Added diagnostic value and impact on antimicrobial therapy of 16S rRNA PCR and amplicon sequencing on resected heart valves in infective endocarditis: a prospective cohort study.

OBJECTIVES: For adequate management and therapy of infective endocarditis (IE), identification of the causative pathogen is crucial but molecular testing results are not currently included in diagnostic criteria. The added diagnostic value and impact on antimicrobial therapy of 16S rRNA PCR and amplicon sequencing (16S rRNA PCR) performed on excised heart valves from patients with IE was evaluated alongside the effect of pre-operative antibiotics on the performance of blood culture (BC), valve culture (VC) and 16S rRNA PCR. METHODS: All patients undergoing valve surgery for definite or possible IE, according to modified Duke Criteria, were prospectively included from July 2013 up to and including June 2016. RESULTS: In all, 127 patients were included. Sensitivity for detecting the causative micro-organism in 120 post-operative definite IE patients was 26% for VC and 87% for BC and 16S rRNA PCR. 16S rRNA PCR, VC and BC were equally sensitive for different valve types and causative pathogens. In 27 (21%) definite IE patients, 16S rRNA PCR clarified discrepant culture results or was the only method identifying the causative pathogen. In 12 (10%) post-operative definite IE cases, molecular testing results influenced antimicrobial therapy. CONCLUSIONS: The very good performance characteristics, added diagnostic value and impact on antimicrobial therapy of molecular testing of heart valves should support the incorporation of molecular testing in diagnostic criteria and guidelines for IE.

Clinical predictive value of real-time PCR quantification of human cytomegalovirus DNA in amniotic fluid samples.

The aim of this study was to evaluate the diagnostic reliability and prognostic significance of the quantification of cytomegalovirus (CMV) DNA in amniotic fluid (AF). We retrospectively reviewed the results for 282 amniotic fluid samples that had been tested for CMV by a quantitative real-time PCR. We observed three cases in which no CMV genomes were detected in the AF but in which the children were nevertheless congenitally infected. Hence, we conclude that a negative result by PCR for CMV in AF cannot rule out the possibility of congenital infection. No false-positive PCR results were observed. A correlation between the CMV viral load in AF and the fetal and neonatal outcomes could not be demonstrated in our study. Instead, a correlation was found between the CMV viral load and the gestational age at the time of amniocentesis.

Multicenter evaluation of the new Abbott RealTime assays for quantitative detection of human immunodeficiency virus type 1 and hepatitis C virus RNA.

The analytical performances of the new Abbott RealTime hepatitis C virus (HCV) and human immunodeficiency virus type 1 viral load assays were compared at nine laboratories with different competitor assays. These included the Abbott LcX, Bayer Versant bDNA, Roche COBAS Amplicor, and Roche COBAS TaqMan assays. Two different protocols used during the testing period with and without a pre-m1000 RNA isolation spin were compared. The difference proved to be nonsignificant. A uracil-N-glycosylase (UNG) contamination control option in the HCV test for previous Roche COBAS Amplicor users was evaluated. It proved to decrease amplicon carryover by 100-fold independent of the amplicon input concentration. The protocol including UNG proved to overcome problems with false-positive negative controls. Comparison with other assays revealed only minor differences. The largest difference was observed between the Abbott HCV RealTime assay and the Roche COBAS Amplicor HCV Monitor version 2.0 assay.

Diagnosis of human immunodeficiency virus infection by a polymerase chain reaction assay evaluated in patients harbouring strains of diverse geographical origin.

Since the development of the highly sensitive polymerase chain reaction, PCR has been increasingly used for the diagnosis of viral infections, including the detection of human immunodeficiency virus (HIV), the causative agent of AIDS. In our laboratory a diagnostic PCR is carried out on proviral HIV-1 DNA using a standardised algorithm based on three HIV-1 primer sets. The three primer sets, amplifying a fragment in the LTR-gag gene, in the pol gene and in the env gene, are situated within conserved regions of the HIV-1 genome. These primers allow us to detect not only HIV strains from Belgian patients but also from African patients, who are, for historical reasons, a substantial part of the HIV-positive patients in Belgium. We are able to detect 1-5 copies of proviral HIV-1 DNA with each of the three nested primer sets. A sensitivity and specificity of 92 and 100%, respectively, were achieved when testing 24 Belgian and African HIV-1 seropositive samples. In our lab, the same PCRs are also used for the detection of viral RNA in cases of a doubtful undetectable viral load when using a commercial HIV-1 viral load assay. This is because present-day commercial assays are not entirely reliable with divergent strains. Both our 'in-house' diagnostic DNA and RNA-PCR can also be used semiquantitatively with limiting dilutions.

Failure to quantify viral load with two of the three commercial methods in a pregnant woman harboring an HIV type 1 subtype G strain.

The level of HIV-1 RNA in plasma has become one of the most important markers in the follow-up of HIV-infected patients. Three techniques are commercially available: both the Amplicor HIV Monitor and the NASBA HIV-1 RNA QT are target amplification methods, whereas the Quantiplex HIV RNA assay is a branched DNA signal amplification technique. Detection in both target amplification techniques is based on a single primer pair and a single probe in the gag region, whereas multiple probes capture the pol region of the viral RNA in the branched DNA assay. We investigated the discrepant observation of an undetectable viral load in an immunodeficient pregnant HIV-1-infected patient of African origin with no prior antiretroviral treatment. Although clinical progression was present in this patient with tuberculosis and a low CD4 cell count, viral load determinations with both the Amplicor Monitor and NASBA assays revealed no detectable RNA levels. The presence of HIV-1 RNA in the plasma of the patient was demonstrated by an in-house RNA-PCR. Subsequent HIV-1 RNA quantification with the branched DNA method revealed a high viremia (460,000 copies/ml). DNA sequence analysis of the gag gene identified a subtype G HIV-1 strain (HIV-1BL). To our knowledge this is the first report of a patient harboring an HIV-1 genotype of the main group with a high viral load as quantified by the branched DNA assay, but undetectable with the two commercial HIV RNA amplification techniques because of genetic divergence. In the case of discrepant low viral loads determined by one amplification technique in patients with advanced clinical stage one should use an alternative quantification technique for confirmation.

Confirmatory strategy of hepatitis C serology based on two screening assays in a diagnostic setting.

One hundred ninety seven successive sera, positive for anti-hepatitis C virus antibody with a third generation screening enzyme immunoassay (MEIA on IMX, Abbott), were tested with alternative third generation screening assays from two different manufacturers (Sanofi and Ortho), with an immunoblot assay (RIBA 3.0, Chiron), and by reverse transcriptase polymerase chain reaction (RT-PCR). Samples positive by RIBA 3.0 or by RT-PCR were considered as true positives. The positive predictive value of a combination of strong positive results in two screening assays was more than 98%. This combination of results has the same predictive value for detectable viraemia as a positive RIBA 3.0 (86.5%). Using a policy of two successive enzyme immunoassays in this clinical diagnostic setting diminishes the need for supplemental assays by more than 85%.