Chronic obstructive pulmonary disease (COPD) is a common condition characterized by persistent respiratory symptoms, often punctuated by acute exacerbations that can lead to hospitalization and a faster decline in lung function (Criner et al., 2015; Global Initiative for Chronic Obstructive Lung Disease, 2021). Different studies, including the observational Acute Exacerbation and Respiratory InfectionS in COPD (AERIS) study, have shown an association between exacerbation state and increased prevalence of airway bacteria, most commonly non-typeable Haemophilus influenzae (NTHi) and Moraxella catarrhalis, with some reports of an association with Streptococcus pneumoniae also (Garcha et al., 2012; Molyneaux et al., 2013; Huang et al., 2014; Millares et al., 2014; Wang et al., 2015; Wilkinson et al., 2017; Jubinville et al., 2018; Mayhew et al., 2018; Weeks et al., 2021). In the AERIS study, changes in the yearly COPD exacerbation rate were associated with changes in H. influenzae colonization (Wilkinson et al., 2019a), and nearly all (99%) H. influenzae isolates were non-typeable (Wilkinson et al., 2017).

A multi-component investigational vaccine was developed to reduce the frequency of acute exacerbations of COPD (AECOPD) associated with NTHi (Leroux-Roels et al., 2016), followed by a second related vaccine (NTHi-Mcat vaccine) to prevent NTHi-and M. catarrhalis-associated exacerbations (Van Damme et al., 2019). Both vaccines contain three conserved surface NTHi proteins, while the NTHi-Mcat vaccine also contains a surface protein from M. catarrhalis (Leroux-Roels et al., 2016; Van Damme et al., 2019). A phase 2 study of the NTHi vaccine (NTHI-004) and phase 2b study of the NTHi-Mcat vaccine (NTHI-MCAT-002) showed immunogenicity and no safety concerns when either vaccine was administered to patients with COPD (Wilkinson et al., 2019b; Andreas et al., 2022). In the NTHI-MCAT-002 study, while vaccination did not reduce the frequency of moderate/severe exacerbations, with no difference between groups in rate of AECOPD associated with NTHi or M. catarrhalis, observations suggested possible reductions in severe exacerbations and related hospitalizations in the vaccinated group versus placebo (Andreas et al., 2022).

In the AERIS, NTHI-004, and NTHI-MCAT-002 studies, sputum samples were collected from patients at regular intervals and at exacerbation (Wilkinson et al., 2017, 2019b; Andreas et al., 2022). Bacteria in freshly collected sputum were identified in local laboratories using conventional culture-based microbiological methods that were not harmonized. In parallel, frozen dithiothreitol (DTT)-treated sputum samples were transported to a central laboratory and analyzed by real-time PCR assay. Various studies have shown that molecular techniques, such as PCR, have better specificity and sensitivity than conventional culture-dependent methods in the detection of airway bacteria in patients with COPD (Eser et al., 2012; Garcha et al., 2012; Bafadhel et al., 2015; Gadsby et al., 2015; Wilkinson et al., 2017). Also, culture-based methods require fresh samples and their reliability can be affected by antibiotic treatment (Oliver, 2010; Wu et al., 2014) as well as the microbiologist’s skills in phenotypic identification methods. In contrast, a PCR assay can be used on frozen samples in a central location where it has been well characterized, thus avoiding the risk of variations in microbiological methods among individual local laboratories.

We now report on the performance of the microbiological assays used in the detection of bacteria in sputum samples in the AERIS, NTHI-004, and NTHI-MCAT-002 studies (Wilkinson et al., 2017, 2019b; Andreas et al., 2022). Quantitative and qualitative H. influenzae, M. catarrhalis, and S. pneumoniae identification results obtained with real-time PCR are compared with those obtained with culture-based methods. Additionally, using data from the NTHI-004 study, we examine the feasibility of freezing sputum samples before bacterial detection by culture in a central laboratory. Qualitative Pseudomonas aeruginosa, Staphylococcus aureus, and Streptococcus pyogenes identification data by PCR assay are also assessed from the three studies.

Figure 1 provides a plain language summary of the findings from these assessments.

We compared the performance of culture-based and PCR assays in identifying bacterial airway pathogens in sputum samples taken in three clinical studies of patients with COPD. In each study, two triplex real-time PCR assays were used, developed by GSK: a qPCR assay to detect and quantify H. influenzae, M. catarrhalis, and S. pneumoniae from sputum total nucleic acids, involving hydrolysis probes to maximize specificity, and a qualitative PCR assay to detect P. aeruginosa, S. aureus, and S. pyogenes. S. pyogenes was not identified in sputum samples from any of the studies. The three studies were conducted over the period 2011 to 2020, during which time the PCR assays were improved, such as in extraction methods for nucleic acids, leading to adjustments in LOD.

Our results show that real-time qPCR has higher specificity and sensitivity than culture-based methods for the detection of the most frequent bacterial species identified: H. influenzae, M. catarrhalis, and S. pneumoniae. The bacterial load analyses for these three species showed that some samples negative by culture contained a significant amount (over 10 million copies/ml) of bacterial DNA. Moreover, for P. aeruginosa and S. aureus, analysis of over 1,700 samples in the NTHI-MCAT-002 study showed significantly higher positivity rates with PCR than with culture-based detection methods for both species, although in the other two studies the difference was only significant for S. aureus in the NTHI-004 study. Overall, however, these data confirm previous reports of higher sensitivity with the PCR assay than with culture-based methods not only in COPD but also other conditions, such as otitis media and cystic fibrosis (Eser et al., 2012; Garcha et al., 2012; Bafadhel et al., 2015; Gadsby et al., 2015; Wilkinson et al., 2017; Gavillet et al., 2022). For example, one study of the detection of P. aeruginosa and S. aureus in cystic fibrosis found significantly lower detection of both lung pathogens by culture, which often did not detect either pathogen despite being found repeatedly by qPCR (Gavillet et al., 2022). In our study, qPCR positivity rates were around two-to four-fold higher for H. influenzae and M. catarrhalis in all three studies and for S. pneumoniae in the NTHI-004 and NTHI-MCAT-002 studies. In the concordance analysis, the lowest percentage positive agreement was for S. pneumoniae in the AERIS study (35%), reflecting the proportion of qPCR-negative samples among the culture-positive ones. Further analysis of this discrepant result led to the re-identification of a significant number of isolates, initially attributed to be S. pneumoniae isolates, as S. pseudopneumoniae or S. mitis by molecular techniques. The reason for this observation in the AERIS study may be related to characteristics of locally circulating strains or the specificity of the culture methods used to identify S. pneumoniae in the AERIS study laboratory; positive agreement in the other two studies was 71%. This suggests the culture-based method may have, in some settings, low specificity for S. pneumoniae.

A lack of specificity with culture-based methods is also demonstrated by the need to discriminate H. influenzae from H. haemolyticus isolates in presumptive H. influenzae-positive samples by using molecular methods (Murphy et al., 2007), as confirmed by qPCR or microarray in the three studies analyzed. Of the bacterial species assessed, the concordance analysis showed the lowest percentage overall agreement (76–82%) for H. influenzae (confirmed samples), due mainly to samples that were negative with culture-based detection but positive with qPCR. However, the presence of culture-negative samples associated with high bacterial DNA loads suggests this may have been partly due to the presence of non-culturable but viable bacteria (Oliver, 2010) or poor sensitivity of the culture method used. For example, use of bacitracin agar increases identification rates for H. influenzae (Harris et al., 2017) but this was not used in all laboratories. There is also a possibility that, although patients were instructed not to take an antibiotic before site visits, antibiotic treatment before sputum collection could have had an impact on the bacterial culture results (Brown and Spiegel, 2019). In this case, bacteria may not have been capable of growing on an agar plate but would have been detected by real-time PCR.

Our data suggest important advantages related to the specificity and sensitivity of real-time PCR over conventional culture-based methods for the assessment of airway bacteria in patients with COPD. Molecular assays can be performed on frozen sputum samples (Zhao et al., 2011; Cuthbertson et al., 2014), whereas culture needs to be done shortly after sputum collection to ensure sample integrity and bacteria viability (Baron et al., 2013). Frozen samples can be processed in a central laboratory with a well characterized PCR-based method, while fresh samples processed via conventional microbiological methods may require multiple local laboratories to avoid loss of viability. These local laboratories may not use exactly the same methods (see Figure 2), affecting the consistency of results (Hogardt et al., 2009). Culture of freeze/thawed samples in a central laboratory was examined in the NTHI-004 study as an option to avoid unharmonized methods across local laboratories. This analysis of the impact of freezing sputum samples before species identification by culture-based methods used STGG as storage medium since preserved H. influenzae, M. catarrhalis, and S. pneumoniae isolate viability had been reported with its use (Kaijalainen et al., 2004). However, the culture-positivity rate in STGG-frozen sputum samples was only around 50% or less of that in fresh samples, showing this is not a feasible option for evaluating sputum samples, thus providing further support for PCR testing of frozen samples in a central laboratory. The ability to freeze sputum samples is a particular advantage in phase 3 clinical trials of COPD patients, which generally require large numbers of sites to enroll a sufficient number of patients (Tashkin et al., 2008; Wedzicha et al., 2008; Calverley et al., 2009; Albert et al., 2011; Wedzicha et al., 2013; Pascoe et al., 2016). However, central analysis of frozen samples is associated with additional shipment costs and there is a risk of inappropriate sample management during the transport or freezing procedure, although there is also a management risk associated with fresh samples, for example, if not processed within the appropriate time window. Additionally, antibiotic usage before sputum sample collection can have an impact on the reliability of bacterial data obtained by culture (Wu et al., 2014; Mammen and Sethi, 2016; Jacobs et al., 2018), and the identification of pathogens by culture can be complicated by species overgrowth and contamination by commensal bacteria, while PCR allows direct species detection irrespective of these circumstances (Scoleri et al., 2016). PCR also has many advantages in terms of easy evaluation of bacterial load, it is relatively inexpensive, and it can be used to process more samples simultaneously than culture. These advantages assume the PCR assay has been appropriately designed (with highly specific oligonucleotide selection) and well characterized in terms of assay parameters, including LOD and quantitation limits, as was done for the PCR assays used in the AERIS, NTHI-004, and NTHI-MCAT-002 studies.

A major strength of these analyses is the large number (totaling 5,003) and multinational origin (Europe and North America) of sputum samples assessed for bacterial detection results from both culture-based and PCR assays. Also, the triplex real-time PCR assays used in each study were essentially the same, with any differences related to improvements made over time. The results of these analyses are limited by the possibility of false-positives resulting from the detection of low amounts of DNA, associated with dead bacteria, by qPCR. However, this was mitigated in these studies by using a LOD for each qPCR target as positivity cut-off (i.e., a sample was considered positive for a pathogen by qPCR if the observed load is equal to or above the corresponding LOD). We found many culture-and PCR-negative samples had PCR signals detected below the positivity cut-off, suggesting that using a PCR positivity cut-off is meaningful to limit the proportion of false-positives. Although qPCR can detect both viable and non-viable bacteria (Rudi et al., 2005), we found the bacterial loads measured by qPCR generally mirrored those measured by culture. Nevertheless, it would have been of interest to determine if samples found negative with culture-based techniques but positive on qPCR assay contained viable bacteria that were not culturable, as reported in other studies (Stenfors and Räisänen, 1992; Oliver, 2010; Lee and Bae, 2018), as well as the rate of non-viable bacteria present in samples recorded as qPCR-positive.

In conclusion, these results encourage the use of real-time PCR assays for the identification of respiratory bacteria in patients with COPD. PCR assay addresses some of the limitations of conventional culture-based methods in terms of specificity and sensitivity in detecting bacterial infection. PCR has additional advantages, including the ability to be performed in a centralized location on frozen samples and the capacity to detect viable but non-culturable bacteria. This supports the use of well characterized molecular methods for the identification and quantification of bacteria in future studies of patients with COPD, especially when working in a multicenter setting requiring sample testing in multiple laboratories.

The datasets presented in this article are not readily available because anonymized individual participant data and study documents can be requested for further research from www.clinicalstudydatarequest.com.

The studies involving human participants were reviewed and approved by the South West Hampshire Research Ethics Committee (Research Ethics Committee reference number: 11/H0502/9) for the AERIS study; the Southampton and South West Hampshire Research Ethics Committee, United Kingdom, and Regional Research Ethics Committee, Gothenburg, Sweden for the NTHI-004 study; the ethics committee of each participating center for the NTHI-MCAT-002 study. The patients/participants provided their written informed consent to participate in this study.

SS, ND, J-MD, LT, SR, AKA, TP, and LM were involved in analysis conception and design. SB, ND, J-MD, LT, SR, AKA, TP, and LM were involved in acquisition and generation of data. All authors performed the data analysis and/or data interpretation. ND and LM provided materials for the analyses. All authors contributed to the article and approved the submitted version.

GlaxoSmithKline Biologicals SA funded the AERIS, NTHI-004, and NTHI-MCAT-002 clinical studies and was involved in all stages of study conduct, including analysis of the data. GlaxoSmithKline Biologicals SA also took in charge all costs associated with the development and publication of this manuscript.

SS, J-LI, SB, ND, J-MD, LT, SR, AKA, TP, and LM are employed by GSK. SS, ND, J-MD, LT, TP, and LM hold shares in GSK. J-MD is also a designated inventor on patents owned by GSK.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.