A novel genotyping method for Cryptosporidium hominis.

Cryptosporidiosis remains the leading protozoan induced cause of diarrhoea-associated mortality worldwide. Cryptosporidium hominis, the anthroponotically transmitted species within the Cryptosporidium genus, contributes significantly to the global burden of infection, accounting for the majority of clinical cases in many countries. This study applied high resolution melting analysis, a post-real-time PCR application, to the differentiation of six globally prevalent C. hominisgp60-subtypes. This novel method targeted three microsatellite, tandem repeat containing genetic markers, gp60, the genetic marker upon which current Cryptosporidium subtype nomenclature is based, MSB, and MSE, by which to differentiate between C. hominis isolates. This multi-locus approach successfully differentiated between all six C. hominisgp60-subtypes studied, some of which, such as IbA10G2, are known to exhibit global ubiquity. Thus, this method has the potential to be universally employed as a sensitive, cost effective and highly reproducible means to rapidly differentiate between C. hominisgp60-subtypes. Such a method would be of particular utility in epidemiological studies and outbreak scenarios, providing cost effective, clinically accessible alternative to DNA sequencing. The success of this preliminary study also supports further analysis of an expanded C. hominisgp60-subtype range and the potential expansion of the multi-locus panel in order to improve the discriminatory power of this approach.

Increased diversity and novel subtypes among clinical Cryptosporidium parvum and Cryptosporidium hominis isolates in Southern Ireland.

Reported incidence rates of cryptosporidiosis in Ireland are consistently among the highest in Europe. Despite the national prevalence of this enteric parasite and the compulsory nature of incidence surveillance and reporting, in-depth analyses seeking to genotype clinical isolates of Cryptosporidium on an intra-species level are rarely undertaken in Ireland. This molecular epidemiology study of 163 clinical Cryptosporidium isolates was conducted in Southern Ireland, from 2015 to 2018, in order to ascertain population subtype heterogeneity. Analysis was conducted via real-time PCR amplification and gp60 gene sequencing, which successfully determined the subtype designation of 149 of the 163 (91.4%) tested isolates. Overall, 12 C. parvum and five C. hominis subtypes were identified, with the incidence of the regionally predominant C. parvum species found to primarily occur during springtime months, while C. hominis incidence was largely confined to late summer and autumnal months. Additionally, one C. parvum and four C. hominis subtypes were newly reported by this study, having not been previously identified in clinical or livestock infection in Ireland. Overall, these data give insight into the diversification of the Cryptosporidium population and emergent subtypes, while also allowing comparisons to be made with clinical epidemiological profiles reported previously in Ireland and elsewhere.

Empirical treatment of urinary tract infections: how rational are our guidelines?

Objectives: This study considers susceptibility test results obtained over a 6 month period for Enterobacteriaceae that caused urinary tract infections (UTIs) in the Cork region of Ireland and uses these results to examine the suitability of Irish empirical treatment guidelines. Patients and methods: UTI-causing Enterobacteriaceae isolates were analysed using EUCAST guidelines to determine resistance to a set of commonly prescribed antimicrobial agents, i.e. ampicillin, amoxicillin/clavulanate, cefalexin, ciprofloxacin, nitrofurantoin and trimethoprim. Patients were categorized by age and patient type, based on origin (hospital inpatients, patients in long-term care facilities and all other non-hospitalized patients). In total, 8999 test results were analysed using the IBM Cognos Analytics Series 7 interrogation tool and Microsoft Office Excel. Results: A variety of resistance patterns were observed. Only one antimicrobial agent, nitrofurantoin, demonstrated a resistance rate of less than 20% for all patient categories considered. Conclusions: Previous studies determined that a resistance rate of >20% renders an antimicrobial agent unsuitable for use as an empirical treatment option. This study demonstrated that this resistance rate is exceeded in many cases, potentially rendering some antimicrobial agents unsuitable for use as empirical treatment. We suggest that the focus on susceptibility when producing surveillance data to create empirical treatment guidelines may inadvertently camouflage resistance rates. The findings of this study highlight the need for laboratory-guided treatment of UTIs and ideally a pre-emptive sample should be obtained for laboratory investigation prior to commencement of antimicrobial therapy.

Impact of BCG vaccination on incidence of tuberculosis disease in southern Ireland.

BACKGROUND: Tuberculosis (TB) is the ninth leading cause of death worldwide and the leading cause from a single infectious agent. Bacillus Calmette-Guerin (BCG) is the only licensed vaccine for TB, yet its efficacy remains debated with variations in vaccine sub-strains, policies, and practices observed across the world. Three BCG vaccination policies were implemented across adjoining regions in the South West of Ireland from 1972; neonatal vaccination (vaccinated Region-A), vaccination of children aged 10-12 years (vaccinated Region-B) and no vaccination (unvaccinated Region-C). The aim of this study is to examine the impact of different BCG vaccination policies on incidence of TB disease in the South of Ireland over a 13-year period. METHODS: Cases of active TB disease from 2003 to 2016 were identified through surveillance data. Residential addresses for each case were geocoded using the Google Maps API. Addresses were linked to 2011 census population data and to Local Health Offices BCG coverage data for study regions A-C. A steady-state population was assumed to calculate the 13-year incidence of TB disease. Using SatScan (v9.4.4), spatial clusters were identified at a small area level with the spatial scan statistic based on the discrete Poisson probability distribution. RESULTS: Of 621 TB disease cases identified, 510 could be linked to the study area based on the reported addresses. The median age was 42 years (range 4 months - 94 years), 65% male and 66% Irish born. The incidence of TB disease was higher in the unvaccinated population, region-C 132/100,000 (95% CI 116-150) versus vaccinated region-A 56/100,000 (95%CI 45-69) and region-B 44/100,000 (95%CI 29-63). A spatial cluster analysis identified a single high-risk cluster in region -C where the relative risk (vs. the areas outside of the cluster) was 4.94 (95% CI 4.03 to 5.96). CONCLUSION: Our study demonstrates significant regional variation in the incidence of TB in demographically similar populations based on BCG vaccination policy. This observation is particularly noteworthy in a country with low TB disease incidence such as Ireland. These findings strengthen existing data demonstrating efficacy of BCG vaccination for primary prevention of TB disease.

Campylobacter corcagiensis sp. nov., isolated from faeces of captive lion-tailed macaques (Macaca silenus).

An investigation of the prevalence of Campylobacter ureolyticus in a variety of animals led to the identification of the strain CIT 045(T), in the faeces of captive lion-tailed macaques (Macaca silenus). Originally, believed to be Campylobacter ureolyticus based on the colony morphology and positive urease test, analysis of 16S rRNA and hsp60 gene sequences of this isolate revealed that the strain differs significantly from other species of the genus Campylobacter described to date. Species-specific primers for 16S rRNA and hsp60 genes were designed and used to identify two additional strains isolated from faeces samples from other macaques. Nucleotide sequence analysis of the 16S rRNA and hsp60 genes revealed ≤95% and ≤82  % sequence similarity to recognized species of the genus Campylobacter respectively. All three isolates formed a distinct group within the genus Campylobacter based on their 16S rRNA and hsp60 sequences and matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) profiles. The unique species status was further supported by phenotypic characteristics of the isolates. All isolates were found to be oxidase-, catalase- and urease-positive, they grew well at 37 °C and 42 °C and produced H2S on TSI (triple-sugar iron) and SIM (sulfide indole motility) media. The name Campylobacter corcagiensis sp. nov. is proposed for this novel species, with the strain CIT 045(T) as the type strain CIT 045(T) ( = LMG 27932(T), CCUG 64942(T)).

Improved detection of bacterial pathogens in patients presenting with gastroenteritis by use of the EntericBio real-time Gastro Panel I assay.

In this study, we evaluated the use of EntericBio real-time Gastro Panel I (Serosep, Limerick, Ireland) for routine use in a clinical microbiology laboratory for simultaneous detection of Campylobacter jejuni, coli, and lari, Shiga toxin-producing Escherichia coli (STEC), Salmonella spp., and Shigella spp. in feces. This system differs from its predecessor (the EntericBio Panel II system, Serosep) in that it allows real-time detection of pathogens directly from feces, without pre-enrichment. It also specifically detects Campylobacter jejuni, coli, and lari rather than all Campylobacter species, as is the case with the previous system. A total of 528 samples from patients presenting with acute gastroenteritis were screened prospectively with this assay, and results were compared with those of the current method, which combines screening the samples with a molecular assay (the EntericBio Panel II assay) and retrospective culture of the specimens in which the target was detected. Discrepancy analysis was conducted using culture and molecular methods. The real-time assay produced 84 positive results, specifically, Campylobacter spp. (n=44); Stx1 and/or Stx2 (n=35); Shigella spp. (n=3); and Salmonella spp. (n=6). Of these, 4 samples represented coinfections with Campylobacter spp. and STEC. The real-time assay showed an increased detection rate for pathogens, apart from Salmonella spp. Four Campylobacter-positive and 6 Stx-positive results remained unconfirmed by any other method used. The isolation rates for PCR-positive samples were as follows: Campylobacter spp., 80%; STEC, 45.7%; Salmonella spp., 100%; and Shigella spp., 66.7%. The sensitivity, specificity, positive predictive value, negative predictive value, and efficiency were 100%, 97.8%, 88.1%, 100%, and 98.1%, respectively.

Effect of Sub-Inhibitory Concentrations of Nitrofurantoin, Ciprofloxacin, and Trimethoprim on In Vitro Biofilm Formation in Uropathogenic Escherichia coli (UPEC).

The purpose of this study was to determine the effect of sublethal concentrations of nitrofurantoin, ciprofloxacin, and trimethoprim on biofilm formation in 57 uropathogenic Escherichia coli strains (UPEC). The minimum inhibitory concentration of nitrofurantoin, ciprofloxacin, and trimethoprim was determined and the biofilm formation for each isolate with and without sub-lethal concentrations of each antibiotic was then quantified. The statistical significance of changes in biofilm formation was ascertained by way of a Dunnett's test. A total of 22.8% of strains were induced to form stronger biofilms by nitrofurantoin, 12% by ciprofloxacin, and 19% by trimethoprim; conversely 36.8% of strains had inhibited biofilm formation with nitrofurantoin, 52.6% with ciprofloxacin, and 38.5% with trimethoprim. A key finding was that even in cases where the isolate was resistant to an antibiotic as defined by EUCAST, many were induced to form a stronger biofilm when grown with sub-MIC concentrations of antibiotics, especially trimethoprim, where six of the 22 trimethoprim resistant strains were induced to form stronger biofilms. These findings suggest that the use of empirical treatment with trimethoprim without first establishing susceptibility may in fact potentiate infection in cases where a patient who is suffering from a urinary tract infection (UTI) caused by trimethoprim resistant UPEC is administered trimethoprim. This emphasizes the need for laboratory-guided treatment of UTI.

Development of a novel, high resolution melting analysis based genotyping method for Cryptosporidium parvum.

This study employed the post-real-time PCR application, high resolution melting (HRM) analysis, in order to differentiate between characterised clinical and reference Cryptosporidium parvum samples obtained from Cork University Hospital (Cork, Ireland) and the Cryptosporidium Reference Unit (Swansea, Wales). A sample set composed of 18 distinct C. parvum gp60-subtypes of the IIa gp60-subtype family (an allele family accounting for over 80% of all cryptosporidiosis cases in Ireland) was employed. HRM analysis-based interrogation of the gp60, MM5 and MS9-Mallon tandem repeat loci was found to completely differentiate between 10 of the 18 studied gp60-subtypes. The remaining eight gp60-subtypes were differentiated into three distinct groupings, with the designations within these groupings resolved to two to three potential gp60-subtypes. The current study aimed to develop a novel, reproducible, real-time PCR based multi-locus genotyping method to distinguish between C. parvum gp60-subtypes. These preliminary results support the further expansion of the multi-locus panel in order to increase the discriminatory capabilities of this novel method.

Development of novel methodology for the molecular differentiation of Cryptosporidium parvum gp60 subtypes via high resolution melting analysis.

Cryptosporidium species subtypes are generally identified via DNA sequencing of the gp60 gene tandem repeat motif region. Due to the immunogenic nature of its glycoprotein products, gp60 is subject to host selective pressures, genetic recombination and evolutionary processes that drive extensive polymorphism at this locus. The elucidation of the polymorphic nature of this gene has led to the current mainstay in Cryptosporidium subtyping nomenclature. This study aimed to develop a real-time polymerase chain reaction based method utilising a post-PCR application, high resolution melting (HRM) analysis, in conjunction with the abovementioned gp60 nomenclature system, in order to differentiate between Cryptosporidium parvum gp60 subtypes. Subtype differentiation is based on the difference between the melting temperatures of individual subtypes conferred by variations in the polymorphic region of gp60. • Nested gp60 primers were designed to amplify a target region of <200 base pairs for effective HRM analysis • This method presents a rapid, sensitive, cost effective alternative to conventional sequencing. • This method is highly flexible and may be applied to other loci in order to facilitate multi-locus analysis and improve the discriminative abilities of the method.

Detection and molecular analysis of Campylobacter ureolyticus in domestic animals.

Previous studies showed the presence of Campylobacter ureolyticus in a large proportion of diarrhoeal samples from patients in Ireland. This emerging gastrointestinal pathogen was the second most common Campylobacter species detected in patients presenting with gastroenteritis, surpassed only by C. jejuni. However, the source of C. ureolyticus infections in humans remains unknown. The aim of this study was to investigate the presence of C. ureolyticus in a range of domestic animals. Over a period of 6 months, 164 samples collected from various domestic animals were tested using molecular method based on detection of the C. ureolyticus specific hsp60 gene. These included canine faeces (n = 44), feline faeces (n = 31) and porcine faeces (n = 89). C. ureolyticus was detected in 32% (10/31) of feline faeces, 9% (4/44) of canine faeces and 18% (16/89) of porcine faeces. Random Amplified Polymorphic DNA (RAPD) analysis of C. ureolyticus isolates showed that an isolate from a cat is genetically similar to a strain isolated from a patient presenting with gastroenteritis. This study reports the first detection and isolation of this organism in domestic animals in Ireland, with a potential source for human infection. Together with the previously reported detection of C. ureolyticus in bovine samples, it is likely that this emerging pathogen has a zoonotic potential.

Campylobacter ureolyticus: a portrait of the pathogen.

Herein, we provide a brief overview of the emerging bacterial pathogen Campylobacter ureolyticus. We describe the identification of the pathogen by molecular as opposed to classical culture based diagnostics and discuss candidate reservoirs of infection. We also review the available genomic data, outlining some of the major virulence factors, and discuss how these mechanisms likely contribute to pathogenesis of the organism.

Draft Genome Sequence of Campylobacter corcagiensis Strain CIT045T, a Representative of a Novel Campylobacter Species Isolated from Lion-Tailed Macaques (Macaca silenus).

Campylobacter corcagiensis CIT045(T) (=CCUG 64942(T), LMG 27932(T)), a new member of the Campylobacter genus, has recently been isolated from lion-tailed macaques in Cork, Ireland. To further characterize this new species and its potential pathogenicity, the genome sequence of C. corcagiensis was determined and is presented here.

Draft Genome Sequence of Campylobacter ureolyticus Strain CIT007, the First Whole-Genome Sequence of a Clinical Isolate.

Herein, we present the draft genome sequence of Campylobacter ureolyticus. Strain CIT007 was isolated from a stool sample from an elderly female presenting with diarrheal illness and end-stage chronic renal disease.

Molecular-based detection of the gastrointestinal pathogen Campylobacter ureolyticus in unpasteurized milk samples from two cattle farms in Ireland.

Campylobacter jejuni and coli are collectively regarded as the most prevalent cause of bacterial foodborne illness worldwide. An emerging species, Campylobacter ureolyticus has recently been detected in patients with gastroenteritis, however, the source of this organism has, until now, remained unclear. Herein, we describe the molecular-based detection of this pathogen in bovine faeces (1/20) and unpasteurized milk (6/47) but not in poultry (chicken wings and caeca). This is, to the best of our knowledge, the first report of the presence of this potential gastrointestinal pathogen in an animal source, possibly suggesting a route for its transmission to humans.

Meningoencephalitis associated with non-tuberculous mycobacteria.

Non-tuberculous mycobacteria are fastidious, difficult organisms to identify, but are increasingly associated with human disease. We report a case of meningoencephalitis associated with Mycobacterium malmoense and Mycobacterium interjectum co-isolation from cerebrospinal fluid. Recognition of these slow growing mycobacteria is important due to differences from standard mycobacterial treatments. We illustrate the rare occurrence of M malmoense as a central nervous system isolate, appearing almost unique among non-tuberculous mycobacteria.

New drugs and treatment for cryptosporidiosis.

PURPOSE OF REVIEW: There is a history of inadequate treatments for cryptosporidiosis and a lack of understanding of the species that cause human disease. Against this background, we review the efficacy of antiparasitic agents, particularly nitazoxanide, which has led to increased treatment options, the potential for immunotherapy, and consider the role of highly active antiretroviral therapy in reducing the incidence of this opportunistic infection. RECENT FINDINGS: Nitazoxanide is effective for cryptosporidiosis in immunocompetent and probably immunocompromised patients (with an alteration in the duration of treatment or the dosing regimen). HIV-infected patients on highly active antiretroviral therapy have a dramatically lower incidence of cryptosporidiosis, attributable to the effects of intestinal immune reconstitution as well as the effect on the CD4 cell count. Protease inhibitors have a direct inhibitory effect on Cryptosporidium infection, suggesting a further reason for the reduction in the incidence of cryptosporidiosis and implying a further possible therapeutic modality. SUMMARY: Cryptosporidiosis remains a significant public health threat. Risk avoidance guidance could be viewed in the more relative terms of risk management depending on the degree of immunosuppression. Of established efficacy in immunocompetent patients, nitazoxanide is also useful for immunocompromised patients. Better prevention and treatment options mean that, in the immunocompromised, this disease is now less common. Immune reconstitution is the key to prevention. Further database mining of the Cryptosporidium genome will assist in the discovery of new genes, biochemical pathways and protective antigens that can be targeted to develop novel therapies for cryptosporidiosis.