PCR has been used as an aid in the diagnosis of invasive aspergillosis for almost 2 decades. A lack of standardization has limited both its acceptance as a diagnostic tool and multicenter clinical evaluations, preventing its inclusion in disease-defining criteria. In 2006, the European Aspergillus PCR Initiative was formed. The aim of the initiative was to provide optimal standardized protocols for the widespread clinical evaluation of the Aspergillus PCR to determine its diagnostic role and allow inclusion in disease diagnosis criteria. Quality control panels were developed and circulated to centers for evaluation of the existing methodology before recommendations based on the initial results were proposed for further panels. The centers were anonymously classified as "compliant" or "noncompliant," according to whether they had followed the proposed recommendations before the performance parameters were determined and meta-regression analysis was performed. Most PCR amplification systems provided similar detection thresholds, although positivity was a function of the fungal burden. When PCR amplification was combined with DNA extraction, 50% of the centers failed to achieve the same level of detection. Meta-regression analysis showed positive correlations between sensitivity and extraction protocols incorporating the proposed recommendations and the use of bead beating, white cell lysis buffer, and an internal control PCR. The use of elution volumes above 100 l showed a negative correlation with sensitivity. The efficiency of the Aspergillus PCR is limited by the extraction procedure and not by PCR amplification. For PCR testing of whole blood, it is essential that large blood volumes (>3 ml) be efficiently lysed before bead beating to disrupt the fungal cell and performance of an internal control PCR to exclude false negativity. DNA should be eluted in volumes of <100 l.

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Different BG assays have similar accuracy for the diagnosis of IFI in hemato-oncological patients. Two consecutive positive antigenemia assays have very high specificity, positive predictive value, and negative predictive value. Because sensitivity is low, the test needs to be combined with clinical, radiological, and microbiological findings.

In a meta-analysis of 10 studies, the BACTEC 960/MGIT and BACTEC 460 systems showed a sensitivity and specificity in detecting mycobacteria (1,381 strains from 14,745 clinical specimens) of 81.5 and 99.6% and 85.8 and 99.9%, respectively. Combined with solid media, the sensitivity of the two systems increased to 87.7 and 89.7%, respectively.Currently, a combination of conventional solid media with a broth-based method is the accepted reference standard for the diagnosis of mycobacterial infection (5, 26). Among the methods utilizing liquid media, the half-automated radiometric BACTEC 460 TB system (BACTEC 460) is widely accepted as the reference standard. This system, however, is also known for some well-established limitations, which include problems with the use of radioactive material, cumbersome manual loading and unloading, potential hazard of needle stick injury, risk of cross contamination, and lack of computerized data management.Recently, the BACTEC Mycobacteria Growth Indicator Tube System (BACTEC 960/MGIT), a newly developed nonradiometric, fully automated, continuously monitoring system, was introduced as an alternative to the radiometric BACTEC 460 for growth and detection of mycobacteria. The results of several comparative studies show that BACTEC 960/MGIT is a suitable tool for the detection of Mycobacterium tuberculosis and other mycobacterial species, though rather wide variations in diagnostic performance have been reported (1-3, 6, 10-14, 16-18, 21, 23-25, 27, 28, 30-35). To provide an evaluation of the quality of the available reports and an overall summary of the diagnostic accuracy of BACTEC 960/MGIT versus that of BACTEC 460, we performed a systematic review and metaanalysis.Literature search. The literature was searched for the period from January 1990 to June 2003 with the Medline, Embase, and Cochrane libraries. The studies were included for analysis if they compared the results of BACTEC 960/MGIT and BACTEC 460TB with and without solid media, they reported data on false-positive (sample flagged as positive without any microscopic or cultural evidence of acid-fast bacilli by subculture at the end of the incubation period), false-negative (negative test samples found positive on subculture), truepositive, and true-negative results separately; the results of the tests were compared against the reference standard, which was defined as a positive culture with at least one of the three systems used, and the comparison between tests was performed prospectively with the same series of patients from a relevant clinical population, defined as a group of individuals requiring routine microbiologic investigations for suspected pulmonary or extrapulmonary mycobacterial infections.Statistical analysis. To evaluate if variations between primary studies in test threshold had an influence on the accuracy of diagnostic procedures, we used receiver operator characteristic (ROC) curves (9,15,20,22).The common value of noncomparative binary outcomes, as sensitivity and specificity, were obtained by transf...

A panel of human serum samples spiked with various amounts of Aspergillus fumigatus genomic DNA was distributed to 23 centers within the European Aspergillus PCR Initiative to determine analytical performance of PCR. Information regarding specific methodological components and PCR performance was requested. The information provided was made anonymous, and meta-regression analysis was performed to determine any procedural factors that significantly altered PCR performance. Ninety-seven percent of protocols were able to detect a threshold of 10 genomes/ml on at least one occasion, with 83% of protocols reproducibly detecting this concentration. Sensitivity and specificity were 86.1% and 93.6%, respectively. Positive associations between sensitivity and the use of larger sample volumes, an internal control PCR, and PCR targeting the internal transcribed spacer (ITS) region were shown. Negative associations between sensitivity and the use of larger elution volumes (>100 l) and PCR targeting the mitochondrial genes were demonstrated. Most Aspergillus PCR protocols used to test serum generate satisfactory analytical performance. Testing serum requires less standardization, and the specific recommendations shown in this article will only improve performance.