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.

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.

Diagnosis and management of Aspergillus diseases: executive summary of the 2017 ESCMID-ECMM-ERS guideline.

The European Society for Clinical Microbiology and Infectious Diseases, the European Confederation of Medical Mycology and the European Respiratory Society Joint Clinical Guidelines focus on diagnosis and management of aspergillosis. Of the numerous recommendations, a few are summarized here. Chest computed tomography as well as bronchoscopy with bronchoalveolar lavage (BAL) in patients with suspicion of pulmonary invasive aspergillosis (IA) are strongly recommended. For diagnosis, direct microscopy, preferably using optical brighteners, histopathology and culture are strongly recommended. Serum and BAL galactomannan measures are recommended as markers for the diagnosis of IA. PCR should be considered in conjunction with other diagnostic tests. Pathogen identification to species complex level is strongly recommended for all clinically relevant Aspergillus isolates; antifungal susceptibility testing should be performed in patients with invasive disease in regions with resistance found in contemporary surveillance programmes. Isavuconazole and voriconazole are the preferred agents for first-line treatment of pulmonary IA, whereas liposomal amphotericin B is moderately supported. Combinations of antifungals as primary treatment options are not recommended. Therapeutic drug monitoring is strongly recommended for patients receiving posaconazole suspension or any form of voriconazole for IA treatment, and in refractory disease, where a personalized approach considering reversal of predisposing factors, switching drug class and surgical intervention is also strongly recommended. Primary prophylaxis with posaconazole is strongly recommended in patients with acute myelogenous leukaemia or myelodysplastic syndrome receiving induction chemotherapy. Secondary prophylaxis is strongly recommended in high-risk patients. We strongly recommend treatment duration based on clinical improvement, degree of immunosuppression and response on imaging.

Scedosporium prolificans osteomyelitis following penetrating injury: A case report.

Scedosporium prolificans are opportunistic moulds that can cause mycetoma following penetrating injuries. This fungus is more virulent than other species and treatment options are limited. Here we describe the first known case in the UK of S. prolificans osteomyelitis, in a 4 year old following penetrating injury. Successful outcome with limb salvage and foot function is achieved after repeated surgical debridement, and combination chemotherapy with voriconazole/terbinafine.

Predictors of large and small for gestational age birthweight in offspring of women with gestational diabetes mellitus.

AIM: To identify predictors of large and small for gestational age in women with gestational diabetes mellitus. METHODS: A retrospective audit of clinical data analysed for singleton births in women diagnosed with gestational diabetes by Australasian Diabetes in Pregnancy Society guidelines from 1994 to 2009. Exclusions were: incomplete data, delivered at < 36 weeks gestation and/or last recorded weight > 4 weeks pre-delivery. We assessed: pre-pregnancy BMI, ethnicity, total maternal weight gain, weight gain before and after treatment initiation for gestational diabetes, HbA(1c) at gestational diabetes presentation and treatment modality (diet or insulin) and smoking. Birthweight was assessed using customized percentile charts (large for gestational age > 90th; small for gestational age < 10th percentile). Multiple regression analyses were undertaken; statistical significance was p < 0.05. RESULTS: There were 1695 women first seen at (mean ± sd) 28.1 ± 5.3 weeks gestation (range 6-39). Ethnic mix was South-East Asian 36.7%, Middle Eastern 27.6%, European 22.4%, Indian/Pakistani 8.6%, Samoan 1.9%, African 1.5% and Maori 1.1%. Therapy was diet 69.1% and insulin 30.9%. Mean total weight gain was 12.3 ± 6.1 kg, the majority (10.6 ± 6.0 kg), gained before dietary intervention. There were 7.9% small for gestational age and 15.2% large for gestational age births. Significant independent large for gestational age predictors were: weight gain before intervention, pre-pregnancy BMI, weight gain after intervention and treatment type, but not HbA1c or smoking. Significant small for gestational age predictors were: weight gain before intervention, weight gain after intervention, but not pre-pregnancy BMI, HbA(1c) or smoking. CONCLUSION: Conventional treatment for gestational diabetes mellitus concentrates on management of blood glucose levels. The trends identified here emphasize the need to also address pregnancy weight gain stratified by pre-pregnancy BMI.

Bacteraemia following single-stage percutaneous dilatational tracheostomy.

OBJECTIVE: Surgical tracheostomy in critically ill adults has largely been replaced by physicians performing percutaneous dilatational tracheostomy (PDT) at the bedside. Complications associated with this technique include haemorrhage, wound infection, barotrauma, false passage, ruptured tracheal ring and bacteraemia. Prophylactic antibiotics are not generally used with this procedure, however the incidence of bacteraemia following PDT has not been extensively studied. DESIGN: Prospective observational study. SETTING: Adult intensive care unit of a university medical centre. METHODS: Peripheral venous blood cultures were obtained immediately before and after PDT in 118 consecutive patients. Surveillance cultures of potential respiratory pathogens were also recorded using routine non-directed broncholalveolar lavage. RESULTS: Forty-three female and 75 male patients underwent PDT. Fifty-seven patients (48.3%) were not receiving antibiotics on the day of PDT, whilst the remaining 61 individuals (51.7%) were on antibiotic therapy at the time of the procedure. Bacteraemia following PDT occurred in six out of 113 patients (5.3%), five of which occurred in patients not receiving antibiotics (9.2%). Unexpected bacteraemia (positive pre-and post PDT blood cultures) was identified in five patients, two of whom were on antibiotic therapy. CONCLUSION: The incidence of bacteraemia following a single stage PDT was similar to other manipulations of the aerodigestive tract such as intubation, insertion of an LMA or tooth brushing. We suggest that routine antibiotic prophylaxis is unnecessary for this procedure.

Standards of care for patients with invasive fungal infections within the United Kingdom: a national audit.

OBJECTIVE: The objective of this study was to audit the compliance and implementation of the British Society for Medical Mycology standards of care for patients with invasive infections in UK hospitals. METHODS: A multidisciplinary audit questionnaire regarding the processing of microbiology and histopathology specimens, radiology imaging and clinical management of patients with invasive fungal infections was distributed to UK hospitals. RESULTS: The study has shown that speciation of Candida and Aspergillus isolates from sterile sites was performed in 42-98% of hospitals. Microscopy of bronchoscopy specimens was not undertaken in 13 of 62 (21%) laboratories. Cryptococcal culture and antigen were undertaken routinely in abnormal CSF in 40-75% and 31-83% of at-risk patients but only in 12% of abnormal CSFs in patients without risk factors. Detailed fungal morphology was provided by <50% of histopathology departments. Most hospitals provided a timely HRCT or MRI on patients suspected to have an invasive fungal infection, but early treatment failed to occur in 15% of hospitals. In patients presenting with candidaemia, central venous catheters (CVC) were not changed routinely within 48h in 15%. CONCLUSION: Improvement in microbiology and histopathology specimen processing as well as rapid interventions such as initiation of anti-fungal therapy or CVC line removal could improve diagnostic rates and clinical outcomes of invasive fungal infections.

Clinical impact of enhanced diagnosis of invasive fungal disease in high-risk haematology and stem cell transplant patients.

AIMS: To investigate the impact of routine use of biomarkers for diagnosing fungal infection within a care pathway on antifungal usage and clinical outcomes. METHODS: A cohort of high-risk haematology and stem cell transplant patients was entered into a neutropenic care pathway in which targeted diagnostic testing replaced empiric antifungal treatment. Patients were screened twice a week by PCR and antigen testing during fever or when chronic graft versus host disease was present and were followed-up for a minimum of 1 year. RESULTS: No excess morbidity or mortality was seen in patients in whom empiric antifungal treatment was withheld, and there were substantial savings in antifungal drug expenditure. CONCLUSIONS: The introduction of a comprehensive diagnostic surveillance strategy to exclude invasive fungal infection in high-risk patients with haematological malignancy and those undergoing transplantation can result in improvements in clinical management. There are also potential additional benefits of improved patient survival, decreased morbidity and decreased hospital stay.

Sources of Differences in On-Orbital Total Solar Irradiance Measurements and Description of a Proposed Laboratory Intercomparison.

There is a 5 W/m(2) (about 0.35 %) difference between current on-orbit Total Solar Irradiance (TSI) measurements. On 18-20 July 2005, a workshop was held at the National Institute of Standards and Technology (NIST) in Gaithersburg, Maryland that focused on understanding possible reasons for this difference, through an examination of the instrument designs, calibration approaches, and appropriate measurement equations. The instruments studied in that workshop included the Active Cavity Radiometer Irradiance Monitor III (ACRIM III) on the Active Cavity Radiometer Irradiance Monitor SATellite (ACRIMSAT), the Total Irradiance Monitor (TIM) on the Solar Radiation and Climate Experiment (SORCE), the Variability of solar IRradiance and Gravity Oscillations (VIRGO) on the Solar and Heliospheric Observatory (SOHO), and the Earth Radiation Budget Experiment (ERBE) on the Earth Radiation Budget Satellite (ERBS). Presentations for each instrument included descriptions of its design, its measurement equation and uncertainty budget, and the methods used to assess on-orbit degradation. The workshop also included a session on satellite- and ground-based instrument comparisons and a session on laboratory-based comparisons and the application of new laboratory comparison techniques. The workshop has led to investigations of the effects of diffraction and of aperture area measurements on the differences between instruments. In addition, a laboratory-based instrument comparison is proposed that uses optical power measurements (with lasers that underfill the apertures of the TSI instruments), irradiance measurements (with lasers that overfill the apertures of the TSI instrument), and a cryogenic electrical substitution radiometer as a standard for comparing the instruments. A summary of the workshop and an overview of the proposed research efforts are presented here.

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.

A case of sepsis caused by Acidovorax.

This report documents a case of sepsis caused by a recently recognized environmental organism and demonstrates the pathogenicity of this bacterium in the clinical setting.

'Spiking' as a rapid method for differentiation of Candida albicans from other yeast species.

This paper describes a simple and rapid method for the differentiation of Candida albicans from other yeast species in primary cultures based on colonial morphology following incubation in carbon dioxide. The technique has superior sensitivity to the traditional germ-tube method and requires no additional laboratory tests. In a busy laboratory, this can result in significant savings in cost and time, as well as improvements in patient care.

Management and outcome of bloodstream infections due to Candida species in England and Wales.

This two-year prospective hospital population-based study of candidaemia is the first to be conducted in the UK. It was carried out on behalf on the British Society for Medical Mycology (BSMM) as part of the European Confederation of Medical Mycology (ECMM) epidemiological survey of candidaemia. Six hospitals in England and Wales acted as sentinel hospitals. Main outcome measures were hospital population-based incidence and 30-day mortality. There were 18.7 episodes of candidaemia per 100,000 finished consultant episodes or 3.0/100,000 bed days and 45.4% cases occurred in intensive care unit (ICU) patients. Candida albicans was isolated in 64.7% of confirmed cases. The majority of isolates were sensitive to standard antifungal agents, including fluconazole. The overall 30-day mortality was 26.4% and removal of the central venous catheter was associated with a significant reduction in mortality. In conclusion, the incidence of candidaemia in England and Wales is similar to that of the USA, the majority of isolates remain sensitive to commonly used antifungal agents and mortality associated with this infection appears to be falling.

Severe infections after bone marrow transplantation.

Bone marrow transplantation and stem cell transplantation have become standard therapies offering potential cures for a number of hematologic malignancies and immunologic disorders. Severe infection remains a life threatening complication after transplantation, contributes significantly to morbidity, and may necessitate admission to the ICU. It is estimated that between 20 and 40% of patients receiving bone marrow transplant will require ICU admission in the initial posttransplantation phase. Historically, survival rates after admission to the ICU are dismal, particularly if mechanical ventilation is required for respiratory failure. Other organ involvement worsens the prognosis still further and has led to proposals for rationing or restricting access to critical care units and supportive measures. Recent studies have reported small but significant improvements in outcome after critical illness. Whether this improvement is a result of changes in levels of supportive care or a more defined patient selection is uncertain. Moreover, risk factors identifying patients who will benefit most from intensive support are poorly defined. However, it is generally accepted that respiratory failure requiring invasive mechanical ventilation is associated with a poor prognosis in this patient group. Early involvement of intensivists in the management of critical illness in transplant recipients is likely to continue to improve survival in this group of patients.

Detection of meningococcal antigen by latex agglutination.

Meningococcal meningitis and septicemia are serious infections with significant morbidity and mortality. A sensitive affordable test is required to provide evidence of meningococcal disease at the earliest opportunity to improve local management and give early warning of potential outbreaks of disease. Culture of organisms is considered the gold standard for diagnosis but is slow (24 h or more) and increasingly influenced by prior antibiotic treatment. Recently, the development of polymerase chain reaction (PCR) has improved diagnosis but this sensitive assay is costly, is not available at most primary care institutions and is not feasible for developing countries. Conventional latex agglutination (LA) enables rapid detection of bacterial antigen in cerebrospinal fluid (CSF) (1,2) and can also be used to test specimens of blood (3,4) or urine (5) and for serogroup determinations on primary cultures (6,7). We discuss here test-card agglutination and also describe a new technique based upon LA in an ultrasonic standing wave that retains the speed of direct antigen testing while significantly increasing sensitivity.

Effects of Spectral Bandpass on SeaWiFS-Retrieved Near-Surface Optical Properties of the Ocean.

A simple correction method to remove the spectral bandpass effects of the Sea-viewing Wide Field-of-view Sensor (SeaWiFS) on the derived normalized water-leaving radiances and ocean-near-surface chlorophyll concentration is developed and implemented in the SeaWiFS data-processing system. SeaWiFS has not only in-band response structures but also significant sensor out-of-band contributions. The effects of the SeaWiFS out-of-band contribution at the green bands is particularly significant for the derived normalized water-leaving radiances and therefore for the retrieved ocean-near-surface chlorophyll concentration. With the sensor spectral bandpass corrections, the low chlorophyll concentration is even lower in the clear ocean regions, whereas there are almost no changes for the oceans with a chlorophyll concentration of >0.2 mg/m(3).

Calibration of SeaWiFS. I. Direct techniques.

We present an overview of the calibration of the Sea-viewing Wide Field-of View Sensor (SeaWiFS) from its performance verification at the manufacturer's facility to the completion of its third year of on-orbit measurements. These calibration procedures have three principal parts: a prelaunch radiometric calibration that is traceable to the National Institute of Standards and Technology; the Transfer-to-Orbit Experiment, a set of measurements that determine changes in the instrument's calibration from its manufacture to the start of on-orbit operations; and measurements of the sun and the moon to determine radiometric changes on orbit. To our knowledge, SeaWiFS is the only instrument that uses routine lunar measurements to determine changes in its radiometric sensitivity. On the basis of these methods, the overall uncertainty in the SeaWiFS top-of-the-atmosphere radiances is estimated to be 4-5%. We also show the results of comparison campaigns with aircraft- and ground-based measurements, plus the results of an experiment, called the Southern Ocean Band 8 Gain Study. These results are used to check the calibration of the SeaWiFS bands. To date, they have not been used to change the instrument's prelaunch calibration coefficients. In addition to these procedures, SeaWiFS is a vicariously calibrated instrument for ocean-color measurements. In the vicarious calibration of the SeaWiFS visible bands, the calibration coefficients are modified to force agreement with surface truth measurements from the Marine Optical Buoy, which is moored off the Hawaiian Island of Lanai. This vicarious calibration is described in a companion paper.