Genetic characterization of a Neisseria meningitidis cluster in Queensland, Australia.

Neisseria meningitidis serogroups B and C have been responsible for the majority of invasive meningococcal disease in Australia, with serogroup B strains causing an increasing proportion of cases in recent years. Serogroup Y has typically caused sporadic disease in Australia. In 2002, a cluster of 4 cases was reported from a rural region in Queensland. Three of these cases were serogroup C, with 1 case diagnosed by molecular detection only, and the fourth case was identified as a serogroup Y infection. Genomic analysis, including antigen finetyping, multilocus sequence typing (MLST), and core genome MLST, demonstrated that the serogroup Y case, though spatially and temporally linked to a serogroup C disease cluster, was not the product of a capsule switch and that one of the serogroup C isolates had a deletion of the entire porA sequence.

Molecular characterization of an Australian serotype 1 Streptococcus pneumoniae outbreak.

Serotype 1 Streptococcus pneumoniae is a cause of invasive pneumococcal disease (IPD) worldwide and has been associated with IPD outbreaks, while carriage is rarely detected in healthy adults or children. This study details an Australian multi-state and territory outbreak of serotype 1 S. pneumoniae IPD between 2010 and 2012. Molecular characterization demonstrated the outbreak was largely due to the clonal expansion of sequence type 306, MLVA type 261 S. pneumoniae serotype 1.

Invasive group A streptococcal disease in children in Queensland.

The epidemiology and clinical features of invasive group A streptococcal (iGAS) disease in Queensland children was investigated in response to anecdotal evidence of an increase in frequency and severity of this condition. A retrospective review of clinical records of all cases of iGAS disease notified to Queensland Health aged 0-18 years during a 5-year period was conducted. The annualized incidence of iGAS was 3·5/100,000 for the total population aged 0-18 and 13·2/100,000 for the Indigenous population of similar age. The annualized incidence was highest in Indigenous infants but no increase in frequency or severity of iGAS infections was observed. Findings included an increased prevalence in Indigenous children particularly in those aged <1 year, a significant male preponderance, lack of seasonal variation and an association with blunt trauma. Further studies are required to confirm and investigate these findings and to define specific risk factors in high-risk groups.

Sporadic Cryptosporidium infection in Nigerian children: risk factors with species identification.

A cross-sectional study was conducted to investigate risk factors for sporadic Cryptosporidium infection in a paediatric population in Nigeria. Of 692 children, 134 (19·4%) were infected with Cryptosporidium oocysts. Cryptosporidium spp. were identified in 49 positive samples using PCR-restriction fragment length polymorphism and direct sequencing of the glycoprotein60 (GP60) gene. Generalized linear mixed-effects models were used to identify risk factors for all Cryptosporidium infections, as well as for C. hominis and C. parvum both together and separately. Risk factors identified for all Cryptosporidium infections included malaria infection and a lack of Ascaris infection. For C. hominis infections, stunting and younger age were highlighted as risk factors, while stunting and malaria infection were identified as risk factors for C. parvum infection.

Identification of Cryptosporidium species and genotypes in Scottish raw and drinking waters during a one-year monitoring period.

We analyzed 1,042 Cryptosporidium oocyst-positive slides (456 from raw waters and 586 from drinking waters) of which 55.7% contained 1 or 2 oocysts, to determine species/genotypes present in Scottish waters. Two nested PCR-restriction fragment length polymorphism (RFLP) assays targeting different loci (1 and 2) of the hypervariable region of the 18S rRNA gene were used for species identification, and 62.4% of samples were amplified with at least one of the PCR assays. More samples (577 slides; 48.7% from raw water and 51.3% from drinking water) were amplified at locus 1 than at locus 2 (419 slides; 50.1% from raw water and 49.9% from drinking water). PCR at loci 1 and 2 amplified 45.4% and 31.7% of samples containing 1 or 2 oocysts, respectively. We detected both human-infectious and non-human-infectious species/genotype oocysts in Scottish raw and drinking waters. Cryptosporidium andersoni, Cryptosporidium parvum, and the Cryptosporidium cervine genotype (now Cryptosporidium ubiquitum) were most commonly detected in both raw and drinking waters, with C. ubiquitum being most common in drinking waters (12.5%) followed by C. parvum (4.2%) and C. andersoni (4.0%). Numerous samples (16.6% total; 18.9% from drinking water) contained mixtures of two or more species/genotypes, and we describe strategies for unraveling their identity. Repetitive analysis for discriminating mixtures proved useful, but both template concentration and PCR assay influenced outcomes. Five novel Cryptosporidium spp. (SW1 to SW5) were identified by RFLP/sequencing, and Cryptosporidium sp. SW1 was the fourth most common contaminant of Scottish drinking water (3%).

Genetic relatedness and virulence gene profiles of Escherichia coli strains isolated from septicaemic and uroseptic patients.

We investigated the relationship between clonality and virulence factors (VFs) of a collection of Escherichia coli strains isolated from septicaemic and uroseptic patients with respect to their origin of translocation. Forty septicaemic and 30 uroseptic strains of E. coli were tested for their phylogenetic groupings, genetic relatedness using randomly amplified polymorphic DNA (RAPD), biochemical fingerprinting method (biochemical phenotypes [BPTs]), adherence to HT-29 cells and the presence of 56 E. coli VF genes. Strains belonging to phylogenetic groups B2 and D constituted 93% of all strains. Fifty-four (77%) strains belonged to two major BPT/RAPD clusters (A and B), with cluster A carrying significantly (P = 0.0099) more uroseptic strains. The degree of adhesion to HT-29 cells of uroseptic strains was significantly (P = 0.0012) greater than that of septicaemic strains. Of the 56 VF genes tested, pap genes was the only group that were found significantly (P < 0.0001) more often among uroseptic isolates. Phylogenetic group B2 contained a significantly higher number of strains carrying pap genes than those in group D. We conclude that uroseptic E. coli are clonally different from septicaemic strains, carry more pap genes and predominantly adhere more to the HT-29 cell model of the gut.

Cercarial dermatitis in the UK.

'Swimmer's itch' or cercarial dermatitis (CD) results from an immunological reaction to free-swimming non-human schistosome parasites released from aquatic snails. Affected bathers develop a self-limiting, pruritic, macular or papular eruption shortly after leaving the water. The condition is well-recognized in continental Europe, Asia and America, but has not to date been recorded in the UK.

Cryptosporidium infection in non-human hosts in Malawi.

Of 1346 faecal samples from the Chikwawa and Thyolo districts of Malawi, analysed for the presence of Cryptosporidium oocysts between October 2001 and May 2003, 61.3% were from cattle (29.8% of these were from calves <6 months old). Cryptosporidium oocysts were detected during all three seasons studied in Chikwawa and Thyolo. In Chikwawa, 13.6% of adult cattle and 11.7% of calves were infected, compared to 28.9% of adult cattle and 36.7% of calves in Thyolo. Dependent on season, between 7.8% and 37.7% (Chikwawa) and 16.7% and 39.3% (Thyolo) of cattle samples contained oocysts. In Chikwawa, the highest percentage of infections occurred in the cool season, whereas in Thyolo, the highest percentage of infections occurred in the dry season. Faecal samples from goats [n=225], pigs [n=92], sheep [n=6]), rabbits, guinea pigs, chickens, ducks, turkeys, doves and guinea fowls were also analysed. Up to 5.6% of goat samples contained oocysts in Chikwawa, compared to between 16.7% and 39.3% in Thyolo. Again, in Chikwawa, the highest percentage of infections occurred in the cool season and the lowest in the rainy season, whereas, in Thyolo, the highest percentage of infections occurred in the dry season and the lowest in the cool season. In pigs, more infections were detected in the dry season in Chikwawa, but infections in the cool season were similar (17.7%), whereas in Thyolo, infections occurred in all three seasons (17.9% in the rainy season, 25% in the cool season and 60% in the dry season). Of ten diarrhoeic, oocyst positive cattle faecal samples collected from Chikwawa and subjected to PCR-RFLP, four oocyst positive samples (two from heifers, one from a cow and one unknown) were amplified at an 18S rRNA and Cryptosporidium oocyst wall protein (COWP) loci. RFLP of the 18S rRNA locus indicated that Cryptosporidium parvum, Cryptosporidium hominis, Cryptosporidium bovis and/or Cryptosporidium ryanae DNA, or a mixture of them was present. Cryptosporidium parvum DNA was identified in one sample that amplified at the COWP locus, indicating the presence of the major zoonotic Cryptosporidium species in Malawi.

Soil-transmitted helminth infections in Nigerian children aged 0-25 months.

The objective of this cross-sectional study was to determine the prevalence and intensity of soil-transmitted helminths (STHs) in children aged 0-25 months and to identify the associated risk factors for Ascaris lumbricoides infections. The study was conducted in three villages outside Ile-Ife, Osun state, Nigeria in May/June 2005. Stool samples (369) were processed by formol-ether concentration. Ascaris lumbricoides (12.2%) was the dominant infection. Age, father's occupation and dog ownership were identified as the significant risk factors in the minimal adequate model for A. lumbricoides. The odds of being infected with A. lumbricoides increased as the children got older. Children aged 12-17 months and 18-25 months were 8.8 and 12.4 times, respectively, more likely to harbour Ascaris than those aged 7-11 months. The odds of harbouring Ascaris for children whose families owned a dog were 3.5 times that of children whose families did not own a dog. Children whose fathers were businessmen were 0.4 times less likely to be infected with Ascaris than those whose fathers were farmers. The findings from this study suggest that many of these young children, who are at a critical stage of development, are infected with Ascaris and that the prevalence of infection with this parasite increases with age. This study has highlighted the need to incorporate preschool children into deworming programmes in endemic regions and to investigate innovative ways of delivering cost-effective deworming treatment to this high-risk age group.

Cryptosporidium--biotechnological advances in the detection, diagnosis and analysis of genetic variation.

Cryptosporidiosis is predominantly a gastrointestinal disease of humans and other animals, caused by various species of protozoan parasites representing the genus Cryptosporidium. This disease, transmitted mainly via the faecal-oral route (in water or food), is of major socioeconomic importance worldwide. The diagnosis and genetic characterization of the different species and population variants (usually recognised as "genotypes" or "subgenotypes") of Cryptosporidium is central to the prevention, surveillance and control of cryptosporidiosis, particularly given that there is presently no broadly applicable treatment regimen for this disease. Although traditional phenotypic techniques have had major limitations in the specific diagnosis of cryptosporidiosis, there have been major advances in the development of molecular analytical and diagnostic tools. This article provides a concise account of Cryptosporidium and cryptosporidiosis, and focuses mainly on recent advances in nucleic acid-based approaches for the diagnosis of cryptosporidiosis and analysis of genetic variation within and among species of Cryptosporidium. These advances represent a significant step toward an improved understanding of the epidemiology as well as the prevention and control of cryptosporidiosis.

Effect of various environmental factors on the viability of Cryptosporidium parvum oocysts.

AIMS: To evaluate individual and combined effects of temperature (4, 18 and 25 degrees C), pH (7 and 10), ammonia (5 and 50 mg l(-1)) and exposure time (1, 2, 4 and 6 days) on the viability of Cryptosporidium parvum oocysts in water. METHODS AND RESULTS: The viability of oocysts was evaluated using the fluorogenic vital dyes assay (4',6-diamidino-2-phenylindole and propidium iodide). All the factors analysed (temperature, pH, ammonia and exposure time) and their interaction were statistically significant (P < 0.005). Exposure of oocysts to pH 10 for 6 days at 25 degrees C reduced oocyst viability from approximately 80% to 51%. Similarly, the exposure of C. parvum oocysts to 5 mg NH(3) l(-1) and 50 mg NH(3) l(-1) for 4 days reduced their viability from between approximately 80% to 41.5% and 14.8%, respectively. CONCLUSIONS: The interaction between pH, temperature and exposure time may have adverse effects on the survival of C. parvum oocysts in water. Low concentrations of ammonia, as commonly found in alga-based wastewater systems, over a long period of time can produce high C. parvum oocyst inactivation rates. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides relevant data on the inactivation of C. parvum oocysts in alga-based wastewater-treatment systems in the northwest of Spain.

Current status and future trends in Cryptosporidium and Giardia epidemiology in Malaysia.

Cryptosporidium and Giardia are major causes of diarrhoeal diseases of humans worldwide, and are included in the World Health Organisation's 'Neglected Diseases Initiative'. Cryptosporidium and Giardia occur commonly in Malaysian human and non-human populations, but their impact on disease, morbidity and cost of illness is not known. The commonness of contributions from human (STW effluents, indiscriminate defaecation) and non-human (calving, lambing, muck spreading, slurry spraying, pasturing/grazing of domestic animals, infected wild animals) hosts indicate that many Malaysian environments, particularly water and soil, are sufficiently contaminated to act as potential vehicles for the transmission of disease. To gain insight into the morbidity and mortality caused by human cryptosporidiosis and giardiasis, they should be included into differential diagnoses, and routine laboratory testing should be performed and (as for many infectious diseases) reported to a centralised public health agency. To understand transmission routes and the significance of environmental contamination better will require further multidisciplinary approaches and shared resources, including raising national perceptions of the parasitological quality of drinking water. Here, the detection of Cryptosporidium and Giardia should be an integral part of the water quality requirement. A multidisciplinary approach among public health professionals in the water industry and other relevant health- and environment-associated agencies is also required in order to determine the significance of Cryptosporidium and Giardia contamination of Malaysian drinking water. Lastly, adoption of validated methods to determine the species, genotype and subgenotype of Cryptosporidium and Giardia present in Malaysia will assist in developing effective risk assessment, management and communication models.

Cryptosporidium and Giardia as foodborne zoonoses.

Cryptosporidium and Giardia are major causes of diarrhoeal disease in humans, worldwide and are major causes of protozoan waterborne diseases. Both Cryptosporidium and Giardia have life cycles which are suited to waterborne and foodborne transmission. There are 16 'valid'Cryptosporidium species and a further 33+ genotypes described. Parasites which infect humans belong to the Giardia duodenalis "type", and at least seven G. duodenalis assemblages are recognised. Cryptosporidium parvum is the major zoonotic Cryptosporidium species, while G. duodenalis assemblages A and B have been found in humans and most mammalian orders. In depth studies to determine the role of non-human hosts in the transmission of Cryptosporidium and Giardia to humans are required. The use of harmonised methodology and standardised and validated molecular markers, together with sampling strategies that provide sufficient information about all contributors to the environmental (oo)cyst pool that cause contamination of food and water, are recommended. Standardised methods for detecting (oo)cysts in water are available, as are optimised, validated methods for detecting Cryptosporidium in soft fruit and salad vegetables. These provide valuable data on (oo)cyst occurrence, and can be used for species and subspecies typing using appropriate molecular tools. Given the zoonotic potential of these organisms, epidemiological, source and disease tracking investigations involve multidisciplinary teams. Here, the role of the veterinarian is paramount, particularly in understanding the requirement for adopting comprehensive sampling strategies for analysing both sporadic and outbreak samples from all potential non-human contributors. Comprehensive sampling strategies increase our understanding of parasite population biology and structure and this knowledge can be used to determine what level of discrimination is required between isolates. Genetic exchange is frequent in C. parvum populations, leading to recombination between alleles at different loci, the generation of a very large number of different genotypes and a high level of resolution between isolates. In contrast, genetic exchange appears rare in Cryptosporidium hominis and populations are essentially clonal with far fewer combinations of alleles at different loci, resulting in a much lower resolution between isolates with many being of the same genotype. Clearly, more markers provide more resolution and high throughput sequencing of a variety of genes, as in multilocus sequence typing, is a way forward. Sub-genotyping tools offer increased discrimination, specificity and sensitivity, which can be exploited for investigating the epidemiology of disease, the role of asymptomatic carriers and contaminated fomites and for source and disease tracking for food and water contaminated with small numbers of (oo)cysts.

Towards standard methods for the detection of Cryptosporidium parvum on lettuce and raspberries. Part 2: validation.

We report the results of interlaboratory collaborative trials of methods to detect oocysts of the protozoan parasite Cryptosporidium parvum on lettuce and raspberries. The trials involved eight expert laboratories in the United Kingdom. Samples comprised 30 g lettuce, and 60 g raspberries. Lettuce samples were artificially contaminated at three levels: low (8.5-14.2 oocysts), medium (53.5-62.6 oocysts), and high (111.3-135.0 oocysts). Non-contaminated lettuce samples were also tested. The method had an overall sensitivity (correct identification of all artificially contaminated lettuce samples) of 89.6%, and a specificity (correct identification of non-contaminated samples) of 85.4%. The total median percentage recovery (from all artificially contaminated samples) produced by the method was 30.4%. The method was just as reproducible between laboratories, as repeatable within a laboratory. Raspberry samples were artificially contaminated at three levels: low (8.5-26.8 oocysts), medium (29.7-65.7 oocysts), and high (53.9-131.3 oocysts). Non-contaminated raspberry samples were also tested. The method had an overall sensitivity (correct identification of all artificially contaminated raspberry samples) of 95.8%, and a specificity (correct identification of non-contaminated samples) of 83.3%. The total median percentage recovery (from all artificially contaminated samples) produced by the method was 44.3%. The method was just as reproducible between laboratories, as repeatable within a laboratory. The results of the collaborative trial indicate that these assays can be used effectively in analytical microbiological laboratories.

Towards standard methods for the detection of Cryptosporidium parvum on lettuce and raspberries. Part 1: development and optimization of methods.

No standard method is available for detecting protozoan parasites on foods such as soft fruit and salad vegetables. We report on optimizing methods for detecting Cryptosporidium parvum on lettuce and raspberries. These methods are based on four basic stages: extraction of oocysts from the foodstuffs, concentration of the extract and separation of the oocysts from food materials, staining of the oocysts to allow their visualization, and identification of oocysts by microscopy. The concentration and separation steps are performed by centrifugation, followed by immunomagnetic separation using proprietary kits. Oocyst staining is also performed using proprietary reagents. The performance parameters of the extraction steps were extensively optimized, using artificially contaminated samples. The fully developed methods were tested several times to determine their reliability. The method to detect C. parvum on lettuce recovered 59.0+/-12.0% (n=30) of artificially contaminated oocysts. The method to detect C. parvum on raspberries recovered 41.0+/-13.0% (n=30) of artificially contaminated oocysts.

Spironucleosis (Hexamitiasis, Hexamitosis) in the ring-necked pheasant (Phasianus colchicus): detection of cysts and description of Spironucleus meleagridis in stained smears.

Trophozoites and cysts of Spironucleus (Hexamita) meleagridis were detected in the intestinal fluid and mucus of pheasant poults with spironudeosis (hexamitiasis, hexamitosis) following staining with Heidenhain iron hematoxylin (HIH) and the Romanowsky-type stain Hemacolor. Their morphology was consistent with that of flagellates of the genus Spironucleus, and bright-field morphologic observations were confirmed by transmission electron microscopy. Cysts occurred mostly within intestinal mucus, which was firmly compressed between microscope slides prior to staining. The internal structures of cysts were similar to those of trophozoites, allowing them to be confidently recognized. Hemacolor provided differential color staining of trophozoites and cysts, allowing accurate identification of S. meleagridis life cycle stages, even in smears in which there was heavy background staining. While HIH often produced dearer and more detailed staining of protozoan structures, in the context of a diagnostic laboratory its use was outweighed by the ease of use, rapidity of results, and differential color staining provided by Hemacolor. The possible significance of a resistant cystic stage in the life cyde of S. meleagridis is discussed.

Cerebral lateralization and handedness and their effects on verbal and spatial reasoning.

Consistent and inconsistent left- and right-handed male subjects completed four tests each of verbal and spatial reasoning. They also participated in tachistoscopic measures of verbal and spatial lateralization--consonant-vowel-consonant recognition and dot enumeration, respectively. The consistent handedness groups displayed significantly more lateralized patterns of cerebral organization that the inconsistent handedness groups. Increased lateralization was associated with superior performance on measures of spatial reasoning, but not on measures of verbal reasoning. Results are interpreted in terms of a competition hypothesis. No differences were found between the handedness groups on either the verbal or spatial reasoning tests.

A paper radioimmunosorbent test (PRIST) for the detection of larva-specific antibodies to Toxocara canis in human sera.

A paper radioimmunosorbent test (PRIST) was shown to be sensitive and reproducible when used with excretory/secretory antigen of Toxocara canis second stage larvae. Whatman No. 50 filter paper (5 mm discs) gave the most consistent and clear results with antigen at a concentration of 100 micrograms/ml, and could be stored for up to 3 weeks in vacuo at -70 degrees C. Antigen coated discs were incubated with test sera at 1:10 dilution for 3 h at room temperature (21 degrees C), reacted with [125I]anti-human IgG for 1 h and counts determined in a gamma counter. Sera from patients with fascioliasis, taeniasis, schistosomiasis, oxyuriasis, trichinellosis and ancyclostomiasis gave counts similar to cord serum controls. Sera from patients with ascariasis gave counts of up to twice as great as controls, but sera from patients with toxicariasis produced counts of 7,000-13,000, a 4-6-fold increase.

The effect of temperature and antimetabolites on antibody binding to the outer surface of second stage Toxocara canis larvae.

In vitro maintained second stage Toxocara canis larvae do not bind antiserum raised to their excretions and secretions (ES) at 37 degrees C as detected by indirect fluorescence. However, when these larvae were incubated at 2 degrees C under the same conditions intense fluorescence on the whole outer surface was observed. This fluorescence remained as long as the larvae were maintained at 2 degrees C. When these larvae were reincubated at 37 degrees C a gradual loss of fluorescence along their outer surfaces occurred. This loss was complete after 3 h. Larvae which were preincubated in antimetabolites at 37 degrees C exhibited intense fluorescence on their outer surfaces as did those incubated at 2 degrees C with antimetabolites. It is concluded that antigens present in ES occur along the whole length of the larval outer surface and turn over at 37 degrees C. This turnover occurs along the whole outer surface and is metabolically dependent. Should this occur in vivo it could afford the parasite with a mechanism for evasion of the immune response.

Emerging parasite zoonoses associated with water and food.

The environmental route of transmission is important for many protozoan and helminth parasites, with water, soil and food being particularly significant. Both the potential for producing large numbers of transmissive stages and their environmental robustness, being able to survive in moist microclimates for prolonged periods of time, pose a persistent threat to public and veterinary health. The increased demands on natural resources increase the likelihood of encountering environments and produce contaminated with parasites. For waterborne diseases, the protozoa, Cryptosporidium, Giardia and Toxoplasma, are the most significant causes, yet, with the exception of Toxoplasma, the contribution of zoonotic transmission remains unclear due to the absence of 'standardised' methods. The microsporidia have been documented in one waterborne outbreak, but the role of animals as the cause of contamination was not elucidated. In foods, surface contamination is associated with the faecal-oral pathogens, and some data are available to indicate that animal wastes remain an important source of contamination (e.g. cattle faeces and apple cider outbreaks), however, further work should focus on examining the source of contamination on fruit and vegetables. Increasing recognition of the burden of human fascioliasis has occurred; it is now recognised as an emerging zoonosis by the WHO. Toxoplasma, Trichinella and Taenia spp. remain important meatborne parasites, however, others, including Pleistophora-like microsporidians may be acquired from raw or lightly cooked fish or crustaceans. With increased international travel, the public health importance of the foodborne trematodiases must also be realised. Global sourcing of food, coupled with changing consumer vogues, including the consumption of raw vegetables and undercooking to retain the natural taste and preserve heat-labile nutrients, can increase the risk of foodborne transmission. A greater awareness of parasite contamination of our environment and its impact on health has precipitated the development of better detection methods. Robust, efficient detection, viability and typing methods are required to assess risks and to further epidemiological understanding.