Evaluation of a protocol for molecular broad-range diagnosis of culture-negative bacterial infections in clinical routine diagnosis

Abstract

Aim: Introduction of a protocol for broad‐range diagnosis of bacterial infections, which remain negative in culture. Methods and Results: The new TaqMan real‐time PCR assay amplifies part of the 16S rRNA gene. Species are identified by subsequent sequencing and phylogenetic blast analysis. The analytical sensitivity showed to be 50 fg DNA per PCR. The lowest detectable bacterial cell concentration in blood was 1000 CFU per 200 μl EDTA‐blood. The utility in clinical routine diagnosis was evaluated by testing 136 clinical specimens. Bacterial pathogens were detected in 33 samples (24·3%) either by culture or molecular diagnosis. In 10 culture negative cases, pathogens such as Mycoplasma timone/orale, Ureaplasma parvum/urealyticum, Treponema pallidum, different streptococci and staphylococci were identified by molecular diagnosis only. Conclusions: The introduced broad‐range real‐time PCR protocol showed to be useful in the clinical routine in cases where bacterial infection was highly anticipated but culture remained negative. However, the obtained data have to be always interpreted with caution and in conjunction with the clinical data, crossing‐point values and with the Blast result of both the sample and the controls. Significance and Impact of the Study: This work introduces a new and well‐evaluated broad‐range real‐time PCR protocol for diagnosis of bacterial infections.

Rapid Real-Time Nucleic Acid Sequence-Based Amplification-Molecular Beacon Platform To Detect Fungal and Bacterial Bloodstream Infections

“…The universal detection of bacteria or fungi in blood using broad-range PCR assays has been described (1,16,22,42), but interpretations can be difficult, as assay contaminants can cause false-positive results (37). The early detection of BSIs requires reliable sensitivity, around 10 CFU/ml of blood for bacteria and fungi, which is below the level of most existing diagnostic platforms.…”

Evaluation of Real-Time 16S rDNA PCR and Pyrosequencing for Routine Identification of Bacteria in Joint Fluid and Tissue Specimens

“…In addition, the sensitivity is reduced by the problem of amplification of background DNA contamination, as seen in the present study. The sensitivity of real-time 16S rDNA PCR and sequencing compares relatively unfavourably to pathogen-specific real-time PCR, being at least 10-fold less sensitive [13]. Therefore, specimens with low bacterial loads will be missed by using this technique; this may be a particular problem in specimens from prosthetic joint infections where bacteria are likely to be present largely in biofilms.…”

“…5 inhibited specimens were considered as PCR negative for the purposes of assay evaluation. As has been well described [2,13,14], negative controls for both extraction and PCR processes were positive using 16S rDNA PCR; Ct values ranged from 34 to 36. Specimens with Ct values ≤ 34 were sequenced to determine positivity.…”

“…Pyrosequencing was carried out using the Pyromark Q24 vacuum workstation and PyroMark Q24 instrument (Qiagen) with a dispensation order of 20(CTGA). The PCR method was adapted for pyrosequencing by use of a 5' biotinylated forward PCR primer and a pyrosequencing primer with the same oligonucleotide sequence as the PCR probe [13]. Sequence reads were used to query the nucleotide collection of the GenBank database using the nucleotide BLAST program with search criteria for highly similar sequences and exclusion of uncultured or environmental isolates.…”

“…Extracts were diluted 1:10 to reduce PCR inhibition. A realtime 16S rDNA PCR assay was used to amplify a 567 bp region at the 5' end of the 16S rRNA gene as previously described [13]. One negative extraction control was added for every batch of 11 -15 samples processed and a negative PCR run control was included for every 10 extracts tested.…”