Direct assessment of possible mutations in the 23S rRNA gene encoding macrolide resistance in Chlamydia trachomatis.

Reports of potential treatment failure have raised particular concerns regarding the efficacy of the single dose azithromycin regimen in the treatment of urogenital and anorectal Chlamydia trachomatis (CT) infections. Several factors have been suggested, including heterotypic resistance. Antimicrobial susceptibility testing in CT requires cell culture with serial dilutions of antibiotics, which is laborious and for which there is no standardized testing methodology. One method to partly overcome these difficulties would be to use a genotypic resistance assay, however most current available assays do still require prior CT culture. In order to facilitate the assessment of genotypic resistance directly from clinical samples, without the need for prior culture, the aim of this study was to develop a CT specific PCR assay for the assessment of resistance associated mutations (RAMs) in the 23S rRNA gene, and to evaluate a sample of clinical cases in which CT PCR's remained positive during follow-up despite azithromycin treatment. Neither the in silico analysis nor the analytical specificity testing demonstrated clinically relevant cross-reactivity with other bacterial species. These results in conjunction with the analytical sensitivity demonstrating consistent CT 23S rRNA gene detection in the range of 10e3 IFU/mL, exemplify the assay's apt performance. Although no known macrolide RAMs were detected in the clinical cases, the described assay allows future culture independent macrolide RAM surveillance in CT, and increases accessibility for other laboratories to engage in screening.

Spatiotemporal prediction of vancomycin-resistant Enterococcus colonisation.

BACKGROUND: Vancomycin-resistant enterococci (VRE) is the cause of severe patient health and monetary burdens. Antibiotic use is a confounding effect to predict VRE in patients, but the antibiotic use of patients who may have frequented the same ward as the patient in question is often neglected. This study investigates how patient movements between hospital wards and their antibiotic use can explain the colonisation of patients with VRE. METHODS: Intrahospital patient movements, antibiotic use and PCR screening data were used from a hospital in the Netherlands. The PageRank algorithm was used to calculate two daily centrality measures based on the spatiotemporal graph to summarise the flow of patients and antibiotics at the ward level. A decision tree model was used to determine a simple set of rules to estimate the daily probability of patient VRE colonisation for each hospital ward. The model performance was improved using a random forest model and compared using 30% test sample. RESULTS: Centrality covariates summarising the flow of patients and their antibiotic use between hospital wards can be used to predict the daily colonisation of VRE at the hospital ward level. The decision tree model produced a simple set of rules that can be used to determine the daily probability of patient VRE colonisation for each hospital ward. An acceptable area under the ROC curve (AUC) of 0.755 was achieved using the decision tree model and an excellent AUC of 0.883 by the random forest model on the test set. These results confirms that the random forest model performs better than a single decision tree for all levels of model sensitivity and specificity on data not used to estimate the models. CONCLUSION: This study showed how the movements of patients inside hospitals and their use of antibiotics could predict the colonisation of patients with VRE at the ward level. Two daily centrality measures were proposed to summarise the flow of patients and antibiotics at the ward level. An early warning system for VRE can be developed to test and further develop infection prevention plans and outbreak strategies using these results.

A spatiotemporal simulation study on the transmission of harmful microorganisms through connected healthcare workers in a hospital ward setting.

BACKGROUND: Hand transmission of harmful microorganisms may lead to infections and poses a major threat to patients and healthcare workers in healthcare settings. The most effective countermeasure against these transmissions is the adherence to spatiotemporal hand hygiene policies, but adherence rates are relatively low and vary over space and time. The spatiotemporal effects on hand transmission and spread of these microorganisms for varying hand hygiene compliance levels are unknown. This study aims to (1) identify a healthcare worker occupancy group of potential super-spreaders and (2) quantify spatiotemporal effects on the hand transmission and spread of harmful microorganisms for varying levels of hand hygiene compliance caused by this group. METHODS: Spatiotemporal data were collected in a hospital ward of an academic hospital using radio frequency identification technology for 7 days. A potential super-spreader healthcare worker occupation group was identified using the frequency identification sensors' contact data. The effects of five probability distributions of hand hygiene compliance and three harmful microorganism transmission rates were simulated using a dynamic agent-based simulation model. The effects of initial simulation assumptions on the simulation results were quantified using five risk outcomes. RESULTS: Nurses, doctors and patients are together responsible for 81.13% of all contacts. Nurses made up 70.68% of all contacts, which is more than five times that of doctors (10.44%). This identifies nurses as the potential super-spreader healthcare worker occupation group. For initial simulation conditions of extreme lack of hand hygiene compliance (5%) and high transmission rates (5% per contact moment), a colonised nurse can transfer microbes to three of the 17 healthcare worker or patients encountered during the 98.4 min of visiting 23 rooms while colonised. The harmful microorganism transmission potential for nurses is higher during weeknights (5 pm - 7 am) and weekends as compared to weekdays (7 am - 5 pm). CONCLUSION: Spatiotemporal behaviour and social mixing patterns of healthcare can change the expected number of hand transmissions and spread of harmful microorganisms by super-spreaders in a closed healthcare setting. These insights can be used to evaluate spatiotemporal safety behaviours and develop infection prevention and control strategies.

Despite Excellent Test Characteristics of the cobas 4800 CT/NG Assay, Detection of Oropharyngeal Chlamydia trachomatis and Neisseria gonorrhoeae Remains Challenging.

Oropharyngeal Chlamydia trachomatis (CT) infections and, especially, Neisseria gonorrhoeae (NG) infections are common, but few commercial nucleic acid amplification tests (NAATs) specify extragenital samples for intended use. The test characteristics of the cobas 4800 CT/NG assay were evaluated for oropharyngeal swabs. The technical validation included analysis of the specificity, sensitivity, dynamic range, linearity, efficiency, and precision. The probability of detection curve combined with historical data enabled the estimation of potentially missed diagnoses. A clinical evaluation was performed on a subset of 2,798 clinical samples available from routine diagnostics. Results of the cobas 4800 were compared with those from in-house C. trachomatis/N. gonorrhoeae PCR assays. Discrepant samples were tested with resolver assays, and these results were considered decisive. No cross-reactivity was seen in the analytical specificity analysis. High linearity (R2 ≥ 0.983), efficiency (89% to 99%), and precision (cycle threshold [CT ] value of 0.1 to 0.9) were seen for both C. trachomatis and N. gonorrhoeae The limit of detection in oropharyngeal samples was 3.2 × 102 inclusion-forming units (IFU)/ml for C. trachomatis and 6.7 × 102 CFU/ml for N. gonorrhoeae Estimates on potentially missed diagnoses were up to 7.2% for C. trachomatis and up to 24.7% for N. gonorrhoeae Clinical sensitivity and specificity were evaluated with 25 C. trachomatis-positive, 86 N. gonorrhoeae-positive, and 264 negative samples, resulting in 100% and 99.6% for C. trachomatis and 100% and 96.7% for N. gonorrhoeae, respectively. The findings in this study demonstrate the utility of the cobas 4800 CT/NG assay for oropharyngeal samples. Despite its being a highly accurate test, the range of reported CT values, especially for N. gonorrhoeae, suggests relatively low oropharyngeal loads. Hence, consistent detection over the full range of oropharyngeal loads could be impaired.

Risk factors for surgical site infections using a data-driven approach.

OBJECTIVE: The objective of this study was to identify risk factors for surgical site infection from digestive, thoracic and orthopaedic system surgeries using clinical and data-driven cut-off values. A second objective was to compare the identified risk factors in this study to risk factors identified in literature. SUMMARY BACKGROUND DATA: Retrospective data of 3 250 surgical procedures performed in large tertiary care hospital in The Netherlands during January 2013 to June 2014 were used. METHODS: Potential risk factors were identified using a literature scan and univariate analysis. A multivariate forward-step logistic regression model was used to identify risk factors. Standard medical cut-off values were compared with cut-offs determined from the data. RESULTS: For digestive, orthopaedic and thoracic system surgical procedures, the risk factors identified were preoperative temperature of ≥38°C and antibiotics used at the time of surgery. C-reactive protein and the duration of the surgery were identified as a risk factors for digestive surgical procedures. Being an adult (age ≥18) was identified as a protective effect for thoracic surgical procedures. Data-driven cut-off values were identified for temperature, age and CRP which can explain the SSI outcome up to 19.5% better than generic cut-off values. CONCLUSIONS: This study identified risk factors for digestive, orthopaedic and thoracic system surgical procedures and illustrated how data-driven cut-offs can add value in the process. Future studies should investigate if data-driven cut-offs can add value to explain the outcome being modelled and not solely rely on standard medical cut-off values to identify risk factors.

First Report of Lasiodiplodia crassispora as a Pathogen of Grapevine Trunks in South Africa.

In 2003 and 2004, a survey of grapevine (Vitis vinifera L.) trunk pathogens was conducted in 30 vineyards in the Western and Northern Cape and Limpopo provinces of South Africa. In each vineyard, 20 visually healthy plants were sampled randomly by removing the distal part of one cordon arm. Isolations were made onto potato dextrose agar (PDA) from the internal wood decay symptoms observed in the cordon samples. Seven Botryosphaeriaceae spp. were identified, including Lasiodiplodia crassispora (1). Other Botryosphaeriaceae spp. are known grapevine trunk pathogens (2). Species identity was confirmed by DNA sequence data of the partial translation factor 1-α gene (1) and sequences deposited in GenBank (GU233658 and GU233659). The L. crassispora isolates (CBS 125626 and 125627) were associated with brown internal necrosis, a known symptom of grapevine Botryosphaeriaceae spp. infection (3), in the cordon arms of Ruby Cabernet grapevines occurring in two vineyards in the Northern Cape Province. L. crassispora was described from cankered wood of Santalum album in Western Australia and endophytically from Eucalyptus urophylla in Venezuela (1). Its grapevine pathogen status was determined using both isolates in a repeated pathogenicity test that included three isolates each of Botryosphaeria dothidea and Neofusicoccum australe as positive controls (2), Trichoderma harzianum as a nonpathogen treatment, and an uncolonized agar plug as a negative control. The Botryosphaeriaceae spp. and T. harzianum were plated on PDA and incubated at 25°C for 7 days. Lignified, 6-month-old shoots of grapevine cv. Chardonnay were excised from grapevines with internodes 4 to 6 used for inoculations. Before wounding, shoots were disinfected by submersion for 1 min in a 1 ml/liter solution of a quaternary ammonium compound (Sporekill; ICA International Chemicals (Pty) Ltd, Stellenbosch, South Africa). Twelve shoots were used for each isolate or control treatment. Wounds were made 2 mm deep on the fifth internode of the shoots with a 5-mm flame-sterilized cork borer (2,3). Wounds were inoculated with a pathogen colonized agar plug (5 mm in diameter) or an uncolonized agar plug and then covered with Parafilm (2,3). Inoculated shoots were incubated in the dark in moist chambers for 14 days at 25°C. After incubation, the bark of the shoots was peeled from the area around the wound and the lengths of any resultant lesions were measured under sterile conditions. The inoculum effect was assessed by analysis of variance and Student's t-test. Results showed that significantly (P < 0.0001) longer lesions were caused by L. crassispora (13.36 mm) compared with N. australe (9.27 mm) and B. dothidea (5.28 mm) and also significantly longer than lesions caused by the nonpathogen and negative controls (3.23 and 2.90 mm, respectively). To determine if lesions were caused by inoculated fungi, isolations were made from the tissue at the edges of the lesions by aseptically removing five 0.5 × 1 mm pieces of wood and placing them on PDA dishes amended with 0.04 g/liter of streptomycin sulfate. Dishes were incubated under normal fluorescent light at 25°C for 14 days before identifying isolated fungi based on morphological and cultural characteristics (1). To our knowledge, this is the first report of L. crassispora as a grapevine pathogen. References: (1) T. I. Burgess et al. Mycologia 98:423, 2006. (2) J. M. van Niekerk et al. Mycologia 96:781, 2004. (4) J. M. van Niekerk et al. Phytopathol. Mediterr. 45:S43, 2006.