The Frequency of Cytomegalovirus and Toxoplasma Among Microcephalic and Hydrocephalic Neonates Admitted to Pediatric Hospital in Bandar Abbas

http Introduction Most women at the age of fertility in lowor moderateincome countries enjoy long-term immunity to prior cytomegalovirus infections. In African, Latin American, and South Asian countries, more than 90% of the public have immunoglobulin G (IgG) antibodies and 40%-60% of the public have IgG antibodies in comparison to highincome countries (1-6). The prevalence of congenital cytomegalovirus (CMV) in high-income countries is 7% of all live births but between 1 and 5% in low-income countries (1, 4, 7). The risk of transmission grows with an increase in gestational age. In fact, among mothers with a primary infection, 35% of transmissions occur in the first trimester while this rate raises to 65% in the third trimester. The majority of neonates afflicted with CMV are asymptotic. However, the common symptoms among neonates are intrauterine growth restriction (IUGR), sensorineural hearing loss, petechial, and jaundice. Further, neural side effects are observed in 60%-90% of those diagnosed with clinical symptoms upon birth. Up to The Frequency of Cytomegalovirus and Toxoplasma Among Microcephalic and Hydrocephalic Neonates Admitted to Pediatric Hospital in Bandar Abbas

Rostamian et al iejm.hums.ac.ir http 15% of asymptotic neonates upon birth could develop the symptoms during childhood (8). According to (9), most women at the age of fertility in Latin America, Central Europe, and West Africa possess specific antibodies against Toxoplasma gondii (IgG type). On the contrary, in the south-east of Asia, China, and South Korea, as well as Scandinavia and the United States, the immunity level is extremely lower (9,10). Transmission from the mother to the child often occurs after affliction with the infection during pregnancy. The global occurrence rate of congenital toxoplasmosis has been estimated at 1.5 cases per 1000 live births. The highest rate of occurrence in some Latin American countries is 3.4 per 1000 live births while its lowest rate is reported in some parts of Europe as 0.5 per 1000 live births (11). Furthermore, the classical consequences of congenital toxoplasma are chorioretinitis, hydrocephaly, intracranial calcification, and epilepsy. It can also cause infant mortality. Contrary to cytomegalovirus, congenital toxoplasma does not transmit the infection to the child if the mother is immune. The risk of fetal infection following pregnancy progress can increase to 60% in the third trimester although the risk of fetal consequences is significantly lowered along the pregnancy progress (12). Microcephaly is a clinical diagnosis upon birth and describes a smaller than normal head. Nonetheless, this diagnosis does not necessarily point to abnormal brain growth as some children afflicted with microcephaly are healthy. Microcephaly is defined as the head occipitofrontal circumference more than 2 standard deviations (SDs) smaller than the mean size for the gestational age and the gender of a properly healthy population (8). Hydrocephaly is the hydrodynamic disorder of the cerebrospinal fluid (CSF) that can increase the volume taken by this fluid in the central nervous system (13). Moreover, congenital hydrocephaly occurs in 1 per thousand births (14). Considering the above-mentioned explanations, the present research aimed to explore the frequency of cytomegalovirus and toxoplasma among patients afflicted with microcephaly and hydrocephaly.

Materials and Methods
The present descriptive, cross-sectional research was conducted on neonates who were diagnosed with hydrocephaly or microcephaly in Bandar Abbas pediatric hospital in 2018. Throughout this year, 5 and 8 neonates were hospitalized for hydrocephaly and microcephaly, respectively. In the computed tomography (CT)-scan result of 2 cases with microcephaly, cerebral dysgenesis was observed which led to the exclusion of these subjects and 11 neonates were included in the research. Informed consent was obtained from all subjects' parents. Before study initiation, an ethical code was obtained from the Ethics Committee of Hormozgan University of Medical Sciences. Blood samples were taken from neonates with microcephaly lower than 3 SD below the head circumference growth curve and hydrocephaly (even mild) that were hospitalized in 2017 with no known infection but suspected of infections with cytomegalovirus and toxoplasma. The samples were then tested for cytomegalovirus and toxoplasma serology. If the result was positive, the CSF was sent for polymerase chain reaction (PCR) for cytomegalovirus and toxoplasma. Ultrasonography and CT scan were used to determine hydrocephaly. To check toxoplasma and cytomegalovirus serology, the luminance quantitative method was used in this research. In this method, minerals such as luminol, isoluminol, and acridinium esters are oxidized in the presence of an oxidant (e.g., hydrogen peroxide), a catalyst (e.g., the microperoxidase enzyme), and metal ions and then the produced light is further stimulated and appears as a sudden flash read by the luminometer. Eventually, microcephaly was set based on Stark's manual of neonatal care.

Statistical Procedures
The data were analyzed using SPSS software (version 19.0) and descriptive tests such as frequency, percentage, mean and, SD. Moreover, the t-test and Fisher's exact test were run to analyze the data and the P value was set at or below 0.05.

Results
The information describing 11 subjects (5 hydrocephaly and 6 microcephaly cases) were analyzed. The description of these patients is summarized in Tables 1 and 2. Table  3 presents the information bout gestational age upon birth and the head circumference of the two groups. The two groups were compared in terms of gestational age at the time of birth. This mean score was 35.8 ± 4.08 and 37.16 ± 1.32 weeks in the hydrocephaly and microcephaly groups, respectively, although the difference between the two groups was not statistically significant (P = 0.507). The comparative information of some qualitative features is provided in Table 4. In addition, anomaly-related information, along with several qualitative features is reported in Table 5.

Discussion
In the present research, heart problems reached 40% and 66.7% in the hydrocephaly and microcephaly groups, respectively. One hundred percent of patients with an anomaly were term cases while 50% with no anomaly belonged to the term group. It seems that anomaly is more associated with term birth. Moreover, 100% of patients with an anomaly reported their parents' close blood relation. However, 50% of patients with no anomaly reported a close family relation. Based on the results, heart problems were more prevalent in the group with an anomaly. Thus, in the case of anomalies, it is recommended that patients should be checked for heart conditions. Congenital toxoplasmosis engagement patterns can be divided into four categories. In the first category, the most severe side effects occur, including cerebral calcification, hydrocephaly, encephalomyelitis, and even abortion. The second category is marked by severe systematic infections, hepatosplenomegaly, pneumonia, myocarditis, and the like. What usually occurs in the third category includes hyperbilirubinemia, epilepsy, purpura, chorioretinitis, lymphadenitis, and fever. Further, subclinical toxoplasmosis or generalized symptoms may occur in the fourth category (15). The congenital infection of cytomegalovirus is asymptotic in the majority of cases and symptomatic only in 11% of them. Even when the secondary maternal infection is the cause, the prevalence of symptoms at birth reaches the rate below 2%. However, in the related body of literature, a correlation has been reported between preterm birth, low birth weight, and the congenital infection of cytomegalovirus. The congenital infection of cytomegalovirus is the main cause of child hearing loss and other neurological disorders such as cognitive and visual disorders. A mild sensory-neural hearing loss is observed in about one-eighth (12.5%) of neonates afflicted with the infection. In a case series study, the hearing problem was reported to be improved after treatment with ganciclovir and valganciclovir. Moreover, research has shown a higher occurrence rate of the congenital infection of cytomegalovirus in congenital hypothyroid cases and Down Syndrome (16). In another Note. SD: Standard deviation. research concerning congenital toxoplasmosis, the most prevalent engagement symptoms were found in the eyes and brain (17).
In the present research, toxoplasma and cytomegalovirus serology were negative in neonates with hydrocephaly and microcephaly. This difference can be explained by the smaller sample size. Concerning the serology of the blood sample, in the present research, the luminance quantitative method was used to check serology. The sensitivity of this test is lower than its specificity which leaves the chance of a false negative.
In another research on 64 neonates born with congenital anomalies, the ELISA method was used to investigate the antibody level against TORCH (Toxoplasma gondii, other, rubella virus, cytomegalovirus, and herpes simplex virus). Based on the findings, 6% of neonates and 14% of mothers showed an anti-toxoplasma immunoglobulin M (IgM), rubella, and positive cytomegalovirus (18). In another investigation for the screening of all neonates, the cytomegalovirus serology was analyzed via the ELISA technique in order to check IgG and IgM and in terms of the DNA virus through PCR. Overall, the prevalence of cytomegalovirus was reported as 12.57% out of which 5.71% pertained to congenital cause and 8.86% was explained by prenatal infections. In neonates with a CMV infection, 80% were milk-fed and 30% had a concomitant congenital anomaly. On the contrary, in neonates with a prenatal infection, 75% were breast-fed and 16.67% were diagnosed with anomalies. It was concluded that low birth weight, congenital anomalies, and breastfeeding were among the risk factors of cytomegalovirus infection in the neonatal intensive care unit (NICU) neonates. The CMV infection was more prevalent in preterm neonates or those of a low birth weight in the NICU. No evidence was found for the transmission of nosocomial cytomegalovirus in the NICU (19).
Based on the findings of another research, the overall prevalence of cytomegalovirus infection in neonates was reported to be 0.49%. In this research, 3 neonates were males and 5 were females. Moreover, 3 cases were born in natural delivery and 5 of them underwent a cesarean section. Additionally, 2 neonates (25%) were preterm and 3 were symptomatic upon birth and were sent for the urine test. Thus, the prevalence was rather lower (16). However, in another study, in which the mother's blood sample and umbilical cord were used for serology via the ELISA technique and urine was cultured, a total number of 988 neonates participated among whom 27 cases showed cytomegalovirus infections. Similarly, 12 cases (1.2%) were found with CMV. The risk of congenital infection in neonates whose mother had a positive serology was 3.4% (20). In another study aimed to explore the prevalence of cytomegalovirus, 75 pregnant women were aminosynthesized and all showed a negative IgM CMV. In addition, 56 cases of 75 mothers (74.7%) were positive at the time of amniocentesis. The amniotic fluid underwent a PCR to check the DNA CMV which revealed no positive cases. Further, 65 fetuses were followed up until birth and their urine samples within the first 2 weeks underwent a PCR but yielded no positive cases (21).
The related body of literature has reported the prevalence of cytomegalovirus in Iran from 0.3% in saliva samples to 4% in the PCR of the sample blood taken from the umbilical cord. The positive serology of the cytomegalovirus antibody of the IgG type in Iranian pregnant women was reported to range from 70% to 98%. However, IgM was found to range between 2.5 and 4.3, indicating that the majority of pregnant women in Iran got afflicted with cytomegalovirus before pregnancy. Nonetheless, several researchers believe that in developing countries, CMV infection is induced by non-preliminary congenital infections. The prevalence of serology and CMV infection among mothers varies across different areas (16).
In an investigation among neonates, the serology of IgG toxoplasma was positive in 35.9% of cases and that of IgM was positive in 3.2% of them. In their research among neonates below one year of age and suspected with intrauterine infection, Khosravi et al (22) found acute toxoplasmosis (IgM) in 10% of cases (23). In another study, the rate of congenital toxoplasmosis was reported to be 1.5%, which is equal to 14.8 cases per thousand live births (17). Based on the findings of another research, the risk of affliction among negative serum mothers during pregnancy was estimated at 14 per thousand cases and the probability of the toxoplasmosis of the neonates of mothers at risk was reported to be 7 per thousand (15). In an investigation exploring the prevalence of cytomegalovirus among neonates whose birth weight was lower (with no specific reason) than their IUGR, PCR was performed on 25 sample placenta which revealed no cases of infection (24). In the present research, the PCR technique was used, which is of high sensitivity and specificity, but the technique was not effective in IUGR neonatal screening. Neither was it economical nor logical. In a study of neonates with encephalopathy, the prevalence  (26). Given that many prenatal infections with severe side effects including cytomegalovirus and toxoplasma are asymptomatic, the present research began to look into the screening of severe infections during pregnancy. If such infections are screened during pregnancy and under certain conditions such as IUGR, microcephaly, and hydrocephaly, severe side effects can be prevented at higher ages. In the current study, the applied laboratory investigation method was luminance quantitative serology and its sensitivity and specificity in examining IgM were 92% and 97.3%, respectively. It seems that the sensitivity of ELISA tests in examining serology or performing PCR is higher. This could be one reason for the negative result.
Therefore, it is suggested that more detailed research be conducted following more reliable serology methods or through PCR and microarray for a more comprehensive outlook. A longer time period needs to be spent on sampling as well.

Conclusion
In general, the frequency of congenital infections, cytomegalovirus, and toxoplasma varies as reported in different investigations conducted in different times and places and using various testing methods. The prevalence of the two target infections among microcephaly and hydrocephaly neonates in the child hospital was found to be zero in the present study. Thus, it seems that investigating and screening neonates affected by microcephaly and hydrocephaly is not economical.