Clinical and x-ray oral evaluation in patients with congenital Zika Virus

Abstract Objective: The aim of this study was to investigate possible malformations in the soft, bone and/or dental tissues in patients with congenital Zika Virus (ZIKV) by clinical and x-ray evaluation. Methodology: Thirty children born with ZIKV and 30 children born without ZIKV (control group) were included in the study. Patients were evaluated over 24 consecutive months according to the variables: sex, age, cleft palates, soft tissue lesions, alveolar ridge hyperplasia, short labial and lingual frenums, inadequate posture of the lingual and perioral muscles at rest, micrognathia, narrow palatine vaults, changes in the teeth shape and/or number, sequence eruption, spasms, seizures and eruption delay were evaluated. Chi-square test, Student's t-test and nominal logistic regression were used (p<0.05). Results: Among the 30 babies examined, the mean age of the first dental eruption was 10.8±3.8 with almost two-thirds of the children (n=18, 60%) experiencing eruptions of their first tooth after 9 months of age, nine children (30%) had inadequate lingual posture at rest, more than half of the children (n=18, 60%) had short labial or lingual frenums. ZIKV babies showed a high prevalence of clef palate (p<0.001), inadequate lingual posture at rest (p=0.004), micrognathia (p=0.002), changes in the shape and/or number of teeth (p=0.006), alteration in sequence of dental eruption (p<0.001) and muscles spasms (p=0.002). The delay eruption was associated with inadequate lingual posture at rest (p=0.047), micrognathia (p=0.002) and changes in the shape and/or number of teeth (p=0.021). The delayed eruption (p=0.006) and narrow palatine vaults (p=0.008) were independently associated with ZIKV. Moreover, female patients showed the most narrow palatine vaults (p=0.010). Conclusions: The children with ZIKV showed a greater tendency to have delayed eruption of the first deciduous tooth, inadequate lingual posture and short labial and lingual frenums.

The known clinical symptoms are: fever, headache, arthralgia, myalgia, and a maculopapular rash, a complex of symptoms that hinder differential diagnosis. 1 The evidence of the relationship between ZIKV infection and cerebral birth abnormalities, namely, microcephaly, was first described in January 2015 and has been growing. 2 The direct cells targeted by ZIKV in the developing human fetus are not clear. Recent studies have shown that a strain of the ZIKV, MR766, which is serially passed from monkey and mosquito cells, efficiently infects human neural progenitor cells (hNPCs) derived from induced pluripotent stem cells. 3 As the face is formed mainly by the first branchial arch, which is divided into maxillary and mandibular processes, some changes in the oral and craniofacial development can occur, because infections, as syphilis, are contracted by the mother during pregnancy. [4][5][6] In addition to microcephaly, other changes related to congenital ZIKV infection have been detected in infected children, such as severe ocular lesions, hearing loss, lack of muscle tone and arthrogryposis.
For this reason, experts have suggested the creation of the term "congenital syndrome of Zika virus". 7,8 In January 2017, the epidemiological bulletin from the Ministry of Health showed a national total of 9,770 cases of microcephaly that were reported from March to October 2016. Of these, 2,334 cases were confirmed to be ZIKV infections, while the others remained under investigation or were discarded. 9 In the state of Ceará, Recently, studies showed alteration in the chronology of the first deciduous tooth eruption in Brazilian children with microcephaly associated with ZIKV. Additionally, it has been suggested that these children have great difficulty in dental care due microcephaly. 11,12 So, considering the current panorama that involves a severe spread of ZIKV syndrome, the aim of this study was to investigate possible malformations in the soft, bone and/or dental tissues in patients with congenital ZIKV by clinical and x-ray evaluation.

Methodology
This study was divided into two phases: an initial cross-sectional observational study that included 30 children born with congenital ZIKV and microcephaly; and a case-control study in which more 30 control children born without congenital ZIKV were analyzed for comparison. The microcephaly diagnosis was based on the guideline of the Brazilian Ministry of Health, which defines microcephaly babies as the newborns with 37 weeks or more of gestational age and cephalic perimeter ≤31.5 cm for girls and ≤31.9 cm for boys. 13 All children were referred from medical services   In this study, a delay in the first dental eruption was considered if children were 9 months old or older at the time of the first eruption. These data were based on studies about the mean ages of eruption: for the lower central incisors the mean age of eruption was between 7 and 9 months; for the lower lateral incisors it was between 12 and 14 months; for the upper central incisors it was between 9 and 11 months; and for the upper lateral incisors it was between 10 and 12 months. [17][18][19] Categorical data were expressed as the absolute and percent frequency and compared using the

Results of the cross-sectional observational study
Among the patients evaluated, 14 (46.7%) were female and 16 (53.3%) were male. At the end of the study, 18 children (60%) were less than 25 months old and 12 children (40%) were 25 months old or older.
The mean age of the first dental eruption of children in this study was 10.8±3.8 months, with almost two-thirds of the children (n=18, 60%) experiencing eruptions of their first tooth after 9 months of age. In all cases, the first eruptions were of the lower central incisors ( Figure 3 and Table 1).
In addition, the last child to present an eruption was 19 months of age. Moreover, three children who were 12 months old had no teeth in their oral cavities at the end of the study. Twelve children in the sample had their first eruptions at up to 9 months of age  Table 1).
The frequency of inadequate posture was 7.3 (95% CI=1.1 -68.9) times higher in patients with delayed

Number of children
Age of child at first dental eruption 12 Between 4 and 9 months   Changes in the dental eruption sequence were found in 4 children, in whom the first deciduous molars erupted before all the lower incisors were present in the oral cavity.
In this study, micrognathia was associated with delays in the first dental eruption. An association 38% higher of micrognathia was observed in patients who presented delay on first dental eruption when compared with patients without delay. In addition, patients with poor dental formation had delays in the first dental eruption that were on average 20% higher compared to patients without this condition. There was no statistically significant difference between the mean time to the first dental eruption and the other variables. Female patients had a high prevalence of narrow palatine vaults when compared to male patients (p=0.010). The other variables showed no association with sex (Table 1).
Patients with short labial and/or lingual frenums were referred to a department specialized in pediatric dentistry to have a frenectomy or a frenotomy performed, when indicated.

Age of the first dental eruption (months)
p-value ZIKV group 10 Table 2).

Results of the multivariate analysis
In multivariate analysis (adjustment by age) the factors most prevalent in babies with congenital ZIKV infection were delayed eruption (p=0.006) and narrow palatine vaults (p=0.008). The adjusted odds ratios were 26.6 (CI 95%=2.5 -279.5) and 24.8 (CI 95%=2.3 -270.1) higher than in control group, respectively. The systemic alterations showed no significant differences in ZIKV and control groups when adjusted by age (Table 3) (Table 4).

Discussion
This is the first two-phase study on ZIKV that consists of a cross-sectional observational study and a case control study that evaluated oral and tooth changes in children with ZIKV and microcephaly.
The major limitation of this study is the difficulty of recruiting large numbers of patients, since the total number of patients in the city of Fortaleza is low (N=56). In addition, these patients present a great difficulty in accessing the health service, as well as difficulty during examinations and x-ray evaluation due to neurological impairment. However, this study showed some relevant initial contributions about the development of oral structures in babies with ZIKV.
The existing Brazilian literature, in agreement with the world literature, indicates that the average age of the first dental eruption is 9 months, and a delay in eruption is considered after this period. 17 In this study, considering congenital ZIKV children, approximately two-thirds of the patients (n=18, 60%) had eruptions of the first tooth after 9 months of age. In the control group, some patients had considerable dental delays, with eruptions at 15, 17 and 19 months.
The most significant result in this study was the delay in tooth eruption, which was 26.6 times more prevalent in ZIKV group than in control group, with a significantly higher average for the first eruption.
The suggestion of this study is that delays in dental eruption are associated with congenital ZIKV syndrome. One of the hypotheses for why delayed tooth eruption occurs is due to failure of the precise mechanisms of dentition development resulting from the infection of human neural progenitor cells by the ZIKV, which culminates in microcephaly and may affect the physiological processes for eruption. 4,6 The narrow palatine vault was 24  In terms of the existence of other abnormalities, an inappropriate lingual posture at rest was observed in 9 of the 30 ZIKV children and was significantly more prevalent than in control group. Of these 9 children, 8 also had delayed dental eruptions (p=0.047) ( Figure   3). This finding may be related to the presence of a more severe neurological condition, such as microcephaly; thus, in addition to an inadequate lingual posture at rest, a delay in the first dental eruption occurs. The structures of the face are mainly composed of cells derived from the neural crest of the embryo. 6,20,21 More than half of the children had short labial or lingual frenums with a prevalence 4.9 times higher in ZIKV group than control group. However, when those variables were adjusted by age in multivariate analysis, the association was not significant. The prevalence of short labial or lingual frenums can occur in newborns, but it is has a significant lower occurrence in older children. 24 Because this study assessed ZIKV patients for a short period of time, the results cannot guarantee that these soft tissue abnormalities will persist throughout the years.
Although there was no significant difference between the control group, 4 children after clinical and x-ray evaluations showed alterations in the shape and/ or number of teeth or change in the sequence of tooth eruption. Such occurrences may also be linked to ZIKV infection of cells that cause the development of dental tissues, thus affecting the correct development of the teeth, both in shape and in number. 4,6 Additionally, considering the other characteristics thus, children will be followed up for medium-and longterm periods to monitor the growth and development of their oral structures. Thus, in the future, a complete skull and facial growth and development profile related to congenital ZIKV syndrome will be proposed.

Conclusions
The results obtained in this study demonstrate that