Persistence of Pneumococcal Carriage among Older Adults in the Community despite COVID-19 Mitigation Measures

ABSTRACT Reported rates of invasive pneumococcal disease were markedly lower than normal during the 2020/2021 winter in the Northern Hemisphere, the first year after the start of the COVID-19 pandemic. However, little is known about rates of carriage of pneumococcus among adults during this period. Between October 2020-August 2021, couples in the Greater New Haven Area, USA, were enrolled if both individuals were aged 60 years and above and did not have any individuals under the age of 60 years living in the household. Saliva samples and questionnaires regarding social activities and contacts and medical history were obtained every 2 weeks for a period of 10 weeks. Following culture-enrichment, extracted DNA was tested using qPCR for pneumococcus-specific sequences piaB and lytA. Individuals were considered positive for pneumococcal carriage when Ct values for piaB were ≤40. Results. We collected 567 saliva samples from 95 individuals (47 household pairs and 1 singleton). Of those, 7.1% of samples tested positive for pneumococcus, representing 22/95 (23.2%) individuals and 16/48 (33.3%) households. Study participants attended few social events during this period. However, many participants continued to have regular contact with children. Individuals who had regular contact with preschool and school-aged children (i.e., 2 to 9 year olds) had a higher prevalence of carriage (15.9% versus 5.4%). Despite COVID-19-related disruptions, a large proportion of older adults continued to carry pneumococcus. Prevalence was particularly high among those who had contact with school-aged children, but carriage was not limited to this group. IMPORTANCE Carriage of Streptococcus pneumoniae (pneumococcus) in the upper respiratory tract is considered a prerequisite to invasive pneumococcal disease. During the first year of the COVID-19 pandemic, markedly lower rates of invasive pneumococcal disease were reported worldwide. Despite this, by testing saliva samples with PCR, we found that older adults continued to carry pneumococcus at pre-pandemic levels. Importantly, this study was conducted during a period when transmission mitigation measures related to the COVID-19 pandemic were in place. However, our observations are in line with reports from Israel and Belgium where carriage was also found to persist in children. In line with this, we observed that carriage prevalence was particularly high among the older adults in our study who maintained contact with school-aged children.

were both 60 years of age or older and who did not have anyone under the age of 60 years living in the household". So why include a singleton in the analysis? 2. Methods and Supplementary figure 2 -"Because many other Streptococci have the lytA gene, a sample was only considered to be positive for pneumococcus if it was positive for piaB". Also, in the SF2 I have noticed that if a sample was piaB positive and lytA negative was considered pneumococcus positive. Why? I was expected pneumococcus positive samples to be piaB and lytA positive. Did these samples were confirmed pneumococcus positive by culture or any other method?
3. Methods -"Pooled bacterial colonies were incubated for 10 159 minutes at 95{degree sign}C on a heating block, then tested in qPCR for piaB and lytA." It is unclear to me why isolated pneumococcal colonies were again tested using qPCR for piaB/lytA. 4. Results-Prevalence of pneumococcus: Are all household pairs male-female? Did you consider to include sex as factor of pneumococcal prevalence and/or transmission within a household?
Reviewer #3 (Comments for the Author): Within this study, the authors aimed at investigating the Persistence of pneumococcal carriage among older adults during the COVID-19 pandemic. They found that a large proportion of older adults continued to carry pneumococcus despite COVID-19 measures being in place. Saliva samples have been used and there are many reasons why this is a good idea in principle. In particular, the authors spent a lot of time in optimizing the sampling and the processing procedures (not only in this but also in other studies). In conclusion, this is important work but perhaps certain points need to be addressed Major points: A recent study investigated oropharyngeal samples to measure pneumococcal carriage in adults (PMID: 32727860). While I fully agree that OP and saliva samples (this study) are different, I am slightly concerned re the performance of piaB and lytA. The authors of the msphere paper wrote that: Using additional targets piaB improved qPCR specificity in OP (as compared to lytA alone), although the PPV (42 to 53%) was still poor (However, they used a microarray rather than cultures as golden standard). I am therefore worried if the cut off for saliva samples 'positive for pneumococcal carriage was set when Ct-values for piaB were {less than or equal to}40'. Especially, as the authors found that only 5 out of 40 samples were culture positive. In line 249, this is explained by 2 studies showing reduced sensitivity of cultures (as compared to qPCR) but I am not sure that this is fully convincing. I suggest discussing the findings of the msphere paper in the discussion. Minor points: Line 119: Why did the authors decide to use saliva samples? NP or OP samples seem to show more pneumococcal carriage, no (see table 1 of PMID: 32877517)? Line 126ff: For future studies, using SP2020 could also be a good idea (PMID: 30824850)? Line 129; Culture enrichment (used in this study): Is broth enrichment in line with the WHO guidelines (PMID: 35314867)? In general, what are the differences of this protocol as compared to the WHO guidelines? Line 161: Optochin susceptible colonies were 'only' serotyped or also species confirmed (with e.g. MALD TOF MS)? Using MALDI TOF MS for species detection (and/or sequencing) could also be useful for samples <40ct. Line 171: Should the statistical analyses address the fact that household pairs were included? Perhaps a regression analysis should be done which also includes other factors? Line 201: The total number of positive samples was 40 (true?), but I think the number of samples with <30 Ct must be smaller, yes? How many? It is kind of visible in figure 1 but did not spot this in the text.

Preparing Revision Guidelines
To submit your modified manuscript, log onto the eJP submission site at https://spectrum.msubmit.net/cgi-bin/main.plex. Go to Author Tasks and click the appropriate manuscript title to begin the revision process. The information that you entered when you first submitted the paper will be displayed. Please update the information as necessary. Here are a few examples of required updates that authors must address: • Point-by-point responses to the issues raised by the reviewers in a file named "Response to Reviewers," NOT IN YOUR COVER LETTER. • Upload a compare copy of the manuscript (without figures) as a "Marked-Up Manuscript" file. • Each figure must be uploaded as a separate file, and any multipanel figures must be assembled into one file. For complete guidelines on revision requirements, please see the journal Submission and Review Process requirements at https://journals.asm.org/journal/Spectrum/submission-review-process. Submissions of a paper that does not conform to Microbiology Spectrum guidelines will delay acceptance of your manuscript. " Please return the manuscript within 60 days; if you cannot complete the modification within this time period, please contact me. If you do not wish to modify the manuscript and prefer to submit it to another journal, please notify me of your decision immediately so that the manuscript may be formally withdrawn from consideration by Microbiology Spectrum.
If your manuscript is accepted for publication, you will be contacted separately about payment when the proofs are issued; please follow the instructions in that e-mail. Arrangements for payment must be made before your article is published. For a complete list of Publication Fees, including supplemental material costs, please visit our website.
Corresponding authors may join or renew ASM membership to obtain discounts on publication fees. Need to upgrade your membership level? Please contact Customer Service at Service@asmusa.org.
Thank you for submitting your paper to Microbiology Spectrum.

Reviewer #1 (Comments for the Author):
This study investigates the rates of pneumococcal carriage among older adults during the 2020/2021 winter in the Northern Hemisphere (one year after COVID19). The study design is easy to follow and clearly explained. I have the following concerns: 1. Methods -Inclusion criteria: "We recruited pairs of individuals residing in the community in the greater New Haven area who were both 60 years of age or older and who did not have anyone under the age of 60 years living in the household". So why include a singleton in the analysis?

Response:
The singleton was enrolled early in the study. We had initially planned to enrol individuals living in assisted living facilities, including singletons in these settings due to their high contact patterns with others over the age of 60 years. The singleton included in this study was an individual living in an assisted living facility who reported high contact with others of 60 years but little contact with those under 60 years. We therefore proceeded with their enrolment but due to the community restrictions in place due to the pandemic, we were unable to engage with others in the facility, or other such facilities in the community and thus were unable to enrol others in such settings. We have clarified this in the Results section under 'Population characteristics'.

Methods and Supplementary figure 2
-"Because many other Streptococci have the lytA gene, a sample was only considered to be positive for pneumococcus if it was positive for piaB". Also, in the SF2 I have noticed that if a sample was piaB positive and lytA negative was considered pneumococcus positive. Why? I was expected pneumococcus positive samples to be piaB and lytA positive. Did these samples were confirmed pneumococcus positive by culture or any other method?

Response:
In previous studies, piaB has been shown to be highly specific for pneumococcus. When implementing the PCR assays in our research lab, we found the chemistry of the piaB assay to be more sensitive than the lytA assay, even following multiple assay modifications. This can be seen in Supplementary Figure 3, where there are a disproportionate number of piaB+/lytAsamples close to the limit of detection. This has resulted in a small discrepancy between the expected piaB and lytA Ct values for a pneumococcus strain. This means that towards the lower limit of detection, samples may test positive for piaB while the lytA PCR assay is not sensitive enough to produce a concordant positive value and instead appears to be negative. When pneumococcus is detected towards this lower limit of detection by PCR, the amount of pneumococcus is a tiny fraction of the total bacteria in the sample, making isolation by culture impossible. To improve the reliability of our PCR results when we could not confirm by culture, when a sample tested >35 Ct for piaB, the DNA template was retested. Only samples that tested positive twice (or two out of three tests should a discrepant result occur) were included as a 'positive' result. We have updated the text in the Methods under 'Sample processing and pneumococcal detection' to clarify this further.