Development and Application of a gp60-Based Typing Assay for Cryptosporidium viatorum.

The apicomplexan intestinal parasites of the genus Cryptosporidium take a major toll on human and animal health and are frequent causes of waterborne outbreaks. Several species and genotypes can infect humans, including Cryptosporidium viatorum, which, to date, has only been found in humans. Molecular characterization of Cryptosporidium spp., critical to epidemiological analyses, is commonly based on gp60 gene analysis, which appears to require bespoke species- or group-specific PCR primers due to extensive genetic diversity across the genus. In this study, we amplified, sequenced, and characterized the gp60 gene of C. viatorum for the first time. Moreover, we developed and validated a gp60 typing assay for this species and applied it to 27 isolates originating from Asia, Africa, and Central America. A single subtype family, XVa, was identified containing multiple alleles.

Cryptosporidium parvum infections in a cohort of veterinary students in Sweden.

In March 2013, a veterinary student tested positive for Cryptosporidium; four classmates reported similar gastrointestinal symptoms. We aimed to identify source(s) and risk factors for Cryptosporidium infection in university persons symptomatic between 21 January and 14 April 2013. Sixty-four (79%) students from a cohort of 81 fourth-year veterinary students completed questionnaires, identifying 13 cases; four were Cryptosporidium parvum GP60 subtype IIaA16G1R1b, two were IIdA24G1, seven did not submit stool samples. Thirteen cases attended the university's field clinic before symptom onset (13/37 attendees, 35%); 11 visited at least one of four farms where students recalled seeing calves with diarrhoea. C. parvum subtype IIaA16G1R1b was identified in calves at one of the farms. Entering pens of calves with diarrhoea [relative risk (RR) 7·6, 95% confidence interval (CI) 1·7-33·5] and eating in clinic cars (RR 9·1, 95% CI 1·3-65·8) were associated with being a case. Washing hands at least twice per farm visit (0 cases, P = 0·03) was protective. This outbreak investigation was notable for rapid and effective collaboration between public health, veterinary and environmental sectors, leading to swift identification of a microbiological and epidemiological link between cases, infected calves and their farms. We recommend frequent hand-washing using proper technique and dissuasion from eating in clinic cars to minimize possible exposure to contaminated surfaces.

Secretory antibodies against Giardia intestinalis in lactating Nicaraguan women.

Secretory IgA (sIgA) antibodies are important in the host defence against the intestinal protozoan parasite Giardia intestinalis. However, few antigens have been identified. In this study 100 milk and saliva samples from lactating women, living in an endemic region (León, Nicaragua), were screened for the presence of antibodies against G. intestinalis. Most milk and saliva samples contained anti-Giardia antibodies (59% and 52%, respectively), with a mean sIgA content 50 times higher in milk than in saliva. The positive samples reacted with trophozoite membrane, flagella and cytoplasmic antigens. Western blot analysis showed that milk and saliva anti-Giardia sIgA recognized up to 16 different Giardia proteins in the molecular weight region 20-165 kDa. Two-dimensional Western blotting showed that the major immunoreactive proteins were the same as the immunoreactive proteins identified by serum from acute giardiasis patients in a non-endemic country. The major difference was a stronger reactivity against the variant surface proteins (VSPs) in the milk samples. Milk sIgAs also recognized recombinant Giardia proteins such as alpha-1 giardin, ornithine carbamoyl transferase, VSP-4EX, arginine deaminase and alpha-enolase. These antigens will be important targets in the development of new immunodiagnostic tools and vaccines.

Bacterial infections of free-living amoebae.

Free-living amoebae are a diverse group of ubiquitous unicellular organisms, some of which cause severe central nervous system infections and keratitis. However, the focus of research has shifted from the direct pathogenic effects of free-living amoebae towards their role as carriers of pathogenic bacteria. Large outbreaks of legionellosis with numerous fatal cases, both in hospitals and in the community, appear to be the visible tip of the iceberg of complex relationships between amoebae and bacteria in biofilms. The recognition of amoebae as reservoirs and vehicles for bacterial spread leads us to public health issues such as the development of pathogenicity, antibiotic resistance, quality of public water supplies, housing standards, sanitation and decontamination measures. In this review we discuss bacterial infections of free-living amoebae from both a "biological" and general "infection control" point of view.

Free-living amoebae protecting Legionella in water: the tip of an iceberg?

Bacteria are a main food source for free-living amoebae inhabiting aquatic systems. Some bacteria however, have the ability to prevent intracellular destruction and can survive and grow in amoebic cells as endosymbionts. Free-living amoebae are well adapted to their hostile environmental conditions and are resistant to both desiccation, elevated temperatures and various disinfectants. For their endosymbionts, amoebae represent perfect vectors, providing both protection against adverse environmental conditions and transportation. There is increasing interest in the potential role of free-living amoebae as reservoirs and vectors of pathogenic bacteria. The best known of such pathogenic bacteria is Legionella, and several studies provide evidence for the importance of the amoeba-bacterium relationship in the biology and epidemiology of pneumonia caused by this pathogen. Although the relative importance of endosymbiosis of this kind is unknown when it comes to other human bacterial infections and the exact role of amoebic hosts in bacterial survival, multiplication and transmission in the environment is still poorly understood, naming free-living amoebae the "Trojan horses" of the microbial world is appropriate.

Secondary amoebic eye infections in mice due to Acanthamoeba sp.

The aim of the present study was to determine the occurrence of eye infections accompanying the infection of the central nervous system and to demonstrate the possible tissue affinity of different strains of amoebae in subsequent infections. The results obtained demonstrate a clear correlation between the occurrence of eye infection and the degree of virulence of the strains. Amoebae isolated from eyeballs and other organs of dead mice did not exhibit any specificity in relation to the organs in subsequent infections. Irrespective of the place of isolation--be it eye or brain--in the subsequent passages, the amoebae were most often found in the brain and lungs, followed only then by the eye of the infected animals.

Acanthamoeba keratitis: increased sensitivity of the detection of parasites by modified cultivation procedure.

The purpose of this study was to improve current methods for the detection of Acanthamoeba spp. in clinical samples. The sensitivity of parasite detection in filtered specimens was evaluated using samples containing different numbers of parasites. They were filtered through cellulose membranes and cultivated on non-nutrient agar plates covered with Escherichia coli. The recovery of amoebae collected by filtration was compared with the cultivation of centrifuged samples. All samples containing 10 parasites per specimen and 65% of samples containing 2 parasites per specimen yielded positive cultures after filtration, while the cultivation results after centrifugation were 8% and 0%, respectively. We conclude that significantly higher sensitivity of parasite detection can be obtained with samples processed by filtration.

Detection of Giardia lamblia cysts in stool samples by immunofluorescence using monoclonal antibody.

The diagnostic potential of indirect immunofluorescence to detect Giardia cysts in stool samples using a cyst-specific anti-Giardia lamblia monoclonal antibody was evaluated in comparison to conventional light microscopy. One hundred fifty specimens from clinically suspected Giardia infections and 50 control samples from microscopically proved Giardia infections were tested. Giardia cysts were found in 15 of 150 (10%) samples tested by light microscopy, whereas immunofluorescence microscopy detected 35 of 150 (23%) positive samples. Forty-six of the 50 reference samples previously shown to contain Giardia cysts were positive. Apparently, the four discrepant samples contained very low numbers of parasites, as none could be detected by conventional microscopy. The results show that Giardia lamblia cysts are detected significantly more frequently using the antibody marker. The doubled number of positive stool specimens and detection of as little as four cysts per sample suggest that microscopical examination of samples can be improved by immunofluorescent staining of Giardia lamblia cysts.