Diagnostic yield of broad-range 16s rRNA gene PCR varies by sample type and is improved by the addition of qPCR panels targeting the most common causative organisms.

Introduction. Molecular techniques are used in the clinical microbiology laboratory to support culture-based diagnosis of infection and are particularly useful for detecting difficult to culture bacteria or following empirical antimicrobial treatment.Hypothesis/Gap Statement. Broad-range 16S rRNA PCR is a valuable tool that detects a wide range of bacterial species. Diagnostic yield is low for some sample types but can be improved with the addition of qPCR panels targeting common bacterial pathogens.Aim. To evaluate the performance of a broad-range 16S rRNA gene PCR and the additional diagnostic yield of targeted qPCR applied to specimens according to a local testing algorithm.Methodology. In total, 6130 primary clinical samples were collected as part of standard clinical practice from patients with suspected infection during a 17 month period. Overall, 5497 samples were tested by broad-range 16S rRNA gene PCR and a panel of targeted real-time qPCR assays were performed on selected samples according to a local testing algorithm. An additional 633 samples were tested by real-time qPCR only. The 16S rRNA gene PCR was performed using two assays targeting different regions of the 16S rRNA gene. Laboratory developed qPCR assays for seven common bacterial pathogens were also performed. Data was extracted retrospectively from Epic Beaker Laboratory Information Management System (LIMS).Results. Broad-range 16S rRNA gene PCR improves diagnostic yield in culture-negative samples and detects a large range of bacterial species. Streptococcus spp., Staphylococcus spp. and the Enterobacteriaceae family are detected the most frequently in samples with a single causative organism, but mixed samples frequently contained anaerobic species. The highest diagnostic yield was obtained from abscess, pus and empyema samples; 44.9 % were positive by 16S and 61 % were positive by the combined 16S and targeted qPCR testing algorithm. Samples with a particularly low diagnostic yield were blood, with 3.3 % of samples positive by 16S and CSF with 4.8 % of samples positive by 16S. The increased diagnostic yield of adding targeted qPCR is largest (~threefold) in these two sample types.Conclusion. Broad-range PCR is a powerful technique that can detect a very large range of bacterial pathogens but has limited diagnostic sensitivity. The data in this report supports a testing strategy that combines broad-range and targeted bacterial PCR assays for maximizing diagnosis of infection in culture-negative specimens. This is particularly justified for blood and CSF samples. Alternative approaches, such as metagenomic sequencing, are needed to provide the breadth of broad-range PCR and the sensitivity of targeted qPCR panels.

Nasopharyngeal Codetection of Haemophilus influenzae and Streptococcus pneumoniae Shapes Respiratory Syncytial Virus Disease Outcomes in Children.

BACKGROUND: The role of nasopharyngeal bacteria in respiratory syncytial virus (RSV) disease has been underestimated. We measured the frequency and burden of respiratory bacteria in the upper respiratory tract of infants with RSV infection over 7 respiratory seasons, and their impact on clinical outcomes. METHODS: Children <2 years old with mild (outpatients, n=115) or severe (inpatients, n=566) RSV infection, and matched healthy controls (n=161) were enrolled. Nasopharyngeal samples were obtained for RSV, Streptococcus pneumoniae, Staphylococcus aureus, Moraxella catarrhalis, and Haemophilus influenzae detection and quantitation by PCR. Multivariable models were constructed to identify variables predictive of severe disease. RESULTS: S. pneumoniae, H. influenzae, and M. catarrhalis, but not S. aureus, were detected more frequently in RSV-infected children (84%) than healthy controls (46%; P<.001). Detection of S. pneumoniae and/or H. influenzae was associated with fever, more frequent antibiotic treatment, worse radiologic findings, and higher neutrophil counts (P<.01). In adjusted analyses, S. pneumoniae/H. influenzae codetection was independentlyassociated with greater odds of hospitalization, higher disease severity scores, need for supplemental oxygen, and longer hospitalization. CONCLUSIONS: Nasopharyngeal codetection of S. pneumoniae and H. influenzae in infants with RSV infection is associated with increased disease severity.

Relationship of Multiplex Molecular Pneumonia Panel Results With Hospital Outcomes and Clinical Variables.

BACKGROUND: Antibiotic treatment decisions in severely ill patients must often be made in the absence of microbiologic results. The recently Food and Drug Administration-cleared BioFire FilmArray Pneumonia Panel (PN) detects 15 bacteria semiquantitatively, 3 atypical pneumonia bacteria, 8 viruses, and 7 antimicrobial resistance markers by multiplex PCR in ~1 hour in the laboratory. Previous reports have shown that the PN Panel bacterial detections are highly accurate, even when routine culture had no growth. METHODS: Consecutive bronchoalveolar lavage and endotracheal specimens submitted for culture between June and September 2018 from 270 patients with sufficient clinical and laboratory data were tested with the PN Panel. Patients were divided into 3 groups: (1) both culture and PN Panel positive, (2) PN Panel positive but culture uninformative (no growth or normal flora), and (3) patients with no PN Panel detections. RESULTS: Groups 1 and 2 had significantly higher maximum temperatures on the day of culture (P = .00036, analysis of variance [ANOVA] with Bonferroni correction), higher levels of an inflammatory response as measured by percent polymorphonuclear leukocytes in bronchoalveolar lavage (P = .00025, ANOVA with Bonferroni correction), and gram stain report of white blood cells, as previously reported [1]. CONCLUSIONS: Both group 1 (culture and PN Panel positive), and group 2 (PN Panel positive but culture uninformative) had higher levels of host response inflammatory responses compared with group 3, which had no targets detected, suggesting that PN Panel detections need to be interpreted in the clinical context, even if cultures are discordant. Depending on laboratory turnaround time, there could be opportunities for improved diagnosis and antibiotic stewardship.

Diagnostic Value of 16S Ribosomal RNA Gene Polymerase Chain Reaction/Sanger Sequencing in Clinical Practice.

BACKGROUND: Accurate microbiologic diagnosis is important for appropriate management of infectious diseases. Sequencing-based molecular diagnostics are increasingly used for precision diagnosis of infections. However, their clinical utility is unclear. METHODS: We conducted a retrospective analysis of specimens that underwent 16S ribosomal RNA (rRNA) gene polymerase chain reaction (PCR) followed by Sanger sequencing at our institution from April 2017 through March 2019. RESULTS: A total of 566 specimens obtained from 460 patients were studied. Patients were considered clinically infected or noninfected based on final diagnosis and management. In 17% of patients, 16S rRNA PCR/sequencing was positive and in 5% of patients, this test led to an impact on clinical care. In comparison, bacterial cultures were positive in 21% of patients. Specimens with a positive Gram stain had 12 times greater odds of having a positive molecular result than those with a negative Gram stain (95% confidence interval for odds ratio, 5.2-31.4). Overall, PCR positivity was higher in cardiovascular specimens (37%) obtained from clinically infected patients, with bacterial cultures being more likely to be positive for musculoskeletal specimens (P < .001). 16S rRNA PCR/sequencing identified a probable pathogen in 10% culture-negative specimens. CONCLUSION: 16S rRNA PCR/sequencing can play a role in the diagnostic evaluation of patients with culture-negative infections, especially those with cardiovascular infections.

Performance of a Semiquantitative Multiplex Bacterial and Viral PCR Panel Compared With Standard Microbiological Laboratory Results: 396 Patients Studied With the BioFire Pneumonia Panel.

BACKGROUND: Microbiologic results are critical to optimal management of patients with lower respiratory tract infection, but standard methods may take several days. The multiplex polymerase chain reaction BioFire Pneumonia (PN) panel detects 15 common bacterial species semiquantitatively as copy number/mL, 8 viral species, and 7 resistance genes in about an hour within the clinical laboratory. METHODS: We tested 396 unique endotracheal or bronchoalveolar lavage specimens with the BioFire Pneumonia panel and compared the bacterial detections to conventional gram stain and culture results. RESULTS: Of the 396 patients, 138 grew at least 1 bacterium that had a target on the PN panel, and 136/138 (98.6%) were detected by the panel. A total of 177 isolates were recovered in culture and the PN panel detected 174/177 (98.3%). A further 20% of patients had additional targets detected that were not found on standard culture (specificity 69%, positive predictive value 63%, and negative predictive value 98.9%). Copy number was strongly related to standard semiquantitative growth on plates reported by the laboratory (eg, 1+, 2+, 3+ growths) and was significantly higher in those specimens that grew a potential pathogen. Both higher copy number and bacterial detections found by the PN panel, but not found in culture, were strongly positively related to the level of white blood cells reported in the initial gram stain. CONCLUSIONS: Higher copy number and bacterial detections by the PN panel are related to the host respiratory tract inflammatory response. If laboratories can achieve a rapid turnaround time, the PN panel should have a significant impact both on patient management and on antibiotic stewardship.

The use of broad-range bacterial PCR in the diagnosis of infectious diseases: a prospective cohort study.

OBJECTIVES: Broad-range PCR has the potential to detect virtually any bacterial species via amplification and nucleotide sequencing of a DNA region common to all bacteria. We aimed to evaluate its usefulness and clinical relevance when applied to a wide variety of primary sterile materials. METHODS: A prospective study including 1370 samples (75 heart valves, 151 joint tissue samples, 230 joint aspirates, 848 whole blood samples and 66 culture-negative cerebrospinal fluid samples) were studied by using a commercial PCR system for detecting 16S rDNA (Molzym). The PCR results were compared with culture and were considered to provide added diagnostic value only if the PCR approach revealed new pathogens that were missed by culture. RESULTS: The added value of PCR was evident in 173 of 555 PCR-positive samples (0.126; 0.109-0.144 (proportion from all tested samples; 95% confidence interval)), most frequently in examinations of heart valves (0.56; 0.448-0.672) and joint tissue samples (0.219; 0.153-0.284). In contrast, the lowest rate of PCR with added value was noted for blood samples, regardless of the patient cohort they had been drawn from (nononcologic patients from intensive care: 0.065; 0.043-0.087, haematooncologic children: 0.048; 0.027-0.070). Moreover, PCR missed up to 7.1% of blood culture findings (0.071; 0.048-0.095) regarded as clinically relevant, which was the second highest failure rate after joint tissue samples (0.099; 0.052-0.147). CONCLUSIONS: Broad-range PCR substantially increases detection rate of pathogens, especially from heart valves and joint samples. However, a concurrent risk of false-negative PCR results justifies the need for parallel culture.

Detection of bacterial contamination in platelet concentrates from Brazilian donors by molecular amplification of the ribosomal 16S gene.

OBJECTIVE: The aim of our work was to establish a semi-automated high-throughput DNA amplification method for the universal screening of bacteria in platelet concentrates (PCs). BACKGROUND: Among cases of transfusion transmission of infectious agents, bacterial contamination ranks first in the number of events, morbidity and mortality. Transmission occurs mainly by transfused PCs. Automated culture is adopted by some blood banks for screening of bacterial contamination, but this procedure is expensive and has a relatively long turnaround time. METHODS: PCs were spiked with suspensions of five different bacterial species in a final concentration of 1 and 10 colony-forming units (CFU) per millilitre. After incubation, the presence of bacteria was investigated by real-time polymerase chain reaction (PCR) and by the Enhanced Bacterial Detection System (eBDS, Pall) assay as a reference method. Real-time PCR amplification was performed with a set of universal primers and probes targeting the 16S rRNA gene. Co-amplification of human mitochondrial DNA served as an internal control. RESULTS: Using the real-time PCR method, it was possible to detect the presence of all bacterial species tested with an initial concentration of 10 CFU mL-1 24 h after contamination, except for Staphylococcus hominis. The PCR assay also detected, at 24 h, the presence of Serratia marcescens and Enterobacter cloacae with an initial concentration of 1 CFU mL-1 . CONCLUSIONS: The real-time PCR assay may be a reliable alternative to conventional culture methods in the screening of bacterial contamination of PCs, enabling bacterial detection even with a low initial concentration of microorganisms.

Louse-borne relapsing fever - report of four cases in Switzerland, June-December 2015.

BACKGROUND: Louse-borne relapsing fever (LBRF) is a neglected disease that has been restricted to East Africa for many decades. Several cases in refugees from the Horn of Africa have been recently diagnosed in four European countries. CASE PRESENTATION: We report four additional cases of LBRF in asylum seekers from Somalia and Eritrea who presented with fever shortly after arriving in Switzerland during a seven-month period. Multiple spirochetes were visualized on stained blood films which were identified as Borrelia recurrentis by 16S rRNA gene sequencing. All patients recovered after antibiotic treatment with ceftriaxone and/or doxycycline. Concurrent infections (malaria and tuberculosis) were diagnosed in half of our patients. Possible modes of transmission and preventive measures are discussed. CONCLUSIONS: These reported cases highlight the ongoing transmission of LBRF in migrants from East Africa. Diagnosis of LBRF cases and prevention of autochthonous transmission in asylum seeker camps are important steps for the near future.

An unexpected cause for cavitary pneumonia and empyema.

Tularemia is an emerging zoonotic disease mainly of the Northern Hemisphere caused by the Gram-negative coccobacillus Francisella tularensis. It is affecting a wide range of animals and causes human disease after insect and tick bites, skin contact, ingestion and inhalation. A 66-year-old man presented to our clinic with cavitary pneumonia and distinct pleural effusion. After failure of empiric antibiotic therapy, thoracoscopic assisted decortication and partial excision of the middle lobe were conducted. Conventional culture methods and broad-range bacterial PCR including RipSeqMixed analysis were performed from the excised biopsies. Culture results remained negative but broad-range PCR targeting the first half of the 16S rRNA gene revealed F. tularensis DNA. This result was confirmed by F. tularensis-specific PCR and by serology. The source of infection could not be explored. To conclude, we report the rare clinical picture of a community-acquired pneumonia followed by pleural effusion and empyema due to F. tularensis. Broad range bacterial PCR proved to be a powerful diagnostic tool to detect the etiologic organism.

Evaluation of the novel microarray-based Prove-it™ Bone&Joint assay for direct detection of pathogens from normally sterile body sites in comparison with culture and broad-range bacterial PCR.

The performance of Prove-it™ Bone&Joint, a novel microarray-based assay for direct pathogen detection from clinical samples was assessed. In culture-positive samples, Prove-it™ performed similarly with broad-range bacterial PCR in osteoarticular (sensitivity 62.9% vs. 57.7%) and non-osteoarticular samples (54.6% vs. 56.8%). Prove-it™ is a rapid tool providing preliminary results while awaiting culture results.