Determining the analytical specificity of PCR-based assays for the diagnosis of IA: What is Aspergillus?

A wide array of PCR tests has been developed to aid the diagnosis of invasive aspergillosis (IA), providing technical diversity but limiting standardisation and acceptance. Methodological recommendations for testing blood samples using PCR exist, based on achieving optimal assay sensitivity to help exclude IA. Conversely, when testing more invasive samples (BAL, biopsy, CSF) emphasis is placed on confirming disease, so analytical specificity is paramount. This multicenter study examined the analytical specificity of PCR methods for detecting IA by blind testing a panel of DNA extracted from a various fungal species to explore the range of Aspergillus species that could be detected, but also potential cross reactivity with other fungal species. Positivity rates were calculated and regression analysis was performed to determine any associations between technical specifications and performance. The accuracy of Aspergillus genus specific assays was 71.8%, significantly greater (P < .0001) than assays specific for individual Aspergillus species (47.2%). For genus specific assays the most often missed species were A. lentulus (25.0%), A. versicolor (24.1%), A. terreus (16.1%), A. flavus (15.2%), A. niger (13.4%), and A. fumigatus (6.2%). There was a significant positive association between accuracy and using an Aspergillus genus PCR assay targeting the rRNA genes (P = .0011). Conversely, there was a significant association between rRNA PCR targets and false positivity (P = .0032). To conclude current Aspergillus PCR assays are better suited for detecting A. fumigatus, with inferior detection of most other Aspergillus species. The use of an Aspergillus genus specific PCR assay targeting the rRNA genes is preferential.

Bronchial colonization and complications after lung cancer surgery.

PURPOSE: Infectious complications occur following pulmonary resections preceded or not by induction chemoradiotherapy. We aimed to investigate whether bacterial colonization of the bronchial tree at the time of surgery was associated with postoperative complications. PATIENTS AND METHODS: A retrospective analysis of all patients who underwent open anatomical pulmonary resections for malignancies at a single center was performed. Demographical data of the included patients, intraoperative data, and data on the postoperative course of patients were collected. Outcome of patients with a positive intraoperative bronchial culture was compared to patients with a negative bronchial culture. Relations between the presence of potential bacterial pathogens in the bronchial tree and other possible risk factors for the development of postoperative infectious and non-infectious complications, were analyzed using uni- and multivariate analysis. RESULTS: Between January 2010 and January 2012, a total of 121 consecutive patients underwent open anatomical pulmonary resections for malignancy, of whom 45 were preceded by induction chemoradiotherapy and 5 by induction chemotherapy. Intraoperative bronchial cultures were taken from 58 patients (48 %). Patients with a positive bronchial culture developed significantly more infectious (88 % vs. 20 %, p < 0.001) and non-infectious complications (63 % vs. 12 %, p = 0.001). Positive intraoperative bronchial cultures showed the strongest association with the development of infectious and non-infectious postoperative complications (OR 24.8 and 12.2, respectively). After multivariate analysis, only BMI less than 20 kg/m(2) and the presence of a positive intraoperative bronchial culture were found to be independent risk factors for the development of infectious complications. Chemoradiotherapy was not associated with postoperative complications in the present study. CONCLUSIONS: Bacterial colonization of the bronchial tree assessed intraoperatively, appears to be associated with higher rates of infectious and non-infectious complications after pulmonary resection. Whether early starting of appropriate antibiotics based on intraoperative-taken culture findings will reduce the infectious complication rate in a subcategory of patients needs to be investigated.

Galactomannan detection for invasive aspergillosis in immunocompromised patients.

BACKGROUND: Invasive aspergillosis is the most common life-threatening opportunistic invasive mycosis in immunocompromised patients. A test for invasive aspergillosis should neither be too invasive nor too great a burden for the already weakened patient. The serum galactomannan enzyme-linked immunosorbent assay (ELISA) seems to have the potential to meet both requirements. OBJECTIVES: To obtain summary estimates of the diagnostic accuracy of galactomannan detection in serum for the diagnosis of invasive aspergillosis. SEARCH METHODS: We searched MEDLINE, EMBASE and Web of Science with both MeSH terms and text words for both aspergillosis and the sandwich ELISA. We checked the reference lists of included studies and review articles for additional studies. We conducted the searches in February 2014. SELECTION CRITERIA: We included cross-sectional studies, case-control designs and consecutive series of patients assessing the diagnostic accuracy of galactomannan detection for the diagnosis of invasive aspergillosis in patients with neutropenia or patients whose neutrophils are functionally compromised. The reference standard was composed of the criteria given by the European Organization for Research and Treatment of Cancer (EORTC) and the Mycoses Study Group (MSG). DATA COLLECTION AND ANALYSIS: Two review authors independently assessed quality and extracted data. We carried out meta-analysis using the bivariate method. We investigated sources of heterogeneity by adding potential sources of heterogeneity to the model as covariates. MAIN RESULTS: We included 54 studies in the review (50 in the meta-analyses), containing 5660 patients, of whom 586 had proven or probable invasive aspergillosis. When using an optical density index (ODI) of 0.5 as a cut-off value, the sensitivity of the test was 82% (73% to 90%) and the specificity was 81% (72% to 90%). At a cut-off value of 1.0 ODI, the sensitivity was 72% (65% to 80%) and the specificity was 88% (84% to 92%). At a cut-off value of 1.5 ODI, the sensitivity was 61% (47% to 75%) and the specificity was 93% (89% to 97%). None of the potential sources of heterogeneity had a statistically significant effect on either sensitivity or specificity. AUTHORS' CONCLUSIONS: If we used the test at a cut-off value of 0.5 ODI in a population of 100 patients with a disease prevalence of 9% (overall median prevalence), two patients who have invasive aspergillosis would be missed (sensitivity 82%, 18% false negatives), and 17 patients would be treated unnecessarily or referred unnecessarily for further testing (specificity 81%, 19% false negatives). If we used the test at a cut-off value of 1.5 in the same population, that would mean that four invasive aspergillosis patients would be missed (sensitivity 61%, 39% false negatives), and six patients would be treated or referred for further testing unnecessarily (specificity 93%, 7% false negatives). These numbers should, however, be interpreted with caution because the results were very heterogeneous.

Prosthetic joint-associated infections treated with DAIR (debridement, antibiotics, irrigation, and retention): analysis of risk factors and local antibiotic carriers in 91 patients.

BACKGROUND AND PURPOSE: For prosthetic joint-associated infection (PJI), a regimen of debridement, antibiotics, irrigation, and retention of the prosthesis (DAIR) is generally accepted for acute infections. Various risk factors associated with treatment success have been described. The use of local antibiotic carriers (beads and sponges) is relatively unknown. We retrospectively analyzed risk factors in a cohort of patients from 3 hospitals, treated with DAIR for PJI. PATIENTS AND METHODS: 91 patients treated with DAIR for hip or knee PJI in 3 Dutch centers between 2004 and 2009 were retrospectively evaluated. The mean follow-up was 3 years. Treatment success was defined as absence of infection after 2 years, with retention of the prosthesis and without the use of suppressive antibiotics. RESULTS: 60 patients (66%) were free of infection at follow-up. Factors associated with treatment failure were: a history of rheumatoid arthritis, late infection (> 2 years after arthroplasty), ESR at presentation above 60 mm/h, and infection caused by coagulase-negative Staphylococcus. Symptom duration of less than 1 week was associated with treatment success. The use of gentamicin sponges was statistically significantly higher in the success group, and the use of beads was higher in the failure group in the univariate analysis, but these differences did not reach significance in the logistic regression analysis. Less surgical procedures were performed in the group treated with sponges than in the group treated with beads. INTERPRETATION: In the presence of rheumatoid arthritis, duration of symptoms of more than 1 week, ESR above 60 mm/h, late infection (> 2 years after arthroplasty), and coagulase-negative Staphylococcus PJI, the chances of successful DAIR treatment decrease, and other treatment methods should be considered.

Aspergillosis due to voriconazole highly resistant Aspergillus fumigatus and recovery of genetically related resistant isolates from domiciles.

BACKGROUND: Azole resistance is an emerging problem in Aspergillus fumigatus and complicates the management of patients with Aspergillus-related diseases. Selection of azole resistance may occur through exposure to azole fungicides in the environment. In the Netherlands a surveillance network was used to investigate the epidemiology of resistance selection in A. fumigatus. METHODS: Clinical A. fumigatus isolates were screened for azole resistance in 8 university hospitals using azole agar dilution plates. Patient information was collected using an online questionnaire and azole-resistant A. fumigatus isolates were analyzed using gene sequencing, susceptibility testing, and genotyping. Air sampling was performed to investigate the presence of resistant isolates in hospitals and domiciles. RESULTS: Between December 2009 and January 2011, 1315 A. fumigatus isolates from 921 patients were screened. A new cyp51A-mediated resistance mechanism (TR46/Y121F/T289A) was observed in 21 azole-resistant isolates from 15 patients in 6 hospitals. TR46/Y121F/T289A isolates were highly resistant to voriconazole (minimum inhibitory concentration ≥16 mg/L). Eight patients presented with invasive aspergillosis due to TR46/Y121F/T289A, and treatment failed in all 5 patients receiving primary therapy with voriconazole. TR46/Y121F/T289A Aspergillus fumigatus was recovered from 6 of 10 sampled environmental sites. CONCLUSIONS: We describe the emergence and geographical migration of a voriconazole highly resistant A. fumigatus that was associated with voriconazole treatment failure in patients with invasive aspergillosis. Recovery of TR46/Y121F/T289A from the environment suggests an environmental route of resistance selection. Exposure of A. fumigatus to azole fungicides may facilitate the emergence of new resistance mechanisms over time, thereby compromising the use of azoles in the management of Aspergillus-related diseases.

Old and new biomarkers for predicting high and low risk microbial infection in critically ill patients with new onset fever: a case for procalcitonin.

OBJECTIVES: Fever suggests the presence of microbial infection in critically ill patients. The aim was to compare the role of old and new biomarkers in predicting absence or presence of microbial infection, its invasiveness and severity in critically ill patients with new onset fever. METHODS: We prospectively studied 101 patients in the intensive care unit with new onset fever (>38.3 °C). Routine infection parameters, lactate, procalcitonin (PCT), midregional pro-adrenomedullin (MR proADM), midregional pro-atrial natriuretic peptide (MR proANP) and copeptin (COP) were measured daily for three days after inclusion. Likelihood, invasiveness (by bloodstream infection, BSI) and severity of microbial infection were assessed by cultures, imaging techniques and clinical courses. RESULTS: All patients had systemic inflammatory response syndrome; 45% had a probable or proven local infection and 12% a BSI, with 20 and 33% mortality in the ICU, respectively. Only peak PCT (cutoff 0.65 ng/mL at minimum) was of predictive value for all endpoints studied, i.e. BSI, septic shock and mortality (high risk infection) and infection without BSI, shock and mortality (low risk infection), at areas under the receiver operating characteristic curves varying between 0.67 (P = 0.003) and 0.72 (P < 0.001). In multivariable analysis, the combination of C-reactive protein and lactate best predicted high risk infection, followed by PCT. For low risk infection, PCT was the single best predictor. CONCLUSIONS: In critically ill patients with new onset fever, plasma PCT as a single variable, among old and new biomarkers, best helps, to some extent, to predict ICU-acquired, high risk microbial infection when peaking above 0.65 ng/mL and low risk infection when peaking below 0.65 ng/mL.

Clinical implications of azole resistance in Aspergillus fumigatus, The Netherlands, 2007-2009.

The prevalence and spread of azole resistance in clinical Aspergillus fumigatus isolates in the Netherlands are currently unknown. Therefore, we performed a prospective nationwide multicenter surveillance study to determine the effects of resistance on patient management strategies and public health. From June 2007 through January 2009, all clinical Aspergillus spp. isolates were screened for itraconazole resistance. In total, 2,062 isolates from 1,385 patients were screened; the prevalence of itraconazole resistance in A. fumigatus in our patient cohort was 5.3% (range 0.8%-9.5%). Patients with a hematologic or oncologic disease were more likely to harbor an azole-resistant isolate than were other patient groups (p<0.05). Most patients (64.0%) from whom a resistant isolate was identified were azole naive, and the case-fatality rate of patients with azole-resistant invasive aspergillosis was 88.0%. Our study found that multiazole resistance in A. fumigatus is widespread in the Netherlands and is associated with a high death rate for patients with invasive aspergillosis.

Characterization of DIM-1, an integron-encoded metallo-beta-lactamase from a Pseudomonas stutzeri clinical isolate in the Netherlands.

A carbapenem-resistant Pseudomonas stutzeri strain isolated from a Dutch patient was analyzed in detail. This isolate produced a metallo-beta-lactamase (MBL) whose gene, with 43.5% GC content, was cloned and expressed in Escherichia coli. beta-Lactamase DIM-1 (for Dutch imipenemase) was weakly related to other Ambler class B beta-lactamases, sharing <52% amino acid identity with the most closely related MBL, GIM-1, and 45% identity with IMP-type MBLs. The beta-Lactamase DIM-1 significantly hydrolyzed broad-spectrum cephalosporins and carbapenems and spared aztreonam. This MBL gene was embedded in a class 1 integron containing two other gene cassettes, encoding resistance to aminoglycosides and disinfectants, that was located on a 70-kb plasmid.

Enrichment broth improved detection of extended-spectrum-beta-lactamase-producing bacteria in throat and rectal surveillance cultures of samples from patients in intensive care units.

We evaluated the use of a trypticase soy broth (TSB) for improving detection of extended-spectrum-beta-lactamase-producing (ESBL(+)) bacteria. Preenrichment of throat and rectal swabs in TSB prior to inoculation on solid medium doubled the number of ESBL(+) bacteria detected in samples obtained from patients in our intensive care unit.

Galactomannan detection for invasive aspergillosis in immunocompromized patients.

BACKGROUND: Invasive aspergillosis (IA) is the most common life-threatening opportunistic invasive mycosis in immunocompromized patients. A test for IA needs to be not too invasive and not too big a burden for the already weakened patient. The serum galactomannan ELISA seems to have potential for both requirements. OBJECTIVES: To obtain summary estimates of the diagnostic accuracy of galactomannan detection in serum for the diagnosis of IA. SEARCH STRATEGY: We searched MEDLINE, EMBASE and Web of Science with both Medical Headings and text words for both aspergillosis and the sandwich ELISA. We checked reference lists of included studies and review articles for additional studies. SELECTION CRITERIA: Cross-sectional studies, case-control designs and consecutive series of patients assessing the diagnostic accuracy of galactomannan detection for the diagnosis of IA in patients with neutropenia or patients whose neutrophils are functionally compromised were included. The reference standard was composed of the criteria given by the European Organization for Research and Treatment of Cancer (EORTC) and the Mycoses Study Group (MSG). DATA COLLECTION AND ANALYSIS: Two review authors independently assessed quality and extracted data MAIN RESULTS: Thirty studies were included in the meta-analyses, with a median prevalence of IA (proven or probable) of 7.7%. Seven of these (901 patients) reported results for an Optical Density Index (ODI) of 0.5 as cut-off value. The overall sensitivity in these studies was 78% (61% to 89%) and overall specificity was 81% (72% to 88%). Twelve studies (1744 patients) reported the results for cut-off value of 1.0 ODI, overall sensitivity was 75% (59% to 86%) and mean specificity 91% (84% to 95%). Seventeen studies (2600 patients) reported the results for cut-off value 1.5 ODI, sensitivity was 64% (50% to 77%) and mean specificity 95% (91% to 97%). AUTHORS' CONCLUSIONS: At a cut-off value 0.5 ODI in a population of 100 patients with a disease prevalence of 8% (overall median prevalence), 2 patients who have IA, will be missed (sensitivity 78%, 22% false negatives), and 17 patients will be treated or further referred unnecessarily (specificity of 81%, 19% false negatives). If we use the test at cut-off value 1.5 in the same population, that will mean that 3 IA patients will be missed (sensitivity 64%, 36% false negatives) and 5 patients will be treated or referred unnecessarily (specificity of 95%, 5% false negatives). These numbers should however be interpreted with caution, because the results were very heterogeneous.

Extended-spectrum-beta-lactamase production in a Salmonella enterica serotype Typhi strain from the Philippines.

A Salmonella enterica serotype Typhi strain was cultured from blood and fecal samples from a 54-year-old man with fever and diarrhea. He had returned from travel to the Philippines a few days earlier. Phenotypic and genotypic analysis confirmed the production of the SHV-12 extended-spectrum beta-lactamase.

Characteristics of clinical Helicobacter pylori strains from Ecuador.

In Ecuador, Helicobacter pylori infections are highly prevalent. A total of 42 H. pylori clinical isolates from 86 patients attending the outpatient clinic of the gastroenterology department of the university hospital of Guayaquil in Ecuador were characterized. Their susceptibility, and cagA and vacA status were determined. Resistance to metronidazole and clarithromycin was found in 80.9% and 9.5% of strains, respectively. Neither amoxicillin- nor tetracycline-resistant strains were found. The most prevalent genotype was the cagA(+), vacA s1b,m1 type. This genotype was associated with gastric cancer and peptic ulcer. Typing by random amplified polymorphic DNA showed no genetic relationship among the strains.

Superior antimicrobial activity of trisodium citrate over heparin for catheter locking.

BACKGROUND: Haemodialysis catheters used for vascular access are frequently complicated by infection and catheter-related thrombosis. Improvement of interdialytic locking solutions could reduce these problems. Trisodium citrate (TSC) has been advocated in recent years because it might have antimicrobial qualities. METHODS: Antimicrobial efficacy of four concentrations of TSC (2.2, 7.5, 15 and 30%) was compared with three equi-osmolal sodium chloride (NaCl) concentrations, unfractionated heparin 5000 IU/ml and a solution of gentamicin 1 mg/ml in TSC 7.5%. We analysed antimicrobial properties by two classical in vitro susceptibility tests. All tests were performed in triplicate by incubation of test fluids with Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, Pseudomonas aeruginosa and Candida albicans. RESULTS: Increasing TSC concentrations effectively killed the staphylococcal strains in both assays. For E.coli and P.aeruginosa complete killing was achieved only with TSC 30%. TSC 30% was also the only solution that significantly inhibited growth of C.albicans. Heparin manifested no antimicrobial effect of any significance. Adding gentamicin to TSC provided superior bacterial growth inhibition but had no effect on yeast growth. TSC solutions manifested superior antimicrobial activity compared with iso-osmolal NaCl solutions in both assays. CONCLUSION: This in vitro study demonstrates superior antimicrobial activity of TSC, especially in higher concentrations, in contrast to heparin. The mechanism seems to differ from hyperosmolality. Ca(2+) and Mg(2+) chelating effects are probably more important. Adding gentamicin provided the most potent antimicrobial solution. However, for reasons concerning development of bacterial resistance and sensitization of the patient, continuous exposition to aminoglycosides seems not advisable.

Nosocomial spread of a Staphylococcus capitis strain with heteroresistance to vancomycin in a neonatal intensive care unit.

A premature infant in a neonatal intensive care unit (NICU) developed a bloodstream infection caused by coagulase-negative staphylococci (CoNS) sensitive to vancomycin. The infection persisted for 3 weeks, despite therapy with vancomycin and replacement of all intravenous catheters. The neonate died due to necrotizing enterocolitis which developed during the ongoing sepsis. We screened this strain and 216 other strains of CoNS from cultures of blood obtained from neonates between 1997 and 2000 for heteroresistance to vancomycin. Forty-eight isolates, including the strain that caused ongoing sepsis, proved heteroresistant. All isolates were identified as Staphylococcus capitis and were identical, just as their resistant stable subcolonies were, when they were genetically fingerprinted by amplified-fragment length polymorphism analysis. The heteroresistant phenotype of this endemic strain was confirmed by population analysis. We conclude that heteroresistance to vancomycin occurs in S. capitis and might be the cause of therapeutic failures in NICUs. Moreover, heteroresistant strains can become endemic in such units.