PONE-D-24-46089Postnatal cytomegalovirus infection and its effect on hearing and neurodevelopmental outcomes among infants aged 3 – 10 months: a cohort study in Eastern Uganda.PLOS ONE

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Partly

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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5. Review Comments to the Author

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Reviewer #1: The manuscript an important clinical condition that is often missed in low-resource settings, however, the authors need to revise the number of study participants whose results they are presenting.

As per the participant enrolment diagram, they report results from 427 of the 438 recruited participants. They lost 10 participants to follow-up and one participant died.

Yet

Parity is reported for 434 participants

Known HIV status is reported for 413 participants

Presence of flu-like symptoms among mothers is reported for 436 participants

Hearing assessment is reported for 424 participants

Comparison of clinical outcomes is reported for 438 participants including those lost to followup and the participant who died.

Reviewer #2: Thank you for the opportunity to review this insightful and valuable study exploring the association between postnatal CMV infection and hearing as well as neurodevelopmental outcomes in infants. The research addresses an important gap in understanding the impacts of postnatal CMV on early childhood health in resource-limited settings, and the use of a community-based cohort is commendable. The study's strengths include the rigorous matching of cases and controls, thorough statistical analysis, and comprehensive assessments for neurodevelopmental and hearing outcomes. I do suggest some minor revisions, which will make the article even more robust and impactful:

- Wording: The use of the term "positive result" in the context of the hearing screening is somewhat confusing, as this may be interpreted as "positive otoacoustic emissions" (i.e. "pass" result), please consider using a different wording, e.g. "failed or abnormal test result"

- The authors mention in the introduction section that "infectious and endemic illnesses, such as HIV/AIDS and malaria, (..) can complicate the immune response in infected individuals and impact viral shedding", but only data on maternal HIV status are reported in the results section. Do the authors have any information on active or dormant malaria cases during pregnancy/lactation in the population?

- Could the authors provide more detailed information on infant feeding practices in both cohorts? (esp. breastfeeding rates?). This sort of information would be very interesting to the readership, especially bearing in mind potential (and likely) routes of viral transmission.

- Figure 1: Study flowchart. I congratulate the authors on the high follow-up rates in this challenging setting! Could the authors please specify the causes of infant mortality? (total no. of 5 babies died).

- Could the authors provide a rationale for screening in saliva and confirmatory testing in urine? (and not vice versa?)

- Reporting of medication use in the first 3 months of life: Did this include over-the-counter medication?

- Do the authors have any information on examination results in infants referred to ENT? Or could they provide a rough estimate of the number of infants actually suffering from hearing loss?

- Out of curiosity: Are the authors planning on conducting a study with longer-term follow-up in the same cohort?

Reviewer #3: Postnatal CMV (pCMV) infection is known to be associated with morbidity in pre-term < 32 weeks or <1500 gram babies. A systematic review reports a possible impact on (late) neurodevelopment in this group but long term hearing loss was not found to be an association (reference 29, Stark AT et al, 2021). Whether pCMV infection is associated with neurodevelopmental compromise or (sensorineural) hearing loss in TERM babies is however less investigated. In general, term babies are believed to not suffer clinical consequences due to their relative maturity.

This is a sub-study that used a cohort recruited for their (ongoing) primary study called the Babygel Study. This Babygel Study is investigating the impact of improving hand hygiene in rural Ugandan households using locally-produced alcohol-based hand gel. The BabyGel study’s primary outcome is the rates of severe infant illness or death in the first 90 days of life. We are not provided details about BabyGel Study but their study protocol is cited (ref 24).

This current (sub) study is a case control study investigating whether or not pCMV infection in TERM babies impact on neurodevelopment or hearing (in the first year of life). The study compared age matched term using validated tools (the Malawi Development Assessment Tool and the Hammersmith Infant Neurological Examination) and hearing ‘loss’ was screened for by Otoacoustic Emission (OAE) testing. Note that absent OAEs could be due to hearing loss (HL) which could be either conductive or sensorineural in origin, wax in the ears, fluid or infection in the middle ear or a malformed inner ear. Formal audiology was not performed.

The study found that neurodevelopment between the two groups were similar, but babies with postnatal CMV failed their hearing screen at a higher rate. The authors appropriately point out that failing a hearing screen does not equate to hearing loss and that a formal hearing assessment is needed to establish actual hearing loss. The study did not provide formal audiological assessments (ABR), so it remains unknown if the babies had hearing loss (conductive or sensorineural). In addition, if formal audiology had been performed, we may have found the hearing loss to be mainly conductive hearing loss, which in young babies is closely related to middle ear infections (otitis media) and not permanent.

Overall, I have concerns about the methodology of this paper and the conclusion. Firstly, this is a sub- group in a study investigating the impact on infection rates by providing hand hygiene to households via a RCT. The randomisation status of participants in the study (so intervention arm (i.e hand hygiene) vs non intervention arm) is not provided nor included in the analysis. Hand hygiene may contribute to less viral infections in the household or baby, which may then result in less middle ear infections, which in turn is associated with less hearing loss. We do not know from this group if there was a predominance of the non-PCMV group in the BabyGel ‘intervention” arm, which may have been a factor in less hearing loss (indirect effect of less viral rep infections).

The Table 1 reporting the parental socio-demographic characteristics and household hygiene should have had a comment about whether there were any statistical differences between the groups (although eyeballing the figures suggests the groups were similar).

The infant data which is really the ‘confounders’ for risks for hearing loss, should have been a separate table and should include factors that predispose to recurrent infections in the household/ child and include whether or not mothers were randomised to “Babygel” , babies’ vaccination status, breast feeding status, the number of respiratory tract infections the babies have had, with a statical significance provided (p values).

Overall, whilst I appreciate you have compared to controls, I am not confident that pCMV contributed to the failed hearing screens as insufficient analysis of risk have been performed and there may have been bias in the groups.

Some specific comments for clarification:

1) Abstract: In the conclusion, what do the authors mean by “Exploration into the feasibility of incorporating hearing and CMV screening into routine care will play a vital role in improving early diagnosis of CMV and hearing impairment in resource-limited settings.’ .The sentence is sweeping

2) Introduction: A brief explanation of BabyGel Study should have been provided in the text. I appreciate you have referenced citation no. 24, but a brief explanation here provides the requisite context. Also. you should clarify what sort of hearing loss you are concerned about. The assumption is you are concerned about sensorineural hearing loss which is permanent, not conductive, which s reversible

3) Methods

1. How were the babies sourced and recruited?

2. Were the babies only assessed by the 5 research assistances? S - no clinical follow up by a medical team (this could be citied as a limitation? )

3. Were babies all breast fed? The assumption is yes in the LMIC setting but this is an important confounder (breast feeding protective against respiratory tract infections and middle ear infections)

4. Reference 27 (for methods) not available as yet in literature (so unable to access)

5. Can you clarify if a ‘failed hearing screen ‘ is based on one screen? was a second screen done ? Failing a hearing screen could be due to a temporary blocked ear that resolves on a second screen

6. Table 1: demographic / epidemiological data: p values would be useful (understand this is a ‘Table 1’, but this is not an RCT and there could be differences between the groups )

7. BabyGel randomisation status of the mothers?

8. Baby data: Should be a separate table. Needs confounders like whether or not these babies had more respiratory infections/ viral upper respiratory tract infections - a risk factor for otitis media, the vaccination status of babies, breast fed, number of children in the household, etc

4) Conclusion. Confusion with ‘public health policy’ need and the aims of this study. I agree that hearing screening is an important public health measure in infancy. However, I found linking the 2 concepts of attempting to link hearing loss with postnatal CMV and the need for a hearing screening because of postnatal CMV confusing and perhaps misleading. The study sends a message that postnatal CMV in term babies can be associated with hearing loss. As outlined above, the there were confounders for hearing loss in term babies not included in the investigations and thus the conclusions are left somewhat questionable

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Reviewer #1: Yes: Musa Sekikubo, Department of Obstetrics and Gynaecology, School of Medicine, College of Health Sciences, Makerere University, Kampala Uganda

Reviewer #2: No

Reviewer #3: No

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Postnatal cytomegalovirus infection and its effect on hearing and neurodevelopmental outcomes among infants aged 3 – 10 months: a cohort study in Eastern Uganda.

PONE-D-24-46089R1

Dear Dr. Okalany,

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Kazumichi Fujioka

Academic Editor

PLOS ONE

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