Thanks for your comments.
Reviewer 1
The introduction is somewhat lengthy, consider making it more concise particularly
pages 1-2.
The introduction section has been reduced as recommended
Where can this 'preestablished protocol' be found? Was it published or registered?
If not, then unfortunately, you cannot claim it was preestablished.
The protocol of the systematic review was registered at PROSPERO under number CRD42020151754
on 10/27/2019 and is accessible at the link: https://www.crd.york.ac.uk/prospero/display_record.php?RecordID=151754
Line 144- a comprehensive search strategy does not necessary reduce publication bias
since the databases used were traditional journal databases. Was grey literature explored?
In addition to conducting an extensive search of the available scientific literature,
we also explored the grey literature, but no documents were retrieved according to
the inclusion and exclusion criteria established as described in line 106.
“Additionally, manual search was performed for bibliographic references of the selected
articles and grey literature databases were also included to minimize publication
bias [51–55]
Page 7- Please include a PICO table with exclusion and inclusion criteria for the
search as Table 1 in the methods.
The elements and the search strategy used according to the acronym PICO are presented
in the supplementary material. The description was included in the methods section,
line 99.
We used PICO (population, intervention, comparison, outcome) as a search strategy
tool as described in S1 Table. Line 108
Page 7- The excluded studies are a bit unclear- why exclude research on vaccines and
prevention if this might be epidemiological or clinical research with data pertaining
to the question at hand? Same with molecular biology research- these criteria are
vague and broad, it would be best to specify the study designs to be excluded and
not the overall research area.
The following types of publication were excluded: “editorials, letters, guidelines,
and reviews.” (line 118). Studies were not included if they did not meet the inclusion
criteria: “report data describing signs and symptoms of fetal growth impairment and
altered development in children exposed to Zika virus during pregnancy, among laboratory-confirmed
mothers and/or children <=2 years old”. (line 126)
Line 167- Data extraction is usually performed by 2 independent reviewers and cross-checked.
This is a substantial limitation.
Data extraction was performed by two independent reviewers (DFA and WB), and reviewed
by the other (MDW) as described in line 125
Line 169- 'elaborated' is not the right word here
It was replaced by “made”. (line 137)
Line 174- 179- The way by which quality assessment was performed is confusing. Was
this done at the study level or outcome level? It seems that for some studies this
was at the study level assessing risk of bias and for others, this was done at the
outcome level using the GRADE approach. And which items were selectively excluded
from the GRADE measure? I think it would be best to find a more suitable measure to
assess risk of bias in your studies rather than adapting an existing one to suit your
types of studies.
Line 215: The quality of articles was assessed with the MINORS instrument for all
observational studies and with the Grading of Recommendations, Assessment, Development,
and Evaluation (GRADE) approach- were both methods used for all studies? The way this
is written is unclear.
Line 218- If MINORS is used for non-comparative studies, how did you assess study-level
quality for the 3 comparative studies? GRADE cannot be used for this purpose as it
is an outcome-level assessment.
Line 218- what is meant by 'most' did not report adequately.. how many?
To assess the quality of case reports we used JBI (Joanna Briggs Institute) critical
appraisal checklist for case reports, which we considered more suitable to our studies.
(Line 145). MINORS was used for the following study designs: cohort, case-control,
sectional and case series. Please see in Methods and in Results.
Methods: …“Assessment of the methodological quality of the observational studies was
based on the Methodological Index for Non-Randomized Studies (MINORS) [53]. The instrument
consists of 12 items, the first eight being specific for noncomparative studies. To
assess the quality of case reports, we used JBI (Joanna Briggs Institute) critical
appraisal checklist for case reports [54]. (line 142)
Results … “Among the articles, more than 90% presented end points appropriate to their
aim and consecutive inclusion of patients according to Minors. Less than 20% of the
studies reported adequately on the following criteria: “loss to follow-up less than
5%”, “prospective calculation of the study size”, “an adequate control group”, “Contemporary
groups”, “baseline equivalence of groups” and “adequate statistical analyses” (Fig
2). From all case reports 36 % did not describe patient´s history adequately. The
other items were adequate in more than 90% of studies and two were not applicable
(Fig 3).”
We also replaced figures 2 and 3. (line 163)
Line 204- A 'description was performed' is grammatically incorrect.
The sentence has been modified to:
…“The studies were described according to country, study and sample characteristics,
clinical examination, imaging tests, autopsy findings….” (Line 151)
Line 207- If quantitative data were available, why was a meta-analysis not performed?
It was not possible to perform a meta-analysis because most of the studies included
were case reports and case series. The remaining four comparative studies presented
different variables and outcomes.
Line 220- 'care' reports?
The word was misspelled, it was “case”. However, the text was excluded because we
have modified the quality assessment method.
Line 221- What is a sufficiently long follow up defined as?
The text was excluded because we have modified the quality assessment method.
Figure 2 is titled 'Quality assessment of comparative studies (MINORS)' whereas in
line 176 you state that the first 8/12 items on the scale are for noncomparative studies.
It appears the tool used here is inappropriate. Please use a more suitable tool.
“Quality assessment of the studies included in the systematic review based on Methodological
Index for Non-Randomized Studies (MINORS).”
MINORS is used to assess the quality of non-randomized studies. We used this method
to evaluate cohort, case-control, cross-sectional and case series studies. The instrument
has 12 items, the last four apply only to comparative studies.
As directed by Reviewer 1, some of the table language is not in English- please correct
and proof this (PAÍS? Mather?)
We reviewed all the text of the manuscript. Portuguese terms were excluded.
The symbols used in Table 1 for diagnostic method are missing in some parts and difficult
to follow- suggest including the words/ abbreviations instead or finding a better
way of presenting this information.
The symbols were reviewed and some were replaced (line 185)
Line 263 onwards- please include referencing for the studies discussed (this is done
in other sections e.g. line 325-330, but not here and should be consistent).
It seems there are sufficient data for a meta-analysis.
Line 334-336 and onwards- Include references for the studies here and throughout.
The references had been inserted in the tables only, but we included all the references
cited in the text. (lines 215-217, 219-222, 227-231, 242-243, 245-246, 267-276, 278-285,
289-292, 300-302)
Line 334- Correct bracket/ parantheses
The brackets were removed.
“Lesions in other organ systems were also observed, to a lesser extent: cardiovascular
system, [16], genitourinary system (ambiguous genitalia [72] and bilateral cryptorchidism
[37,81]), and gastrointestinal system (dysphagia [16,68]) (Table 2). Other clinical
features included small for gestational age (SGA) in six studies [16,20,66,81,82,87],
hearing abnormalities in four [16,56,60,84], and unilateral diaphragmatic paralysis
in three articles [37,67,70] (Table 2).” (line 241)
Line 396-400: Can this be presented in a table to show each sign and symptom and how
many studies reported on this being associated with ZKV (and their references)?
We included tables presenting the most frequent signs associated with ZIKV reported
in the studies, regardless the diagnostic method, in the supplementary material.
The manuscript text in lines 396-400 was modified to “…In our review, the main signs
and symptoms associated with congenital Zika virus syndrome were microcephaly, parenchymal
or cerebellar calcifications, ventriculomegaly, CNS hypoplasia or atrophy; arthrogryposis;
ophthalmic findings mainly focal pigmentary retina mottling, chorioretinal atrophy
and/or coloboma, pallor, atrophy, increased excavation, hypoplasia and/or coloboma
of optic nerve and abnormal visual function and low birthweight for gestational age”
(line 312)
Line 401: how many studies reported on microcephaly?
Forty one
Microcephaly was the most frequent sign found in neonates exposed to ZIKV during pregnancy
among the articles in our review [25,35–38]. Forty one studies reported microcephaly
[16,20,37,42,55–57,59–64,66,67,67,68,70–73,76–84,86–91,93–98]. (line 318)
The terms ZIKV, ZKV and Zika virus or Zika syndrome are all used. Please use the abbreviation
specified in the introduction and keep this consistent throughout
The terms were corrected in the manuscript.
Line 401-448: This entire section of the discussion just reiterates the results (as
does most of the discussion). I would summarise and place into context- why is this
important? What is new here?
In agreement with Reviewer 1, more thought it needed to describe what this review
adds to the literature and what the limitations of the literature are.
What are the limitations of the review itself? You need a section describing the strengths
and limitations, potential for bias in your results and issues with methodological
rigour.
The manuscript would be improved with more careful thought in the discussion and presenting
results in light of study limitations as well as discussion of the implications of
the findings. At present, the discussion just describes the results again and there
is a need for further synthesis/ summarising and description of what this study adds
to what is already known.
The discussion has been rewritten. The changes will be described at the end of this
letter.
Page 19 line 374 - "There were calcifications in five articles" change to "Calcifications
were reported in five studies"
The text has been modified.
“Parenchymal or cerebellar calcification were described in 37 studies of our review
[16,20,37,42,56,59–68,70–73,75–82,84,87,88,90,91,93–95,97,98] and also in other review
which reported a prevalence of 42.6% [44]. These findings may also be found in congenital
infections known by the mnemonic TORCH (Toxoplasmosis, Rubella, Cytomegalovirus and
Herpes. However, the distribution of intracranial calcifications differs in each congenital
infection: typically larger, denser and subcortical in congenital ZIKV, punctate and
periventricular or cortical in CMV, diffuse and widely distributed in congenital toxoplasmosis
and at basal ganglia in rubella [38,106–108]. Besides that it should be noted that
some neurological changes (calcifications and cerebral atrophy) in congenital Zika
syndrome are similar to other syndromes of infectious and also genetic etiology, such
as Aicardi-Goutières syndrome [109].” (line 325)
Page 22 lines 450-451 - Unformatted reference? (ROBINSON et al., 451 2018)
The reference was formatted …“The pathophysiology of ZIKV infection and the mechanism
of the virus’ passage across the placental barrier are still under investigation [45].
(line 383)
Table 1 - In the caption, it should be "...characteristics of the study SAMPLE" not
POPULATION it has been corrected
Table 1 - Words in Portuguese are scattered throughout the table (examples, País,
imunohistoquimica)
Table 1 - change title of "POPULATION" column to "SAMPLE SIZE"
Table 1 - Correct typo in the column "MOTHER'S", not "MATHER'S"
Table 1 - What is "null" in Carvalho et al (2016) row, column CEPHALIC PERIMETER?
Table 1 - In DIAGNOSTIC METHOD(ZIKV) column some legend symbols are rendering as white
squares. I suggest changing to letters or numbers if possible.
Table 1 - In English, decimal separators are points (.) not commas (,). Change all
instances throughout Table 1
It has been corrected throughout the document.
Figure 3 - Improve quality of this figure. The numeric axis is unreadable and the
text in the quality assessment categories are truncated. Font color should be black,
not grey.
Figure 3 has been reviewed and modified.
Table 3 - Instead of "Associated factors" it should be "outcomes". Again, change "population"
to "SAMPLE SIZE"
Table 3 - Why "associated factors" are repeated in this table? Reformat to reduce
visual polution
Table 3 - Why some studies have the wording case/control and others have exposed/unexposed?
Standardize nomenclature.
Table 3 - Why studies in the bottom part of the table 3 have contingency tables of
case/control/whatever groups and the upper part does not?
Table 3 - Why "Not calculated"? If you have the contingency tables (as it seems to
be the case) you could calculate yourselves.
Table 3 has been reviewed and modified: the association measures were calculated for
Brazil (2016); the spreadsheet data were presented according to the measure of association
used in the analysis: odds ratio for the case-control study and relative risk for
cohort studies. Zin, 2016 was excluded because the comparative groups were missing.
References – Words in Portuguese (excluding the titles) are scattered throughout citations
It has been corrected throughout the document.
Reviewer 2
Well written systematic review on Congenital Zika Syndrome (CZS).
The introduction could be strengthened to expand on international travelers.
Zika as arboviral disease in pregnancy should be briefly compared to other arboviral
diseases in pregnancy, and this paper could be referred to:
Dengue, Zika and chikungunya during pregnancy: pre- and post-travel advice and clinical
management.
Vouga M, Chiu YC, Pomar L, de Meyer SV, Masmejan S, Genton B, Musso D, Baud D, Stojanov
M. J Travel Med. 2019 Dec 23;26(8):taz077. doi: 10.1093/jtm/taz077. PMID: 31616923
Free PMC article. Review.
The authors would do well highlighting how Zika could still re-emerge post 2016 outbreak
in Asia and Africa where seroprevalence, and hence herd immunity, is not so high as
in the Americas:
Zika in Angola and India.
Hamer DH, Chen LH. J Travel Med. 2019 Jun 11;26(5):taz012. doi: 10.1093/jtm/taz012.
Incidence of laboratory-confirmed Zika in Israeli travelers to Thailand: 2016-2019.
Leshem E, Lustig Y, Brosh-Nissimov T, Paran Y, Schwartz E. J Travel Med. 2019 Oct
14;26(7):taz057. doi: 10.1093/jtm/taz057.
We included in the discussion, line 307
“The ZIKV reemergence in regions with autochthonous transmission and the introduction
of the virus in areas with established vector mosquito infestation may increase the
risk of the development of congenital Zika syndrome in all regions of the world, especially
in Africa, the Americas, Southeast Asia and the Western Pacific [158].
In the absence of effective vaccines, licensed to date [164,165], and considering
the complexity of effective vector control, innovative intersectoral strategies that
transcend exclusive vector chemical control actions should be incorporated in areas
with viral circulation [159–164]. Prevention strategies such as the use of repellents
by pregnant women and condoms by sexual partners, both for travelers to regions with
ZIKV circulation, and for residents of risk areas, as well as the implementation of
strict screening protocols in the donation system of blood should be implemented.
In addition, pregnant women and those intending to become pregnant may be advised
to avoid unnecessary travel to endemic regions [165].” (line 468)
The introduction should also briefly highlight that Guillain-Barre Syndrome is the
other feared complication of Zika.
It was described in the introduction:
“Most individuals infected with the Zika virus either do not develop symptoms or have
mild and self-limited signs [12–14]. However, the disease has been linked to several
neurologic manifestations in children and adults such as Guillain-Barré syndrome and
peripheral nerve involvement, and ophthalmic complications such as retinal and optic
nerve abnormalities.” (line 66)
The methods section should highlight the need for a good case definition:
Zika virus infection in pregnancy: Establishing a case definition for clinical research
on pregnant women with rash in an active transmission setting.
Ximenes RAA, Miranda-Filho DB, Brickley EB, Montarroyos UR, Martelli CMT, Araújo TVB,
Rodrigues LC, de Albuquerque MFPM, de Souza WV, Castanha PMDS, França RFO, Dhália
R, Marques ETA; Microcephaly Epidemic Research Group (MERG). PLoS Negl Trop Dis. 2019
Oct 7;13(10):e0007763. doi: 10.1371/journal.pntd.0007763. eCollection 2019 Oct. PMID:
31589611 Free PMC article.
Not sure whether I overlooked it, but the issue of asymptomatic Zika infections in
pregnancy and their fetal outcomes needs to be highlighted.
The need for a good case definition is highlighted in the introduction section:
“...The laboratory diagnosis of ZIKV infection is limited by the high cost and cross-reaction
with other flaviviruses [161,162], and then protocols for clinical diagnosis, in the
context of simultaneous infection by other arboviruses, need to be implemented to
define cases of ZIKV infection among pregnant women who have a rash” (lines 62-65).
The issue of asymptomatic Zika infections is described in the discussion:
…“Congenital Zika syndrome is a serious public health problem, both because of the
clinical severity of the cases and the extent of functional impairment. Absence of
signs and symptoms at birth in exposed infants does not rule out their appearance
later in childhood, thus highlighting the importance of structuring healthcare networks
for comprehensive monitoring and care of these children. The high prevalence of asymptomatic
cases at birth (65 to 83%) [5,157] can delay the identification of the association
between congenital syndromes (with or without late onset) and ZIKV infection in the
mother during pregnancy. Efforts are needed to provide care and support for all the
needs of children with congenital Zika syndrome and their families, as well as effective
organization of healthcare and social services.” (lines 457-466)
The discussion should end with the need for bringing a vaccine to the market and underpin
the hurdles and challenges to indeed achieve this.
It is described in the discussion
…“In the absence of effective vaccines, licensed to date [164,165], and considering
the complexity of effective vector control, innovative intersectoral strategies that
transcend exclusive vector chemical control actions should be incorporated in areas
with viral circulation [159–164]. Prevention strategies such as the use of repellents
by pregnant women and condoms by sexual partners, both for travelers to regions with
ZIKV circulation, and for residents of risk areas, as well as the implementation of
strict screening protocols in the donation system of blood should be implemented.
In addition, pregnant women and those intending to become pregnant may be advised
to avoid unnecessary travel to endemic regions [165].” (line 471)
Other changes made to the text:
Abstract
� We included the word usually in line 28
The signs and symptoms of Zika virus infection are usually mild and self-limited.
• We deleted ophthalmic and in line 29
• We included and; and also to in line 30
• We deleted prevalence of in line 32
• We deleted the word preestablished in line 35
� We modified “..microcephaly, abnormal brain findings detected by imaging studies,
significant changes in head circumference, low birthweight, low weight for gestational
age, adverse pregnancy outcomes, central nervous system anomalies, corpus callosal
anomalies, cerebral hyperechogenicity, and intrauterine fetal death.” lines 40-43.
To: “...parenchymal or cerebellar calcifications, ventriculomegaly, CNS hypoplasia
or atrophy, arthrogryposis, ocular findings in the posterior and anterior segments,
abnormal visual function and low birthweight for gestational age.” (lines 40-42).
� We modified “…It is thus necessary to develop effective preventive measures such
as the use of specific repellents for pregnant women in areas of viral circulation
and the establishment of effective public vector control measures” in lines 47-49.
To Our findings outline the disease profile in newborns and infants and may contribute
to the development and updating of more specific clinical protocols. (lines 46-47).
Introduction
� We replaced “…Zika virus (ZIKV) is a flavivirus of the family Flaviviridae, isolated
in 1947, initially in non-human primates in Uganda, Africa, in humans in 1954 in Nigeria,
in Aedes aegypti mosquitoes in 1969 in Malaysia, and in humans in 1977 in Indonesia
[1–4]” in lines 52-54.
� With “...Zika virus (ZIKV) is a flavivirus of the family Flaviviridae, isolated initially
in non-human primates in Uganda (1947), and in humans (1954) in Nigeria, Africa[1–4]
in lines 52-53.
� We replaced “…introduction of ZIKV in Brazil, in 2013 or 2014 [7], the first cases
of the disease were only reported in May 2015 [8]” in lines 59-60.
� With “...In Brazil, the first cases of the disease were reported in May 2015 [6]”
in lines 55-56.
• We replaced the word “perinatal” in line 64 with “vertical” in line 60
We included in line 62 The laboratory diagnosis of ZIKV infection is limited by the
high cost and cross-reaction with other flaviviruses [12,13], and then protocols for
clinical diagnosis, in the context of simultaneous infection by other arboviruses,
need to be implemented to define cases of ZIKV infection among pregnant women who
have a rash [14].
� We replaced “…However, the disease has been linked to ophthalmic and neurological
complications such as Guillain-Barré syndrome and peripheral nerve involvement” in
lines 67-69.
� With “...However, the disease has been linked to several neurologic manifestations
in children and adults such as Guillain-Barré syndrome and peripheral nerve involvement,
and ophthalmic complications such as retinal and optic nerve abnormalities” in lines
67-70.
• We replaced “besides various” in line 70 with “and also with” (line 71)
• We deleted lines 71-75
In Brazil, from 2015 to 2017, there were 433 suspected cases of ZIKV in fetuses involving
miscarriages and stillbirths [30]. In 2018, 3,332 cases were reported in children
with impaired growth and development. Among fetal, neonatal, or infant deaths related
to ZIKV and other infectious etiologies, 357 cases were reported [31].
• We deleted lines 81-83
“…Because of the increase in reported cases of microcephaly and the appearance of
other signs and symptoms in fetuses and newborns exposed to the Zika virus during
pregnancy…”
• We replaced the word appearance in line 79 with onset (line 75)
• We replaced the word from in line 84 with in (line 79)
• We deleted”...until 84 November of the same year...” in line 84-85.
� We replaced “...The detection occurred by clinical examination and imaging tests,
which also reported brain calcifications. Children born to women infected with ZIKV
between gestational weeks 16 and 18 presented more severe microcephaly (CDC, 2016).
A cohort study of pregnant women conducted in Brazil described spontaneous abortions
and other neurological changes related to ZIKV infection. There were four infants
with microcephaly, 49 children had alterations in the clinical examination, in the
imaging tests, or both, and 31 (63%) had abnormal neurological tests. The authors
concluded that ZIKV infection during pregnancy is harmful to the fetus and is associated
with fetal death, intrauterine growth restriction, and a spectrum of central nervous
system abnormalities such as brain calcifications, atrophy, ventricular enlargement,
and hypoplasia of central nervous system (CNS) structures [13,17,35] in lines 90-100.
� With “...Detection of ZIKA infection during pregnancy has been found to be harmful
to the fetus and can lead to fetal death and other abnormalities in newborns [12,16,34]
in lines 85-87.
• We deleted lines 101-106
“Other clinical findings have been described in the literature, including craniofacial
disproportion, brainstem dysfunction, seizures, irritability, limb contractures, spasticity,
auditory and ocular abnormalities, and dysphagia, in addition to various abnormal
neuroimaging findings [14,21,29,36–44]. These findings can also occur in similar congenital
syndromes associated with other infectious diseases, such as toxoplasmosis, syphilis,
varicella, parvovirus B1, rubella, cytomegalovirus, and herpes simplex [41,45–47].”
• We deleted lines 112-115
“Importantly, most of the published studies have been case reports and case series.
They are usually based on early assessments which may help to describe the clinical
picture, establish the natural history, and depict potential association of the virus
with congenital syndromes.”
� We replaced “...The characterization of congenital Zika syndrome is still challenging,
mainly due to its recent discovery and the lack of robust studies with scientific
evidence. This review aimed to determine the prevalence of signs and symptoms and
characterize the congenital Zika syndrome based on a systematic review of the scientific
literature” in lines 120-123.
� With “...This review aimed to determine the signs and symptoms that characterize
the congenital Zika syndrome and contribute to a more accurate and timely diagnosis”
in lines 95-97.
Methods
� We repleced “...This review was performed with a preestablished protocol and described
according to the recommendations of the Preferred Reporting Items for Systematic Reviews
and Meta-Analyses statement [59]” in lines 129-131
With “…This review was performed with a protocol and described according to the recommendations
of the Preferred Reporting Items for Systematic Reviews and Meta-Analyses statement
[50] and is registrered in PROSPERO (CRD42020151754) in 27 October 2019. We used PICO
(population, intervention, comparison, outcome) as a search strategy tool as described
in S1 Table. in lines 104-108.
• We Included lines 114-116
“…Additionally, manual search was performed for bibliographic references of the selected
articles and grey literature databases were also included to minimize publication
bias [48–52].”
• We deleted lines 141-147
Only 4 studies included in our review presented measures of association, therefore,
it was not possible to perform a meta-analysis and to assess publication bias using
statistical procedures. However, to minimize publication bias, we used a comprehensive
search strategy [60,61].
Congenital Zika syndrome is a disease that is still being characterized, so data from
observational studies with a limited number of participants and no measure of association
were included.
• We fixed the typos in line 153 (DAF and WB) (line 125)
• We deleted lines 159-161
“... entomological studies, molecular biology research, and in vitro studies; social
and psychological research, studies in public policy; descriptive studies on epidemiological
profile, research on vaccines and prevention”.
� We replaced “...Data were extracted by one of the authors (DFA) and reviewed independently
by the other (MDW)” in lines 167-168
� With “…Data were extracted by two authors independently (DAF and WB), and reviewed
by the other (MDW)” in lines 135-136.
• We replaced the word elaborated in line 169 with made (line 137)
• We included the word observational in line 175 (line 143)
• We replaced “…To assess the quality of case reports and case series, the Grading
of Recommendations, Evaluation, Development, and Evaluation (GRADE) was used. The
original instrument consists of 8 items, but we used 5 items, because the others are
more relevant for studies on adverse drug events, which was not the purpose of our
review [62] in lines 177-181.
• With “...To assess the quality of case reports, we used JBI (Joanna Briggs Institute)
critical appraisal checklist for case reports [53]” in lines 145-146.
• We replaced the word LG ( line 181) with DAF (line 147)
• We replaced “…A description was performed of the studies regarding...” line 204
• With “...The studies were described according to the country in line 151.
• We deleted line 207-209
Odds ratios (OR) and respective confidence intervals (CI) were presented for studies
that performed multivariate analysis of factors associated with congenital Zika virus
infection [63].
• We Included the word findings in line 152.
• We replaced “…The quality of articles was assessed with the MINORS instrument for
all observational studies and with the Grading of Recommendations, Assessment, Development,
and Evaluation (GRADE) approach for case series and case reports. Comparative studies
(n=3) achieved good quality criteria, while most did not report adequately on the
following criteria: loss to follow-up less than 5% and prospective calculation of
the study size (Fig 2). At least 93% of the care reports and case series showed excellent
quality standards, but 26% had not performed sufficiently long follow-up for outcomes
to occur (Fig 3). Most of the studies were performed in Brazil (59%), the epicenter
of reported cases (Table 1)” in lines 215-223.
• With “…Among the articles, more than 90% presented end points appropriate to their
aim and consecutive inclusion of patients according to Minors. Less than 20% of the
studies reported adequately on the following criteria: “loss to follow-up less than
5%”, “prospective calculation of the study size”, “an adequate control group”, “Contemporary
groups”, “baseline equivalence of groups” and “adequate statistical analyses” (Fig
2). From all case reports 36 % did not describe patient´s history adequately. The
other items were adequate in more than 90% of studies and two were not applicable
(Fig 3)
…Most of the studies were performed in Brazil (59%), the epicenter of reported cases.
Regarding study design, most of the studies were case reports (n=22) or case series
(n=20), and there were two cohort studies, one case-control, and one cross-sectional
study. All the studies reported at least one laboratory method for the diagnosis of
women and/or children exposed to Zika virus during pregnancy. Most of the studies
described signs and symptoms in children exposed to Zika virus in the first and second
trimesters (Table 1)” lines 163-169 and 178-184.
• We replaced figures 2 and 3.
• We replaced Tables 1 and 3
• We included references in lines 215-217, 219-222, 227-231, 242-243, 245-246, 267-276,
278-285, 289-292, 300-302
• We deleted lines 263-264
Neurological signs and symptoms were the ones most frequently described in both
the imaging tests and autopsies
• We replaced “…Four Four articles described the association between congenital ZIKV
infection, microcephaly, and other adverse pregnancy outcomes. One case-control study
demonstrated the association between microcephaly and ZIKV, tested by Zika virus-specific
IgM and quantitative RT-PCR in serum and cerebrospinal fluid of neonates with microcephaly
and two controls, while maternal serum samples were tested by plaque reduction neutralization
assay for Zika virus and dengue virus. In addition to microcephaly, De Araújo et al.
(2016) [21] detected congenital malformations through abnormal brain findings on imaging
tests in children with virus in the serum or cerebrospinal fluid and serum only, and
anthropometric alterations such as significant changes in head circumference, birth
weight, and weight for gestational age. Brasil et al. (2017) described adverse pregnancy
outcomes, including cases of fetal loss in every trimester and the need for emergency
cesarean sections [17]. In a case series of children exposed to ZIKV, an association
was found between ocular changes and first-trimester infection, microcephaly, arthrogryposis,
and other central nervous system (CNS) lesions [65] (Table 3). ZIKV infection was
associated with CNS anomalies, corpus callosum anomalies, cerebral hyperechogenicity,
and intrauterine fetal death [41]” in lines 266-282.
• With “...Three articles described the association between congenital ZIKV infection,
microcephaly, and other adverse pregnancy outcomes [16,20,55]. One case-control study
demonstrated the association between microcephaly and ZIKV in neonates tested by Zika
virus-specific IgM and quantitative RT-PCR in serum and cerebrospinal fluid. In addition
to microcephaly, this study detected congenital malformations through abnormal brain
findings on imaging tests in children with virus in the serum or cerebrospinal fluid
and serum only [20]. One cohort study described adverse pregnancy outcomes, including
cases of fetal loss in every trimester [16]. Another cohort study identified an association
between ZIKV infection and CNS anomalies, cerebral hyperechogenicity, and intrauterine
fetal death [55] (Table 3)” in lines 247-256.
• We replaced “…A frequently observed clinical sign was arthrogryposis, reported in
12 articles. Other osteoskeletal signs included clubfoot and hip dysplasia. Morphological
changes of the head were described in ten articles and overriding sutures or closed
fontanels in five.
• The most frequent ophthalmic changes were in the posterior segment, found in 22%
of the reviewed articles that presented abnormalities of the retina and optic nerve
[29,65,67,73,73,74,88,91,96]. Abnormalities of visual function were described in 11%
of the articles [67,73,84,86,88], as well as extrinsic ocular motility [65,67,74,84].
Abnormalities in the anterior segment appeared in 9% of the articles [74,91,98] and
refractive error in 4% [67,91] (Table 2)” in lines 322-330.
• With “...A frequently observed clinical sign was arthrogryposis, reported in 12
articles [37,58,66,68,70,72,77,82–84,87,89]. Other osteoskeletal signs included clubfoot
[82,84] and hip dysplasia [16,68]. Morphological changes of the head were described
in ten articles [16,37,62,66,68,70,77,80,81,84] and overriding sutures or closed fontanels
in five [67,68,77,80,81].
The most frequent ophthalmic abnormalities occurred in the posterior segment, found
in 22% of the reviewed articles which included lesions of the retina ( focal pigmentary
retina mottling, chorioretinal atrophy and/or coloboma) and optic nerve (pallor, atrophy,
increased excavation, hypoplasia and/or coloboma) [28,57,59,66,66,67,81,84,89]. Abnormalities
of visual function were described in 11% of the articles [59,66,77,79,81], as well
as extrinsic ocular motility (nystagmus and strabismus)[57,59,67,77]. Abnormalities
in the anterior segment (cataract and glaucoma) appeared in 9% of the articles [67,84,91]
and refractive error (myopia, hyperopia and astigmatism) in 4% [59,84]. Lines 218-231
• Lesions in other organ systems were also observed, to a lesser extent: cardiovascular
system, [16], genitourinary system (ambiguous genitalia [72] and bilateral cryptorchidism
[37,81]), and gastrointestinal system (dysphagia [16,68]) (Table 2). Other clinical
features included small for gestational age (SGA) in six studies [16,20,66,81,82,87],
hearing abnormalities in four [16,56,60,84], and unilateral diaphragmatic paralysis
in three articles [37,67,70] (Table 2)” in lines 241-246.
• We replaced titles:
Table 2: Clinical signs of children exposed to ZIKV in pregnancy.
Table 4: Signs detected on imaging tests of children exposed to Zika virus (ZIKV)
in pregnancy.
Table 5: Autopsy findings of children exposed to Zika virus (ZIKV) in pregnancy.
• We deleted in line 332: (congenital heart disease [n=1])
• We deleted in line 333: [n=1], [n=2] and in line 334 [n=2].
• We deleted in line 342: increased fluid spaces
• We replaced “…The following other lesions were observed on imaging tests in a cohort
study” in line 369
• With The following other lesions were observed on imaging tests in one study [16]”
in line 285.
� We deleted line 375-376 “…in three and two studies, respectively.”
Discussion
� We replaced “...In our review, the main signs and symptoms associated with congenital
Zika virus syndrome were microcephaly, abnormal brain findings detected by imaging
studies (calcifications, CNS hypoplasia, ventriculomegaly, corpus callosum anomaly,
cerebral hyperechogenicity), low birthweight for gestational age, adverse pregnancy
outcomes (miscarriage, emergency cesarean section, fetal loss), and intrauterine fetal
death in lines 396-400.
� With “...In our review, the main signs and symptoms associated with congenital Zika
virus syndrome were microcephaly, parenchymal or cerebellar calcifications, ventriculomegaly,
CNS hypoplasia or atrophy; arthrogryposis; ophthalmic findings mainly focal pigmentary
retina mottling, chorioretinal atrophy and/or coloboma, pallor, atrophy, increased
excavation, hypoplasia and/or coloboma of optic nerve and abnormal visual function
and low birthweight for gestational age” in lines 313-317.
� We deleted the word also in line 413
� We deleted words likewise and other in line 414
� We replaced “...Parenchymal or cerebellar calcification was a common sign described
by the studies in the review, similar to the characteristics found in congenital cytomegalovirus
(CMV) infection. However, the distribution of intracranial calcifications differs,
typically subcortical in congenital ZIKV and periventricular in CMV [51,117,118].
It should be noted that the neurological changes (calcifications and cerebral atrophy)
in congenital Zika syndrome show similarities to other syndromes of infectious and
also genetic etiology, such as Aicardi-Goutières syndrome [119] in lines 423-429.
• With “...Parenchymal or cerebellar calcification were described in 37 studies of
our review [16,20,37,42,56,59–68,70–73,75–82,84,87,88,90,91,93–95,97,98] and also
in other review which reported a prevalence of 42.6% [44]. These findings may also
be found in congenital infections known by the mnemonic TORCH (Toxoplasmosis, Rubella,
Cytomegalovirus and Herpes. However, the distribution of intracranial calcifications
differs in each congenital infection: typically larger, denser and subcortical in
congenital ZIKV, punctate and periventricular or cortical in CMV, diffuse and widely
distributed in congenital toxoplasmosis and at basal ganglia in rubella [38,106–108].
Besides that it should be noted that some neurological changes (calcifications and
cerebral atrophy) in congenital Zika syndrome are similar to other syndromes of infectious
and also genetic etiology, such as Aicardi-Goutières syndrome [109] lines 325-335.
• We included lines 353-356
In short, these findings can also occur in similar congenital syndromes associated
with other infectious diseases, such as toxoplasmosis, syphilis, varicella, parvovirus
B1, rubella, cytomegalovirus, and herpes simplex [20,39,55,115] but the signs and
symptoms presented by CZS seem to be more serious.
• We replaced “...The osteoskeletal system was the second most frequently altered
organ system in newborns exposed in utero to ZIKV, featuring arthrogryposis [13,17,66,68,83,89–431
91,113]” in lines 430-432.
• With “...The osteoskeletal system was the second most frequently altered organ system
in newborns exposed in uterus to ZIKV and arthrogryposis was described in 18 studies
reviewed [16,37,58,66–68,70–72,77,78,82–84,87,89,94,116], as observed by other studies,
mainly in more severe cases [12,44,76,110] lines 357-360.
• We replaced …“Ocular findings were described in 15 articles as anatomical changes,
and five of these articles also reported functional changes. However, many of these
anatomical changes may impact the child’s visual function in the future, since they
affect prime areas of the eye. Although ocular alterations have been detected in children
with microcephaly and other brain anomalies [65], Zin (2017) identified ophthalmic
lesions in 7% of children who had no CNS abnormalities, suggesting that ocular abnormalities
may be key findings in the syndrome. Importantly, since the child’s first contact
with the environment is through eyesight, children with visual impairment commonly
experience delayed neuropsychomotor development, which, alongside the syndrome’s other
signs and symptoms, hinder the child’s ability to integrate with his or her surroundings
[120]. Posterior segment ocular alterations have been described as possible features
of the severe form of the syndrome [17], as described in other studies [121,122].
Additionally, Ventura identified posterior segment alterations in 44% of children
and abnormal visual function in 100%, showing that cerebral cortical involvement is
also an important cause of visual impairment in these children [67]. Alterations in
the anterior segment were reported by 9% of the selected studies, also observed in
others [123] “lines 433-448.
• With …“Anatomical [57,59,66–68,70,75,81,84,89] and functional [59,60,68,77,81] changes
of the eye were described in 12 articles, such as posterior and anterior segment abnormalities,
extrinsic eye motility and abnormal visual function. Many of these anatomical changes
may impact the child’s visual function in the future, since they affect prime areas
of the eye. Importantly, since the child’s first contact with the environment is through
eyesight, children with visual impairment commonly experience delayed neuropsychomotor
development, which, alongside the syndrome’s other signs and symptoms, hinder the
child’s ability to integrate with his or her surroundings [117]. These ocular findings
were described also in other studies descripted [44,118–120]” in lines 361-369.
• We included in lines 370-382
“TORCH infections have also been associated with serious ophthalmic alterations. In
congenital toxoplasmosis infection, chorioretinal lesions are usually bilateral and
can present with active lesions or regressed scar [57,121]. Congenital cytomegalovirus
and herpes virus infections can also manifest with active ocular inflammation. In
Zika virus infection, however, there has been no active ocular inflammation cases
reported so far [121]. There is usually not pigmentary mottling seen outside of areas
of chorioretinal atrophy in congenital toxoplasmosis infection, as seen in zika virus
infection [57]. In congenital rubella infection, the pigment mottling is usually
diffused compared with the focal pigment mottling seen in zika virus infection [57].
Congenital cytomegalovirus can present with chorioretinal lesions similar to congenital
toxoplasmosis but less heavily pigmentated and with pale or small optic disc [122,123].
Optic nerve hypoplasia, commonly seen in ZIKV congenital infection, is rarely seen
in rubella, toxoplasmosis and herpes congenital infections [57].”
• We included lines 444-445
“Recent studies have identified neurological disorders such as delay in neurodevelopment,
mainly in the domain of language in children exposed to ZIKV who were asymptomatic
at birth. [124,146].”
� We excluded (ROBINSON et al., 2018) in lines 450-451
We replaced “Eight studies in our review reported placental alterations [41,50,72,85,88,94,97,106]”
in line 453.
� With “Placental alterations were reported in eight studies [37,55,65,78,81,87,90,100]”
in line 387.
� We included the following sentencs in lines 406-413:
“…possibly due to the limited access to those exams in some settings. Obstetric ultrasound
proved to be useful for tracking ZIKV-related brain injury in prenatal care in pregnant
women exposed to ZIKV. Although less accurate than CT and MRI it is available in many
health facilities [141,142].”
“…One point to highlight in our review is the anatomopathological analysis. It was
possible to notice either the severity of the systemic impairment of the cases that
died, especially those whose maternal infection occurred in the first trimester of
pregnancy and the correspondence of clinical and anatomopathological findings.”
� We replaced …”Several studies reported structural changes in the genitourinary system
[41,50,79,143]. Villamil-Gómez (2019) detected severe bilateral renal hypoplasia in
fetal autopsies [144]. Recent studies in older children have detected bladder impairment
and possible kidney damage such as very low bladder capacity, bladder hyperactivity
with increased consistency, high bladder pressure during the filling phase, and high
postvoid residual volume (PVR) and/or recurrent urinary tract infection [145]. Congenital
cardiovascular anomalies were identified in children exposed to ZIKV [41,68,104,130].
Other studies have also described cardiological symptoms such as complex congenital
heart disease, echocardiographic abnormalities, and cardiac overload [146–149]” in
lines 490-498.
With “...Structural changes in the genitourinary system were reported [40,58,75,148]
and were also described by Villamil-Gómez (2019) in fetal autopsies [149]. Bladder
impairment and possible kidney damage such as very low bladder capacity, bladder hyperactivity
with increased consistency, high bladder pressure during the filling phase, and high
postvoid residual volume (PVR) and/or recurrent urinary tract infection were reported
in older children [150]. Congenital cardiovascular anomalies were identified in children
exposed to ZIKV such as cardiomyopathy, hyperechogenicity of aortic valve, mitral
valve, and aortic root [19,58,64,100]. Other studies have also described cardiological
symptoms and imaging findings such as complex congenital heart disease, echocardiographic
abnormalities, and cardiac overload [151–154]. Lines 422-432
� We replaced “Recent studies have identified neurological disorders in children exposed
to ZIKV who were asymptomatic at birth [137,151]. Such findings are relevant, since
there is evidence that ZIKV can continue to replicate in the infant’s brain after
birth [152], and that cerebral growth of infants exposed during pregnancy can decelerate,
even after birth [73]”. lines 511-515.
� With “…Recent studies have identified neurological disorders such as delay in neurodevelopment,
mainly in the domain of language in children exposed to ZIKV who were asymptomatic
at birth. [131,153]. Such findings are relevant, since there is evidence that ZIKV
can continue to replicate in the infant’s brain after birth [154], and that cerebral
growth of infants exposed during pregnancy can decelerate, even after birth [66] “in
lines 444-448.
• We included the following sentences in lines 468-480
…“The ZIKV reemergence in regions with autochthonous transmission and the introduction
of the virus in areas with established vector mosquito infestation may increase the
risk of the development of congenital Zika syndrome in all regions of the world, especially
in Africa, the Americas, Southeast Asia and the Western Pacific [158]. “ …In the absence
of effective vaccines, licensed to date [164,165], and considering the complexity
of effective vector control, innovative intersectoral strategies that transcend exclusive
vector chemical control actions should be incorporated in areas with viral circulation
[159–164]. Prevention strategies such as the use of repellents by pregnant women and
condoms by sexual partners, both for travelers to regions with ZIKV circulation, and
for residents of risk areas, as well as the implementation of strict screening protocols
in the donation system of blood should be implemented. In addition, pregnant women
and those intending to become pregnant may be advised to avoid unnecessary travel
to endemic regions [165]”.
• We deleted lines 539-541
� We replaced” …One limitation to the study was the lower number of observational clinical
studies. The inclusion of case series and case reports, with lower levels of scientific
evidence, is justified for the investigation of new and rare diseases such as congenital
Zika syndrome, since evidence based on clinical case reports is necessary to understand
the illness process [157] in lines 535-539
� With “…One limitation to the study was the low number of comparative clinical studies.
The inclusion of case series and case reports, with lower levels of scientific evidence,
is justified for the investigation of new and rare diseases such as congenital Zika
syndrome, since evidence based on clinical case reports is necessary to understand
the natural history of a new illness [166]” in lines 481-485.
- Attachments
- Attachment
Submitted filename: Response to Reviewers.pdf
https://orcid.org/0000-0002-5460-8316
