As a new disease, many questions still need to be clarified about COVID-19 and one of these refers to the possible different methods of transmission, besides the respiratory route.
Later case of clinical manifestation of COVID-19 in a neonate  called the attention to the possible vertical transmission of SARS-CoV-2, even in a small percentage of cases compared to the number of infected mothers.
In order to determine a possible vertical transmission of SARS-CoV-2 from the mother to the fetus during pregnancy, we selected 3 pregnant women with confirmed laboratory diagnosis for COVID-19. It is considered to be vertical transmission not only during pregnancy, with the invasion of virus in placenta through hematogenous route, but also during delivery through transcervical route and postpartum infection through environmental exposure [5,12]. In order to exclude contamination by hematogenous or transcervical routes, we selected three cases of pregnant women whose delivery occurred by cesarean section to ensure that a possible transmission has occurred via transplacental route.
Serological antibody-based test for SARS-CoV-2 was performed as a screening test in the totality of pregnant women admitted to the HNMD for delivery to identify those one who had COVID-19 or was in the course of the disease. Thus, it was identified one case with positive results for both IgM and IgG (case 3), indicating current or recent infection for COVID-19 and two cases of positive IgM and negative IgG (case 1 and 2), indicating current infection . Antibodies seroconverction can occur between less than 1 week and more than 6 weeks after the emergence of symptoms . In fact, obstetric patient case 3 related to had symptoms of headache, runny nose, nausea, myalgia, malaise, joint pain 2 months before the hospitalization date. In one case, serological test was positive for IgM even with no symptoms related by the patient (case 2). This finding corroborates with another study that identified 13.7% asymptomatic obstetric patients through RT-PCR screening test for SARS-CoV-2, pointing out to the importance of carrying out screening diagnostic tests in order to identify infected patients and monitor them more carefully, as well as their infants .
Following, RT-PCR tests of the maternal nasopharyngeal swabs identified two positive cases. In case 2, positive result was in the selected pregnant women. Positive results for SARS-CoV-2 was identified in case 2 and 3, indicating the presence of RNA of SARS-CoV-2 virus. Positive RT-PCR test can be observed in the incubation period prior to the beginning of COVID-19 symptoms. (as seen in case 2), (in case 2, for example) and last until the resolution of symptoms (in case 3, for example) . SARS-CoV-2 was not detected in case 1, being compatible with positive IgM serological test and the oneset of symptoms two months before testing.
The probability of occurrence of infectious vertical transmission through transplacental route increases with increased gestational age  and the results of serological and molecular tests performed in the three cases showed that they were infected in the third trimester of pregnancy. In 5 cases of fetal death, SARS-CoV-2 was detected in the amniotic fluid or placenta and vertical transmission was attested to had occurred during the third trimester of gestation .
To verify the presence of SARS-CoV-2 in the neonates of our study, RT-PCR was performed in the nasopharyngeal swabs collected shortly after labor and before contact with their mothers and the results turned out to be negative. Although RT-PCR is considered the gold standard for diagnoses of SARS-CoV-2 infection, diagnostic efficiency of this test in newborns has not been stablished yet . This can be explained by the fact that the airways are not functional during intrauterine life and that proliferation of SARS-CoV-2 in the upper respiratory tract seems to be irrelevant for the infection of the fetus .
Because the hematogenous route is the most probably mechanism for vertical viral infection , serological test of the umbilical cord blood was performed and the results were different in the three cases. IgM and IgG antibodies for SARS-CoV-2 were negative in case 2 and IgG was detected in the newborn of the case 1, probably of maternal origin that was transferred to the fetus by the placenta. In case 3, positive results for both IgM and IgG in the cord blood immediately after birth is a highly indication that vertical infection occurred, since IgM is a macroglobulin that can not cross the placenta from mother to the fetus . The presence of IgM in the cord blood with negative result for the detection of SARS-CoV-2 in swab samples of the newborn is rare but can be observed , which confirms that RT-PCR analysis of nasopharingeal swab samples may not be a gold standard for diagnosis of COVID-19 in neonates .
Detecting of SARS-CoV-2 was also performed in amniotic fluid and umbilical cord blood samples of the 3 cases. A systematic review identified 51 amniotic fluid samples tested but none of them was positive for SARS-CoV-2 RNA . In our study, first RT-PCR was also not able to detect the virus, then nested RT-PCR was considered for these types of samples. In fact, CDC guideline attests RT-PCR analysis to be performed solely in upper respiratory swab specimens. We could successfully detect SARS-CoV-2 in all of the amniotic fluid and cord blood samples by nested RT-PCR. This
This technique could nested polymerase chain reaction (nested PCR) normalyis used in situations in which it is necessary to increase the sensitivity and/or specificity of PCR . The product of the first amplification reaction (all negative for SARS-CoV-2) was used as the template for the second PCR. Additionally, we used the reverse primers for reverse transcription, generating a specific ssDNA for the region of interest. The use of nested PCR methodology allows to increase the sensitivity of the test up to 100 folds , thus allowing a differentiated research potential for viral infection switch a low number of copies of initial genetic material.
Regarding the analysis of the placentas, there was no evidence of abnormality that could be observed macroscopically, except the small size of the placenta of case 2. Microscopic analysis revealed maternal and fetal vascular malperfusion of the placentas, corroborating with a systematic review of histopathological lesions observed in third semester placentas of COVID-positive mothers . Maternal vascular malperfusion can be observed in placentas of SARS-CoV-2 infected mothers, even though the virus itself was is not identified by RNA in situ hybridization [22,23]. The observed lesions associated with coagulation in the placentas of this study may be related to any inflammatory disease, and SARS-CoV-2 can not be excluded. Hypertension and preeclampsia can also be the associated with vascular thrombotic disease , but we can exclude these hypotheses in the three cases presented.
Histopathological analysis of the placentas showed some extension of old infaction and thrombi, lesions associated with coagulation. Vascular thrombotic disease may be associated with hypertension and preeclampsia , pathologies not observed in the mothers of this study. Another possibility is an associated inflammatory disease to be the cause of the vascular lesions, and SARS-Cov-2 can not be excluded. Also, the placentas from the three cases presented a mild fibrin deposit as well as foci of hemorrages and calcifications in the intervillous space. It is worth mentioning that only in the placenta of case 2 it was noticed a mild focal acute inflammatory infiltrate (mild acute intervilositis), which may be related to a viral infection. Regarding the umbilical cord, in case 2 it was also observed extensive foci of hemorrage in the jelly and hemopoiesis. It is important to notice that so far, there is no specific pathology characteristic of SARS-CoV-2 that could be observed in the placenta .
We are still facing the uncertainties that follows the emergence of a novel virus. As the pandemic spreads around the world, more case reports of COVID-19 accumulate, which allow us to conclude that SARS-CoV-2 is a virus capable of cross the placenta and infect the fetus, even at low rates. We conducted different diagnostic tests that add value information in order to confirm vertical virus transmission via the transplacental route. Also, the results of serological and PCR tests obtained of maternal and neonatal samples indicated that RT-PCR of infant nasopharingeal swab samples is not an option for detection of SARS-CoV-2 vertical infection. Instead, amniotic fluid or umbilical cord blood are non invasive samples that can be analyzed by serological or virological tests.
So far, there is no standard protocol that states the best sample to collect or diagnostic test to run in order to determine the occurrence of vertical transmission . The results here presented show that a single analysis test may not be enough for a diagnostic conclusion and we strongly suggest to conduct different analysis in order to confirm a possible infection of the neonate.