PREreview of Coxsackievirus A24: A Causative Agent of Acute Haemorrhagic Conjunctivitis Outbreak in Dar es Salaam, Tanzania, between January and February 2024

Brief summary of the study

The study reports about determination of the causative agent(s) of an outbreak of acute hemorrhagic conjunctivitis (AHC) in Dar es Salaam, Tanzania, between January and February 2024 using molecular techniques. The study further does NGS sequencing and compares the resulting viral sequences to known sequences in public databases.

The authors address a timely public health concern by investigating a rapidly spreading conjunctivitis outbreak. They combine classical microbiological techniques (bacterial culture) with modern molecular diagnostics and next-generation sequencing. Ethical approval was obtained, and strong data transparency is demonstrated through open sharing of sequencing data. The authors acknowledge study limitations, particularly the small sample size. The study is highly relevant to the context.

Major comments 

Comments on the validity or strength of the methodology, experiments and analyses, strength of the conclusions

• The conclusion drawn from this study appears overgeneralized, which may lead to misinterpretation of the outbreak’s cause. Although Coxsackievirus A24 variant (CVA24v) was identified in a subset of PCR-positive samples, it is important not to assume that this was the sole agent responsible for the outbreak. The study included only 25 patients with clinical symptoms, and just 36% of the samples tested positive for enterovirus, with CVA24v confirmed in even fewer cases. Due to the small sample size and limited detection, the conclusion should be presented more cautiously. Using terms like “likely,” “may be,” or “suggestive of” would better reflect the possibility that other viral pathogens may have contributed to the clinical symptoms.

• The claim that most cultures showed no bacterial growth or only normal flora like Staphylococcus epidermidis seems reasonable. However, the study would be stronger if it included images of typical MacConkey and blood agar plates, or provided results from confirmatory biochemical tests and RT-PCR. This would improve transparency and make the findings more reliable. It would also better support the conclusion that the outbreak wasn’t caused by bacteria, making the diagnosis more convincing. Including visuals would also make the paper more appealing and help capture the reader’s attention.

• The RT-PCR and sequencing workflows description does not mention the inclusion of positive and negative controls, which are critical for ensuring reliability and ruling out false positives/negatives. Even if these controls were used as part of the commercial kits, this should be explicitly stated to assure readers and reviewers of the technical validity of the molecular data.

• Important information is missing in the methods section (more details in section-wise comments)

• There are large discrepancies in results reporting as well as what is expected given the methods described

Minor comments

Clarifications to statements in the text, interpretation of the results, presentation of the data/figures

• When referring to the diseases outbreak, it is better to be specific with the outbreak i.e outbreak of the acute hemorrhagic conjunctivitis.

• Language and flow of information need to be improved

Conflicts of interest of reviewers

• None declared

Data and code availability

• Data was deposited in a public repository and accession number given

• The authors should consider depositing the code used for the analysis in repository and sharing it

Ethical clearance and approval

• Ethical clearance was done, and certificate number is given

• Information on consent from study participants is missing

Comments by section

Abstract

1. The background to the study should also describe what Acute Haemorrhagic Conjunctivitis is and not end by mentioning the study date

2. Indicate how many clinics were involved in this study

3. Results of bacterial culture results are missing, they should be added

Introduction

This section reads more like an introduction to a review paper or textbook rather than that of an original research article. For better clarity, the section would benefit from a concise overview of acute conjunctivitis and its causes and its epidemiology in East Africa and especially in the study region. The introduction should also cover the public health impact of the disease and burden with supporting statistics, introducing the recent Tanzania outbreak earlier and clearly stating the existing knowledge gap regarding pathogen identification. This will set a better stage for the study’s goal.

Results

In general, the result section is too brief and gives only overviews of demographics and detection results. It is not well supported with figures, tables and other visual aides.

Specific comments:

1. Demographics: Add detailed demographic information including the summary statistics, ideally presented in a table. Give more details of the study participants including their age, sex, education, district, urban or rural, vital signs and any other useful and relevant health records that were taken.

2. Bacterial culture and PCR

   1. Please present the results with enough evidence to show that the work was actually done. e.g you cannot say that you did PCR without providing a gel image. Culture plates can also be shown. Presenting the results in tables and/or graphs and describing them would significantly improve the section.

   2. The statement “9 (36%) samples were positive for human enterovirus by Real-time PCR” is contrarily to what was stated in the methods section. If 9 samples were human enterovirus positive by Real Time PCR, why were only 4 samples sequenced? What criteria was used to select these 4 samples?

   3. Sequencing results: Please rewrite this section starting from the Lab and report on the sequence result from the lab before moving to the bioinformatics. Again, using tables and charts makes it easier to follow than text alone.

1. Figures:

   1. Figure 1 is missing, the only figure present is named figure 2. Therefore, either add figure 1 or rename figure 2 to figure 1

   2.  Improve clarity of Figure 2 and text size for readability. Also improve the caption to make it easier to understand the figure

Methods

1. There is missing information that is important to understand the study but also for reproducibility. Such as:

   1. State the clinics in Dar es Salaam region that samples were collected and indicate if these are private or public clinics. Also, how were these clinics selected?

   2. It is stated under "Isolate sampling" subsection that viral analysis was done but a detailed description including reagents, standard protocols followed and any references for the protocol are missing. These should be added

   3. Describe your bacterial culture in details and state which established standards followed. This will greatly improve reproducibility

   4.  Describe the sequencing method before the generation of the FastQ files 

1. The methodology section is really confusing, especially the molecular part, and it needs improvement. For example:

   1. 4 samples tested positive for 1 virus (enterovirus) among 4 tested. Sequencing was done on the 4 positive samples of enterovirus on Illumina Miseq. These were PCR products, but authors talk about host sample removal in the data analysis stage, where would these come from  if the PCR products only were sequenced?

   2. Under Sequencing and sequence assembly subsection, the first sentence says “Four human enterovirus positive samples with cycle threshold less than 30 were subjected to genomic sequencing using Illumina Miseq”, but later in the same paragraph there are these statements “These three samples were assembled with rnaviralSPAdes (23). The fourth sample (27YS) showed presence of Streptococcus pneumoniae, a known cause of bacterial conjunctivitis”. How is it 4 samples one time, then 3 samples, but also where is the Streptococcus pneumoniae positive sample coming from?

   3. In the BLAST/alignment description, the authors report that “Two of the three samples (28A0 and 26HB) yielded high quality hits against a Coxsackievirus A24 genome” but do not state what happened to the third sample

2. Sample collection:

   1. If paired eye swabs were collected from the same individuals, how did the authors decide that:

      1. The sample for eye A is suitable for virology while sample for eye B is suitable for bacteriology? 

      2. Did they consider co-infection?

   2. Describe how consent was obtained from study participants as well as permission from the clinics before the sample collection

   3. Describe the indicative symptoms of the disease that were used to recruit the patients from whom the samples were collected

 4. Clarify discrepancies between the abstract and methods section about bacterial culture and molecular detection (abstract) and bacterial and viral analysis (methods). Authors should reconcile the two since an abstract is a summary of the main work therefore such differences are not expected

1. The molecular detection method section should be broken into two: Section 1: RNA extraction details. Section 2: PCR analysis details

2. Sequencing and sequence assembly

   1. Break the section into the following sub-sections:

• Section 1: Describe in detail the sample quality controls, library preparation and the sequencing procedures.

• Section 2: Describe the bioinformatics analysis and tools from quality controls, mapping/alignment, variant calling, variant detection etc.

• Section 3: Describe the statistical methods and tools employed.

1. While this statement “Four human enterovirus positive samples with cycle threshold less than 30 were subjected to genomic sequencing using Illumina Miseq (Illumina)”,  implies that these 4 samples were selected from the 25, it will be clearer and easier for audience to comprehend if it was stated that "Out of the 25 samples, 4 PCR confirmed positive samples with human enterovirus infection were selected for genomic sequencing using Illumina Miseq"

2. Describe the parameters you set for the trimming. Including adapter information and trimming

3.  67 full length genome sequences is a lot of samples to align and draw a tree with two samples from the study

Discussion

1. Discuss the negative results of bacterial culture and the 1 sample that had Bacillus spp

2. This statement needs better reasoning and references/citations “It’s worth noting that laboratory-negative results are common in viral conjunctivitis outbreaks and can be attributed to various factors, including the timing of specimen collection.”

3. Regarding the concluding statement in the discussion section “the aetiological agent responsible for viral conjunctivitis in Dar es Salaam in January 2024.”, while the study has certainly added knowledge to the body of science, we do not think that it has demonstrated that the cause of AHC is Coxsackievirus A24. The reason is that less than 50% of the samples tested positive for Coxsackievirus A24 and given that samples were collected from “patients presenting with symptoms of acute conjunctivitis at clinics in Dar Es Salaam region”, it is thus likely that the causative agent for AHC may be something else. This statement and/or conclusions from the study need to be revised.

Competing interests

The authors declare that they have no competing interests.