The impact of anti-mould prophylaxis on Aspergillus PCR blood testing for the diagnosis of invasive aspergillosis.

BACKGROUND: The performance of the galactomannan enzyme immunoassay (GM-EIA) is impaired in patients receiving mould-active antifungal therapy. The impact of mould-active antifungal therapy on Aspergillus PCR testing needs to be determined. OBJECTIVES: To determine the influence of anti-mould prophylaxis (AMP) on the performance of PCR blood testing to aid the diagnosis of proven/probable invasive aspergillosis (IA). METHODS: As part of the systematic review and meta-analysis of 22 cohort studies investigating Aspergillus PCR blood testing in 2912 patients at risk of IA, subgroup analysis was performed to determine the impact of AMP on the accuracy of Aspergillus PCR. The incidence of IA was calculated in patients receiving and not receiving AMP. The impact of two different positivity thresholds (requiring either a single PCR positive test result or ≥2 consecutive PCR positive test results) on accuracy was evaluated. Meta-analytical pooling of sensitivity and specificity was performed by logistic mixed-model regression. RESULTS: In total, 1661 (57%) patients received prophylaxis. The incidence of IA was 14.2%, significantly lower in the prophylaxis group (11%-12%) compared with the non-prophylaxis group (18%-19%) (P < 0.001). The use of AMP did not affect sensitivity, but significantly decreased specificity [single PCR positive result threshold: 26% reduction (P = 0.005); ≥2 consecutive PCR positive results threshold: 12% reduction (P = 0.019)]. CONCLUSIONS: Contrary to its influence on GM-EIA, AMP significantly decreases Aspergillus PCR specificity, without affecting sensitivity, possibly as a consequence of AMP limiting the clinical progression of IA and/or leading to false-negative GM-EIA results, preventing the classification of probable IA using the EORTC/MSGERC definitions.

Interlaboratory evaluation of Mucorales PCR assays for testing serum specimens: A study by the fungal PCR Initiative and the Modimucor study group.

Interlaboratory evaluations of Mucorales qPCR assays were developed to assess the reproducibility and performance of methods currently used. The participants comprised 12 laboratories from French university hospitals (nine of them participating in the Modimucor study) and 11 laboratories participating in the Fungal PCR Initiative. For panel 1, three sera were each spiked with DNA from three different species (Rhizomucor pusillus, Lichtheimia corymbifera, Rhizopus oryzae). For panel 2, six sera with three concentrations of R. pusillus and L. corymbifera (1, 10, and 100 genomes/ml) were prepared. Each panel included a blind negative-control serum. A form was distributed with each panel to collect results and required technical information, including DNA extraction method, sample volume used, DNA elution volume, qPCR method, qPCR template input volume, qPCR total reaction volume, qPCR platform, and qPCR reagents used. For panel 1, assessing 18 different protocols, qualitative results (positive or negative) were correct in 97% of cases (70/72). A very low interlaboratory variability in Cq values (SD = 1.89 cycles) were observed. For panel 2 assessing 26 different protocols, the detection rates were high (77-100%) for 5/6 of spiked serum. There was a significant association between the qPCR platform and performance. However, certain technical steps and optimal combinations of factors may also impact performance. The good reproducibility and performance demonstrated in this study support the use of Mucorales qPCR as part of the diagnostic strategy for mucormycosis.

Improved Standardization of the Bio-Rad Platelia Aspergillus Galactomannan Antigen Sandwich Enzyme Immunoassay Using the DS2 (Dynex) Enzyme-Linked Immunosorbent Assay (ELISA) Processing System.

The galactomannan enzyme immunoassay (GM-EIA) is widely utilized for the diagnosis of invasive aspergillosis (IA). There is inconsistent reproducibility of results between centers when the assay is processed manually. Automation of EIAs can reduce variation. This study investigated the semiautomation of the GM-EIA on the DS2 (Dynex) platform in the following three stages: (i) DS2 GM-EIA method validation with experimental samples, (ii) DS2 retesting of case-defined clinical samples, and (iii) a 12-month audit of DS2 GM-EIA performance. In stage i, Bland-Altman analysis demonstrated a reduced variance between optical density index (ODI) values for samples processed on two DS2 platforms (mean difference, -0.02; limits of agreement [LOA], -0.19 to 0.14) compared with the variance between samples processed manually and on a DS2 platform (mean difference, 0.02; LOA, -0.25 to 0.3). In stage ii, 100% (14/14 samples) qualitative agreement was observed for serum samples from patients with IA, with no significant change in the ODI values when samples were processed on the DS2 platform. A significant decrease in ODI values was observed for control serum samples on the DS2 platform (difference, 0.01; P = 0.042). In stage iii, a significant reduction in the frequency of equivocal results, from 5.56% (136/2,443 samples) to 1.56% (15/961 samples), was observed after DS2 automation (difference, 4.0%; 95% confidence interval [CI], 2.7 to 5.2%; P < 0.01), with an equivalent increase in negative results. This study demonstrates that GM-EIA automation may reduce intersite variability. Automation does not have an impact on the repeatability of truly positive results but contributes to a reduction in false-positive (equivocal) GM-EIA results, reducing the need to retest a significant proportion of samples.

Outbreak of Salmonella enterica serotype I 4,5,12:i:- infections: the challenges of hypothesis generation and microwave cooking.

We investigated an outbreak of 396 Salmonella enterica serotype I 4,5,12:i:- infections to determine the source. After 7 weeks of extensive hypothesis-generation interviews, no refined hypothesis was formed. Nevertheless, a case-control study was initiated. Subsequently, an iterative hypothesis-generation approach used by a single interviewing team identified brand A not-ready-to-eat frozen pot pies as a likely vehicle. The case-control study, modified to assess this new hypothesis, along with product testing indicated that the turkey variety of pot pies was responsible. Review of product labels identified inconsistent language regarding preparation, and the cooking instructions included undefined microwave wattage categories. Surveys found that most patients did not follow the product's cooking instructions and did not know their oven's wattage. The manufacturer voluntarily recalled pot pies and improved the product's cooking instructions. This investigation highlights the value of careful hypothesis-generation and the risks posed by frozen not-ready-to-eat microwavable foods.

Determination of the Prevalence of Triazole Resistance in Environmental Aspergillus fumigatus Strains Isolated in South Wales, UK.

Background/Objectives: Azole resistance in Aspergillus fumigatus associated with the TR34/L98H mutations in the cyp51A gene have been increasingly reported. Determining the environmental resistance rate has been deemed important when considering front-line therapy for invasive aspergillosis. The aim of the study was to determine prevalence of azole resistance in environmental A. fumigatus isolates across South Wales. Methods: Over 5 months in 2015, 513 A. fumigatus isolates were cultured from 671 soil and 44 air samples and were screened for azole resistance using VIPcheck™ agar plates containing itraconazole, voriconazole and posaconazole. Resistance was confirmed by the CLSI M38-A2 methodology. The mechanism of resistance was investigated using the PathoNostics AsperGenius® Assay. Results: Screening by VIPcheck™ plate identified azole-resistance in 30 isolates, most of which (28/30) harbored the TR34/L98H mutation, generating a prevalence of 6.0%. Twenty-five isolates had a MIC of ≥2 mg/L with itraconazole, 23 isolates had a MIC of ≥2 mg/L with voriconazole and seven isolates had a MIC ≥0.25 mg/L with posaconazole. All isolates deemed resistant by VIPcheck™ plates were resistant to at least one azole by reference methodology. Conclusions: There is significant environmental azole resistance (6%) in South Wales, in close proximity to patients susceptible to aspergillosis. Given this environmental reservoir, azole resistance should be routinely screened for in clinical practice and environmental monitoring continued.

Antifungal susceptibility of Candida species using the Clinical and Laboratory Standards Institute disk diffusion and broth microdilution methods.

There are conflicting reports on the agreement between the Clinical and Laboratory Standards Institute disk diffusion (M44-A) and reference broth microdilution (M27-A) methods for determination of antifungal susceptibility of yeasts. The antifungal susceptibility of 541 yeasts, the majority of which were from the oral cavity, was determined using these two methods and the accuracy of the disk diffusion method assessed for clinical testing of various Candida species. Of the strains tested, Candida albicans predominated (390 out of 541). The classification of susceptibility determined by the disk diffusion method was largely in concordance with that obtained using the broth dilution method, regardless of species within Candida genus. The overall observed agreement between these two methods was 94.7% for fluconazole and 96.7% for voriconazole was with a 'very major' discrepancy level of 1.5% and 1.7% respectively. This study demonstrates a strong agreement of the simple disk diffusion method with the more labour intensive 'gold standard' broth microdilution method. These findings would support the use of the disk diffusion method in a routine mycology service.

Polymerase chain reaction aids in the diagnosis of an unusual case of Aspergillus niger endocarditis in a patient with acute myeloid leukaemia.

Endocarditis secondary to Aspergillus niger has not been described in a leukaemic patient. We describe a case of A. niger endocarditis in a patient with acute myeloid leukaemia and refractory fever. The microbiological cause of his endocarditis was initially misdiagnosed because he fulfilled the Duke criteria for enterococcal endocarditis. A polymerase chain reaction test utilizing pan-fungal primers detected a product from an Aspergillus sp. The DNA was subsequently sequenced and was found to have 100% homology with A. niger. A postmortem revealed fungal endocarditis secondary to disseminated aspergillosis, without evidence of bacterial endocarditis. The patient was found to have a lung aspergilloma that was possibly occupationally acquired, and may have been long standing.

Perinatal asphyxia II: Clinical management of multisystemic sequelae.

This paper discusses the management of multisystemic sequelae of perinatal asphyxia. Appropriate intrapartum and subsequent adequate neonatal monitoring are an absolute necessity for the early detection of systemic dysfunctions. Prophylactic and therapeutic measures applicable to the various organ malfunctions are described.

Perinatal asphyxia I: Pathogenesis of multisystemic sequelae.

This paper describes the clinical and pathologic sequelae of perinatal asphyxia manifested by 17 neonates treated at Howard University Hospital over an 18-month period. Multiple systemic complications, occurring in 76.5 percent of the patients, were the rule rather than the exception. All vital organs were involved, singly or in combination. Understanding the pathogenesis and extent of these complications is of utmost importance not only to those rendering health care to acutely ill newborns, but also to those responsible for prenatal and maternal intrapartum care.

Strategies to control Salmonella and Campylobacter in raw poultry products.

Foodborne illness is a major public health concern. The largest number of foodborne illness cases attributed to poultry and poultry products are caused by paratyphoid serotypes of Salmonella and by Campylobacter jejuni. The effective prevention of foodborne disease requires an understanding that contamination can be introduced into foods at numerous points along the food chain. Since multiple entry points exist for foodborne pathogens, multifaceted intervention approaches are required to successfully control contamination of poultry during the various phases of the growth period and processing procedure of broiler chickens. Strategies during the grow-out period (the period during which day-old chicks are raised to six- to seven-week-old broiler chickens) include sanitation, biosecurity, vaccine and drug therapy, and biological control procedures, such as those aimed at preventing colonisation. There are also many critical control points identified in the processing plant which reduce contamination. These include temperature controls (washer and product), chemical interventions, water replacements and counter-flow technology in the scalder and chiller, and equipment maintenance. Transportation and food handling at retail outlets and by the consumer (i.e., storage at the proper temperature and adequate cooking) are the final critical control points in the farm-to-table continuum. It is important to apply risk reduction strategies throughout the food chain. These include: easing the development and implementation of voluntary animal production 'best management practices', implementing in-plant hazard analysis and critical control point systems, developing effective transportation and refrigeration standards, working to facilitate adoption of the model Food Code in all States and providing educational materials and support for public health activities nationwide.

Comparative accuracy of recent abbreviated methods of gestational age determination.

Fifty healthy newborn infants were studied in an attempt to test the accuracy of several recently reported shortened methods of gestational age determination. The Finnstrom method with seven external characteristics was more accurate than those that contained only five or less criteria. In the statistical analysis it also yielded results that were very similar to those obtained using the original Dubowitz technique which has 21 criteria.

Pathogen Reduction and Hazard Analysis and Critical Control Point (HACCP) systems for meat and poultry. USDA.

The United States Department of Agriculture (USDA) Food Safety Inspection Service (FSIS) adopted Hazard Analysis and Critical Control Point Systems and established finished product standards for Salmonella in slaughter plants to improve food safety for meat and poultry. In order to make significant improvements in food safety, measures must be taken at all points in the farm-to-table chain including production, transportation, slaughter, processing, storage, retail, and food preparation. Since pathogens can be introduced or multiplied anywhere along the continuum, success depends on consideration and comparison of intervention measures throughout the continuum. Food animal and public health veterinarians can create the necessary preventative environment that mitigates risks for food borne pathogen contamination.

Vitamin preparations: proper use in medical practice.

The prescription of vitamin preparations as dietary supplements should be limited to specific instances of need and should be accompanied by appropriate measures to correct poor diets. Similarly, specific vitamins in therapeutic amounts should be prescribed only in the presence of vitamin deficiencies or increased requirements.

Multistate outbreak of multidrug-resistant Salmonella newport infections associated with ground beef, October to December 2007.

In late October 2007, an outbreak of multidrug-resistant Salmonella Newport infections affected 42 case patients in California, Arizona, Idaho, and Nevada. A case-control study implicated ground beef from one chain store. Despite detailed ground beef purchase histories--including shopper card information for several case patients--traceback efforts by both the U.S. Department of Agriculture, Food Safety and Inspection Service and the California Department of Public Health were unable to identify the source of contamination. Case patients consumed multiple types of ground beef products purchased at numerous chain store A retail locations. These stores had received beef products for grinding from multiple beef slaughter-processing establishments. Detailed retail grinding logs and grinding policies that prevent cross-contamination between batches of ground beef products are crucial in the identification of contaminated beef products associated with foodborne illness.