Molecular Epidemiology of Rotavirus A Strains Pre- and Post-Vaccine (Rotarix®) Introduction in Mozambique, 2012–2019: Emergence of Genotypes G3P[4] and G3P[8]
, Benilde Munlela 1,3, Assucênio Chissaque 1,2, Jorfélia Chilaúle 1, Jerónimo Langa 1, Orvalho Augusto 4,5, Simone S. Boene 1,3, Elda Anapakala 1, Júlia Sambo 1,2, Esperança Guimarães 1,2, Diocreciano Bero 1, Marta Cassocera 1,2, Idalécia Cossa-Moiane 1, Jason M. Mwenda 6, Isabel Maurício 2,7, Hester G. O’Neill 8 and Nilsa de Deus 1,9
Round 1
Reviewer 1 Report
MOLECULAR EPIDEMIOLOGY OF ROTAVIRUS A STRAINS PRE- AND POST-VACCINE (ROTARIX®) INTRODUCTION IN MOZAMBIQUE, 2012–2019: EMERGENCE OF GENOTYPES G3P[4] AND G3P[8]
By Joã et al.,
This paper characterizes the RVAs genotypes, pre- (2012-2015) and post-vaccine (2016-2019) introduction in Mozambique, among diarrhoeic children less than five years of age.
There were 5 sentinel sites involved in the study : The primary site, Mavalane General Hospital (HGM), was analysed from 2012-2019, and five sites were involved in country-wide analyses, 2015-2019. During the pre-vaccine period, G9P[8] was the most predominant genotype for both HGM (28.5%) and the country-wide analysis (46.2%). However, in the post-vaccine period analysed, G9P[8] was significantly reduced. G3P[8] was the most common genotype at HGM, while G1P[8] predominated country-wide. Genotypes G9P[4] and G9P[6] were detected for the first time and an emergence of G3P[8] and G3P[4] genotypes occurred during the post-vaccine period. The distribution and prevalence of rotavirus genotypes were distinct in pre- and post-vaccination periods, while uncommon genotypes were also detected in the post-vaccine period. These observations support the need for continued rotavirus surveillance in Mozambique to monitor changes in circulation rotavirus genotypes due to vaccine pressure.
This is, overall, quite a well-written and well-designed paper. The manuscript lacks bioinformatics analysis but I assume there will be a follow on paper, including whole genome sequence studies as implied in the discussion.
There are a few minor errors in the English, most of which I have corrected. The title and summary are brief and to the point. The text is well written, and I have suggested several minor clarifications in my ‘comments for the author’.
I have a few questions and points which need to be addressed, particularly regarding Tables 1 and 6 (see below).
The paper needs some revisions, before being accepted, but it adds to the existing body of knowledge regarding Rotavirus in Mozambique.
Comments for the authors
General points; please check all of the text and Tables to ensure the figures and percentages are in accordance.
Specific points
Abstract
No comments
- Introduction
L63 please correct this to include VP6.
L81-83 I think that this sentence needs rewording slightly to e.g. ‘Before the introduction of rotavirus vaccines, a high rotavirus disease burden was reported in particular the southern Mozambican region, but there was a lack of information in the centre and northern regions of the country [18-20].’
- Results
2.1. Comparison of rotavirus G- and P-types in Mozambique pre- and post-vaccine introduction
No comments
2.2. Comparison of G/P genotype combinations in Mozambique pre- and post-vaccine Introduction
No comments
2.3. Yearly distribution of rotavirus genotypes at the Mavalane General Hospital (HGM) and national surveillance sites
Please revise this section and make sure that the check that the discrepancies between the text and the table are addressed - e.g. in the text, it is stated that G12P[6] (28.6%) and G2P[4] (23.1%) were the most predominant genotype combinations at HGM during 2012-2013, but in the table (Table 6) it seems that G12P[6] was 38.8%, while G2P[4] was 66.7% - please check all of the Tables, throughout the manuscript.
I think that a line graph of this table would add to the paper - where the fluctuations are evident, particularly among the more dominant G/P genotype combinations.
2.4. Geographical distribution of rotavirus genotypes
No comments
- Discussion
L326 - should ‘none’ be changed to ‘non’ ?
L337 - 340 - please note that in Ireland, prior to introduction of vaccination, unusual genotypes e.g. G2P[6], G3P[6], (Cashman et al., 2011, Lennon et al, 2007), G3P[4], G9P[4], G8P[4], G2P[4+8] and G2G4P[8], Collins et al., 2015, Reidy et al., 2005). I have inserted the additional references for consideration and incorporation as appropriate.
- Materials and Methods
No comments
- Conclusions
No comments
Table 1 No comments.
Table 2 No comments.
Table 3 No comments.
Table 4 No comments.
Table 5 No comments.
Table 6 See comments above.
Figure 1 No comments.
Supplementary Tables
Supplementary Table 1 - why was the data from Maputo Central Hospital excluded ?
What was data from Mavalane GH only available?
Supplementary Table 2 - remove the % sign after 43.8
Additional references
- Cashman, P J. Collins, G. Lennon, B. Cryan, V. Martella,, S. Fanning, A. Staines, H. O’ Shea Molecular characterisation of group A rotaviruses detected in children with gastroenteritis in Ireland in the years 2006-2009. Epidemiology and Infection (2011) 14:1-13.
PJ Collins, Emily Mulherin, Helen O'Shea, Olivia Cashman, Grainne Lennon, Eugene Pidgeon, Suzie Coughlan, William Hall, Séamus Fanning. Changing patterns of rotavirus strains circulating in Ireland: Re‐emergence of G2P [4] and identification of novel genotypes in Ireland. Journal of medical virology (2015) 87 (5), 764-773.
- Lennon, N. Reidy, B. Cryan, S. Fanning, H. O'Shea. Changing profile of rotavirus in Ireland: Predominance of P[8] and emergence of P[6] and P[9] in mixed infections. J. Med. Virol. (2008): 80: 524-530.
Reidy, N. O’Halloran, F. Fanning, S Cryan, B. and O’Shea, H. Emergence of G3 and G9 Rotavirus and Increased Incidence of Mixed Infections in the Southern Region of Ireland 2001-2004. J. Med. Virol. (2005). 77; 571-578.
Author Response
"Please see the attachment"
Author Response File: 
Author Response.pdf
Reviewer 2 Report
The manuscript makes a contribution to worldwide rotavirus surveillance. It covers RVA epidemiology in Mozambique in paediatric population during four years before vaccination was introduced and during four years after anti-RVA vaccine introduction. The genotype distribution is described with the emergence of G3P[4] and G3P[8] strains during the reported period.
The manuscript is written quite comprehensibly and the results are documented by six tables and one figure. As the authors mention the emergence of less common genotypes (e.g. G9P[6] or G3P[4]), I would expect there to be more detailed characterization of those strains with the use of sequencing. Also, the results of multiplex RT-PCR genotyping methodology should be confirmed by the sequencing. It was described before (Arana et al., Infect Genet Evol 2016) that strains reported as G12 with the use of RT-PCR genotyping were later sequenced and identified as equine-like G3 strains. To improve the quality of the manuscript, I strongly recommend the authors to verify the genotyped strains with the use of sequencing.
Moreover, there are many mistakes in numbers in the manuscript, especially in all supplementary tables which are often cited. A lot of the mistakes are just minor – incorrect rounding of decimal numbers (e.g. 23.2558% rounded to 23.2% instead of 23.3% in Table 1 – 10/43) but many of them are more substantial. All supplementary tables should be checked and corrected. Some (not all) mistakes in the text of the manuscript are listed below:
P3, line 63: “... which encode six structural viral proteins (VP1, VP2, VP3, VP4, and VP7)...” Those are only five viral proteins, the authors left out protein VP6.
P5, line 107: “A total of 94.0% (440/468)...” This percentage is taken from Supplementary Table 2, where the wrong number of 94.2% is stated. Please, correct the table.
P5, line 110-111: “... a total of 245 samples were genotyped and in the post-vaccination period (2016-2019) 195 samples (Supplementary Table 1).” Numbers in the cited Supplementary Table 1 are the same, however, these should result from numbers of samples genotyped in all sites during the six included years (Supplementary Table 2). When we add numbers of genotyped samples in pre-vaccine years 2014-2015 (174+59+8+5), we get 246 genotyped samples, for post-vaccine period it is 71+90+26+7=194 genotyped samples.
P5, line 118: “...G9 was the most prevalent G type (30.4%) in the pre-vaccine period (n=200)...” In Table 1 it is stated that there were 61 samples typed as G9. 61/200 is not 30.4% but it is 30.5%. Please, correct this mistake.
P7, Table 2: This table is completely confusing – the number of samples in pre-vaccine period in G-genotyping should correctly be 213 (67+2+10+105+29=213), but the number in the same period in P-genotyping is 204 (1+1+182+20=204), not 212. The numbers of typed samples in post-vaccine period are again different (190 and 194, respectively for G- and P-genotyping). When compared with numbers of genotyped samples for years 2015-2018 at five sentinel sites in Supplementary Table 2, we get once again completely different numbers of genotyped samples.
P10, Table 3: Percentage for G12P[6] (13.0) in pre-vaccine period should be also in bold letters, as it is the third most prevalent genotype per period.
P12, line 230: “...G2P[4] (23.1%) were the most predominant...” Once again, the percentage is not correct. It is 22%, as in 2012+2013 there were 4+16=20 samples of this genotype out of total 67+24=91 samples (20/91=0.21978 which is rounded to 22%).
P14, Table 6: In 2018, there were 12 samples genotyped as G3P[4], which is incorrectly calculated as 7.0%. Correct percentage is 37.5% (12/32) which makes this genotype the most prevalent type in 2018. Subsequently, it is necessary to revise the Results and Discussion accordingly, where the G3P[4] prevalence is mentioned (e.g. P14, line 268; P17, line 369; etc.).
Next mistake in the Table 6: the total number of partially G/P typed samples in 2016 was zero, so the percentage should not be 5.1%, but correctly it is 0%.
Last, but not least P15, line 290: “...in Nampula and Quelimane G9P[4] was the most prevalent at 32.7% and 35.7%, respectively.” This is not correct, the second percentage (35.7%) applies to G9P[6] genotype, as is stated in Supplementary Table 4.
Comments for author File: Comments.pdf
Author Response
" Please see attachment."
Author Response File: Author Response.pdf
Round 2
Reviewer 2 Report
The athors addressed all comments and corrected detected mistakes. I now believe that the manuscript is suitable for publication.
Author Response
Dear Reviewer,
Thank you for considering our manuscript for publication. We thank you for your valuables comments, corrections and recommendations that have improved the quality of our manuscript.
