common than C. parvum in favela children and is associated with heavier infections and greater growth shortfalls, even in the absence of symptoms.     

The prevalence of Cryptosporidium species and subtypes in human faecal samples in Ireland

Cryptosporidium is an important cause of diarrhoeal disease worldwide and, as several recent waterborne outbreaks have shown, poses a significant threat to public health in Ireland. We identified the Cryptosporidium spp. in 199 positive human stool samples by PCR-RFLP of the 18S rRNA and COWP gene loci. Subspecies were identified in 104 samples by sequence analysis of the 60 kDa glycoprotein (gp60) gene fragment. Overall C. parvum was identified in 80%, and C. hominis in 20% of cases. No other Cryptosporidium spp. were detected. C. parvum was by far the most common species in the rural, more sparsely populated west of Ireland and exhibited a pronounced spring peak coincident with a peak in the national cryptosporidiosis incidence rate. Our data indicated a trend towards higher proportions of C. hominis in older age groups. Ninety-nine per cent of all subtyped C. parvum isolates belonged to allele family IIa, of which allele IIaA18G3R1 was by far the most common (63%). According to a recent study by Thompson and colleagues [Parasitology Research (2007), 100, 619-624] this allele is also the most common in Irish cattle. Subtyping of the C. hominis isolates indicated that they belonged to a geographically widely distributed allele (IbA10G2) known to have caused several water- and foodborne outbreaks around the world. The predominance of C. parvum, its geographic and seasonal distribution and the IIaA18G3R1 subtype underlines the importance of zoonotic Cryptosporidium transmission in Ireland.     

Subtypes of Cryptosporidium parvum in humans and disease risk

The 2 main species of Cryptosporidium that infect humans are Cryptosporidium hominis and C. parvum. Here, multilocus fragment analysis of 3 microsatellite loci (ML1, ML2, and gp60) was used to subtype strains from sporadic cases of cryptosporidiosis in Wales and northwest England. Of 72 strains of C. parvum, 63 were typeable at all 3 loci, forming 31 subtypes. These strains formed 3 broad clusters, representing 74.6%, 20.6%, and 4.8% of typeable strains. Of 118 C. hominis strains, 106 were typeable at all 3 loci, forming 9 subtypes; however, 90% belonged to the same subtype. Analysis with epidemiologic data found an association between strains from case-patients who reported contact with farm animals and individual C. parvum microsatellite alleles. The strongest association was with ML1; all strains from case-patients that reported farm animal contact had the same allele (ML1-242). Microsatellite typing of C. parvum provides valuable additional information on the epidemiology of this pathogen.     

Serosurvey of Rickettsia spp. in dogs and humans from an endemic area for Brazilian spotted fever in the State of São Paulo, Brazil

The present study provides a rickettsial serosurvey in 25 dogs and 35 humans in an endemic area for Brazilian spotted fever in the State of S?o Paulo, where the tick Amblyomma aureolatum is the main vector. Testing canine and human sera by indirect immunofluorescence against four Rickettsia antigens (R. rickettsii, R. parkeri, R. felis and R. bellii) showed that 16 (64%) of canine sera and 1 (2.8%) of human sera reacted to at least one of these rickettsial antigens with titers >0r= 64. Seven canine sera and the single reactive human serum showed titers to R. rickettsii at least four times those of any of the other three antigens. The antibody titers in these 7 animals and 1 human were attributed to stimulation by R. rickettsii infection. No positive canine or human serum was attributed to stimulation by R. parkeri, R. felis, or R. bellii. Our serological results showed that dogs are important sentinels for the presence of R. rickettsii in areas where the tick A. aureolatum is the main vector of Brazilian spotted fever.