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SPED 620 Early Intervention Critical Analysis Case Study Guidelines - Zika Virus Students will read/view a variety of different sources about Zika Virus(e.g. research articles, newspaper articles, podcasts, videos, etc.) and engage in discussion/activities about each assigned topic. Students will evaluate the quality of scientific information and analyze the connection of scientific research and application to personal, social or ethical issues in the modern world (i.e., Early Intervention, Environmental Sustainability and/or Social Justice). Students will then complete a written critique and reflection for 3 different case studies. Papers should be 3 pages in length, using APA format. Students can earn up to 30 points each paper, per the following: Summary of Topic - 5 points (approximately 1 page) · Provide a brief summary of the environmental risk factor (i.e., the topic): 2 points · Explain what the impact is on perinatal and early development: 2 points · Describe any treatment or other recommendations: 1 point Critique of Current Scientific Evidence - 10 points (approximately 1 page) · Briefly review the current research on the topic; what are the major findings?: 2 points · Provide your own analysis of the quality of the existing research; what are the strengths and weaknesses?: 5 points · Share your recommendations for future studies; what are the priorities?: 3 points Application and Reflection - 10 points (approximately 1 page) · Apply the information about the topic to the field of Early Intervention; what is the impact on young children and families?: 4 points · Reflect individually on what you’ve learned; what are the personal, social and ethical issues this topic raises for you?: 3 points · What are the implications for the field of Early Intervention, policy-makers, global leaders, etc.?: 3 points Format and Proofread - 5 points · 3 double-spaced pages: 1 point · APA format, including in-text citations and reference page: 2 points · Written in a professional manner and is thoroughly edited: 1 point *Please read example and don’t plagiarism! OPIOID EPIDEMIC CASE STUDY 1 Opioid Epidemic Case Study Gabriela M. Anthony SPED 620.02 Brett Collins OPIOID EPIDEMIC CASE STUDY 2 Opioid Epidemic Case Study Summary of Topic: Opioids are often prescribed to patients as pain relievers. This includes drugs such as oxycodone, morphine, codeine and fentanyl. Heroin is a very common illicit opiate that is derived from morphine. Regardless if the opioids are being used under the direction of a doctor or not, prolonged use can lead to dependency and addiction. The misuse of prescription drugs includes taking the medication in order to feel high, taking a prescription that is under another person’s name, and taking the medication in doses other than what has been prescribed. Opioids are highly addictive and prolonged use can have detrimental effects to a person's life. An addiction to opioids can lead to overdose and even death (NIDA). While an overdose can be While pregnancy is often a motivating factor for many women to get treatment for their drug addiction, the reality is this is not the case for everyone. Mothers who are using drugs, especially opioids, during pregnancy are putting their child at risk for developing an addiction. Neonatal Abstinence Syndrome (NAS) is “a drug-withdrawal syndrome that most commonly occurs after in utero exposure to opioids” (Tolia, 2015). The drugs are passed through the mothers blood into the placenta. NAS is diagnosed using a “ standardized scale that scores the infant on the presence and severity of common withdrawal symptoms” (Tolia, 2015). Once these babies are born they will be cut off throm their supply this immediately endearing a state of withdrawal. Their symptoms of withdrawal begin within the first 24-72 hours after being born and often include; high-pitched crying, trembling, trouble sleeping, and many more. Healthcare workers may rely on opiate replacement therapy if the child's symptoms are severe enough. They may turn to “opioids such as methadone or morphine, and then weaned off over days to weeks” (Wachman 2018). OPIOID EPIDEMIC CASE STUDY 3 Critique of Current Scientific Evidence: Despite the increasing prevalence of NAS, the long-term effects of maternal opiate dependence have not been researched fully. No one seems to really be sure of how this can affect a child’s development. Researchers believe long-term effects may include developmental delays, motor problems, and/or behavioural problems however they have yet to find conclusive evidence that would support these theories. Another big issue with the research being done is that “the preponderance of evidence is based on low-quality studies that are uncontrolled, use single-center or retrospective data, have small sample sizes, or use quality improvement methodologies not designed for generalizability” (Wachman, 2018). Tolia et al. article shares that studies are often limited to hospital records and rarely turns to firsthand accounts. One study stated that “To further select infants in whom drug withdrawal was the primary reason for NICU admission, we classified infants as having the neonatal abstinence syndrome only if the queried phrase was assigned in the first 7 days of life” (Tolia et al.). This however may be detrimental to the research because according to Stanford Children's Health NAS symptoms can begin to develop up to 10 days after birth. Additionally, the “severity of withdrawal is estimated using various scoring systems, the most common of which is the Finnegan Neonatal Abstinence Severity Score.” (Logan et al.). The diagnosis itself is often estimated by healthcare providers when urine samples can be done in order to definitively determine whether a newborn has traces of opiate in their system.This too would bring more clarity and allow researchers to have more credible data. OPIOID EPIDEMIC CASE STUDY 4 Application and Reflection: In order to combat NAS more research needs to be done on its short and long-term effects. Neonatal Intensive Care Units (NICU) need more resources to treat children with NAS. While the cases have increased, new methods have not been developed to help treat newborns who are dealing with withdrawals (Tolia et al.). NICUs should be a key source of information regarding the effects of NAS. There needs to be more intervention strategies for both the mother and child. First and foremost there needs to be more focus on the opioid epidemic itself. The government better regulate the distribution of opioids. Additionally, doctors who are prescribing opiates freely need to be investigated, as they are enabling this behavior. There needs to be more education about drug use and abuse. Fear mongering is not always the best way to get people to stay away from things. Being open about the short and long-term effects of drug use people may deter people from using. The rise in Neonatal Abstinence Syndrome means that more women are continuing to abuse drugs while pregnant. Support groups and rehabilitation facilities should be more widely accessible especially to expecting mothers. This should extend after the child is born to help the women with their sobriety. Children of parents with opiate addictions need to have a strong support system whether this be extended family, their own support groups, or involvement in the community. OPIOID EPIDEMIC CASE STUDY 5 References Logan, B. A., Brown, M. S., & Hayes, M. J. (2013). Neonatal Abstinence Syndrome. Clinical Obstetrics & Gynecology, 56(1), 186–192. https://doi.org/10.1097/grf.0b013e31827feea4 National Institute on Drug Abuse. (2021, May 17). Opioids. National Institute on Drug Abuse. https://www.drugabuse.gov/drug-topics/opioids. Neonatal Abstinence Syndrome. Stanford Children's Health - Lucile Packard Children's Hospital Stanford. (n.d.). https://www.stanfordchildrens.org/en/topic/default?id=neonatal-abstinence-syndrome-90- P02387. Tolia, V. N., Patrick, S. W., Bennett, M. M., Murthy, K., Sousa, J., Smith, P. B., Clark, R. H., & Spitzer, A. R. (2015). Increasing Incidence of the Neonatal Abstinence Syndrome in U.S. Neonatal ICUs. New England Journal of Medicine, 372(22), 2118–2126. https://doi.org/10.1056/nejmsa1500439 Wachman, E. M., Schiff, D. M., & Silverstein, M. (2018). Neonatal Abstinence Syndrome. JAMA, 319(13), 1362. https://doi.org/10.1001/jama.2018.2640 www.thelancet.com/infection Vol 16 June 2016 653 Articles Lancet Infect Dis 2016; 16: 653–60 Published Online February 17, 2016 http://dx.doi.org/10.1016/ S1473-3099(16)00095-5 See Comment page 620 *Contributed equally †Contributed equally Instituto Nacional de Infectologia Evandro Chagas, Laboratório de Pesquisa Clínica em Doenças Febris Agudas (G Calvet PhD, P Brasil PhD), Laboratório de Flavivírus, Instituto Oswaldo Cruz (S A Sampaio BSc, A Fabri BSc, E S M Araujo BSc, P C de Sequeira PhD, M C L de Mendonça PhD, F B dos Santos PhD, R M R Nogueira PhD, A M B de Filippis PhD), and Instituto Nacional de Controle e Qualidade (I de Filippis PhD), Fundação Oswaldo Cruz, Rio de Janeiro, Brazil; Departamento de Genética (R S Aguiar PhD, L de Oliveira PhD, C G Schrago PhD, Prof A Tanuri PhD), Instituto de Biologia (D A Tschoeke PhD, F L Thompson PhD), Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil; Instituto de Pesquisa Professor Joaquim Amorim Neto (IPESQ), Campina Grande, Brazil (A S O Melo PhD); and Laboratório de Sistemas Avanç ados de Gestão de Produç ão-SAGE-COPPE, Centro de Gestão Tecnológica-CT2, UFRJ, Rio de Janeiro, Brazil (F L Thompson) Correspondence to: Dr Ana M B de Filippis, Laboratório de Flavivírus, Instituto Oswaldo Cruz, Fundação Oswaldo Cruz, Rio de Janeiro 21040–900, Brazil [email protected] Detection and sequencing of Zika virus from amniotic fl uid of fetuses with microcephaly in Brazil: a case study Guilherme Calvet*, Renato S Aguiar*, Adriana S O Melo, Simone A Sampaio, Ivano de Filippis, Allison Fabri, Eliane S M Araujo, Patricia C de Sequeira, Marcos C L de Mendonça, Louisi de Oliveira, Diogo A Tschoeke, Carlos G Schrago, Fabiano L Thompson, Patricia Brasil, Flavia B dos Santos, Rita M R Nogueira, Amilcar Tanuri†, Ana M B de Filippis† Summary Background The incidence of microcephaly in Brazil in 2015 was 20 times higher than in previous years. Congenital microcephaly is associated with genetic factors and several causative agents. Epidemiological data suggest that microcephaly cases in Brazil might be associated with the introduction of Zika virus. We aimed to detect and sequence the Zika virus genome in amniotic fl uid samples of two pregnant women in Brazil whose fetuses were diagnosed with microcephaly. Methods In this case study, amniotic fl uid samples from two pregnant women from the state of Paraíba in Brazil whose fetuses had been diagnosed with microcephaly were obtained, on the recommendation of the Brazilian health authorities, by ultrasound-guided transabdominal amniocentesis at 28 weeks’ gestation. The women had presented at 18 weeks’ and 10 weeks’ gestation, respectively, with clinical manifestations that could have been symptoms of Zika virus infection, including fever, myalgia, and rash. After the amniotic fl uid samples were centrifuged, DNA and RNA were extracted from the purifi ed virus particles before the viral genome was identifi ed by quantitative reverse transcription PCR and viral metagenomic next-generation sequencing. Phylogenetic reconstruction and investigation of recombination events were done by comparing the Brazilian Zika virus genome with sequences from other Zika strains and from fl aviviruses that occur in similar regions in Brazil. Findings We detected the Zika virus genome in the amniotic fl uid of both pregnant women. The virus was not detected in their urine or serum. Tests for dengue virus, chikungunya virus, Toxoplasma gondii, rubella virus, cytomegalovirus, herpes simplex virus, HIV, Treponema pallidum, and parvovirus B19 were all negative. After sequencing of the complete genome of the Brazilian Zika virus isolated from patient 1, phylogenetic analyses showed that the virus shares 97–100% of its genomic identity with lineages isolated during an outbreak in French Polynesia in 2013, and that in both envelope and NS5 genomic regions, it clustered with sequences from North and South America, southeast Asia, and the Pacifi c. After assessing the possibility of recombination events between the Zika virus and other fl aviviruses, we ruled out the hypothesis that the Brazilian Zika virus genome is a recombinant strain with other mosquito-borne fl aviviruses. Interpretation These fi ndings strengthen the putative association between Zika virus and cases of microcephaly in neonates in Brazil. Moreover, our results suggest that the virus can cross the placental barrier. As a result, Zika virus should be considered as a potential infectious agent for human fetuses. Pathogenesis studies that confi rm the tropism of Zika virus for neuronal cells are warranted. Funding Consellho Nacional de Desenvolvimento e Pesquisa (CNPq), Fundação de Amparo a Pesquisa do Estado do Rio de Janeiro (FAPERJ). Introduction Since 2015, Brazil has been facing a public health emergency regarding the dramatic increase in the number of newborn babies with microcephaly. Epidemiological data indicate that up to Jan 6, 2016, 4783 cases were reported in 21 states in the North, Northeast, South, and Southeast Regions of Brazil.1 This incidence of microcephaly is 20 times higher than in previous years, reaching 99·7 per 100 000 livebirths, and including 76 deaths of neonates as of Jan 6, 2016.1 When diagnosed prenatally by ultrasound imaging, congenital microcephaly is a strong predictor of adverse neurological outcomes.2 As defi ned by WHO, microcephaly occurs whenever the occipital frontal circumference of the head of the newborn child or fetus is 2 standard deviations smaller than the mean for the same age and sex.3 A brain size that is signifi cantly diff erent to that in the normal range is an important risk factor for cognitive and motor delay.4 Microcephaly is associated with several causes, including genetic disorders (eg, autosomal recessive microcephaly, Aicardi-Goutières syndrome, chromosomal trisomy, Rett syndrome, and X-chromosomal microcephaly); drug and chemical intoxication of the pregnant mother (eg, use of alcohol, cocaine, or antiepileptic drugs, lead or mercury intoxication, or radiation); maternal mal- nutrition; and transplacental infections with viruses or bacteria.5 Maternal viral infections, including rubella, http://crossmark.crossref.org/dialog/?doi=10.1016/S1473-3099(16)00095-5&domain=pdf Articles 654 www.thelancet.com/infection Vol 16 June 2016 cytomegalovirus, herpes simplex, varicella zoster virus, HIV, and arboviruses such as chikungunya, have also been associated with microcephaly in neonates.5,6 Epidemiological evidence suggests that Zika virus infection of pregnant women in Brazil might be associated with the increasing numbers of congenital microcephaly cases reported in the country. Several mosquito species have been found to be naturally infected with Zika virus, including Aedes africanus, Aedes luteocephalus, Aedes hensilli, Aedes polynesiensis, Aedes dalzielii, Aedes albopictus, Aedes apicoargenteus, and Aedes aegypti among others, but little is known about their vector competence.7–10 A aegypti is the over- whelmingly predominant mosquito species found in Brazil, and is also associated with other arboviruses already reported in Brazil, such as the dengue and chikungunya viruses. Zika virus was fi rst isolated from human beings in Nigeria7 during studies undertaken in 1954. Further cases were reported in other African countries11 (Uganda, Tanzania, Egypt, Sierra Leone, Gabon, Nigeria, Côte d’Ivoire, Cameroon, Senegal, and Central African Republic), in Asian countries (India, Pakistan, Malaysia, Philippines, Thailand, Cambodia, Vietnam, and Indonesia), in several islands of the Pacifi c region since 2007 (Federated States of Micronesia, Cook Islands, French Polynesia, New Caledonia, Guam, Samoa, Vanuatu, and Solomon Islands), and since about early 2015 in the Americas (Chile, Colombia, El Salvador, Guatemala, Mexico, Paraguay, Suriname, Venezuela, Canada, and the USA).9–15 Outbreaks of Zika virus infection on Yap Island (in 2007) and in French Polynesia (2013–14), with further spread to New Caledonia, the Cook Islands, and Easter Island, have shown the propensity of this arbovirus species to spread outside its usual geographical range and to cause large outbreaks. The fi rst autochthonous cases of Zika virus in Brazil were confi rmed in May, 2015.16 Since then, as of Jan 6, 2015, 21 states have confi rmed virus circulation, with a higher prevalence in the Northeast Region.17 Reports of microcephaly incidence in Brazil geographically overlap with Zika virus reports; most of the mothers whose infants were diagnosed with micro- cephaly complained during their pregnancies of clinical manifestations, such as low-grade fever, headache, and cutaneous rashes, that might have been symptoms of Zika virus infection or infection with any other arbovirus species that is prevalent in the region. In the face of this potential association between Zika virus infection and the increasing number of cases of microcephaly, the Brazilian Ministry of Health and WHO have recommended that pregnant women should take precautions to avoid contact with all potential vectors, since no specifi c antiviral treatment for Zika virus infection exists.1 Small fragments of the genome of the Zika virus strain circulating in Brazil have been sequenced and phylogenetic analysis has indicated that the virus is similar to the one that circulated in French Polynesia in 2013.16,17 However, evidence linking the high incidence of microcephaly to the presence of Zika virus is scarce. In January, 2016, our group reported ultrasound image evidence of two cases of fetal microcephaly in women who had complained of Zika-like virus symptoms during pregnancy, and we reported brief preliminary PCR fi ndings, confi rming the presence of Zika virus in their amniotic fl uid.18 In this case study, we expand upon these previously reported fi ndings, and describe how we used quantitative reverse transcription PCR (RT-qPCR) and Research in context Evidence before this study Many cases of microcephaly in newborn babies in Brazil have occurred in regions where infections of Zika virus and other arboviruses have also been detected. We searched PubMed with the search terms “Zika”, and “microcephaly” for articles published up to Feb 5, 2016. We found 11 articles that suggested a possible relation between Zika virus and microcephaly in neonates. A short case report by our group, reporting the ultrasound evidence of the two fetal microcephaly cases reported here, has been published previously. Our search found no other clear evidence that Zika virus could cross the placental barrier and infect the human fetus. Added value of this study This study presents the virological and genetic data implicating Zika virus in the two cases of fetal malformation that we described briefl y in our previous case report. We used quantitative reverse transcription PCR and viral metagenomics technology applied to samples of amniotic fl uid obtained from the two pregnant women carrying fetuses with microcephaly, and obtained sequences of the Zika virus genome. The study of these cases provides empirical evidence for the association between Zika virus infection during pregnancy and fetal microcephaly. Furthermore, we isolated the whole genome of Zika virus directly from the amniotic fl uid of two pregnant women, and provided evidence to support previous fi ndings indicating that the origin of the virus is French Polynesia. Implications of all the available evidence On the basis of our fi ndings, Zika virus should be regarded as a possible causative agent in cases of microcephaly, especially during Zika virus outbreaks in endemic regions. Our work emphasises not only the importance of controlling the Aedes aegypti mosquito population while no vaccine or antiviral is available, but also the need for further studies to understand the mechanisms of immunopathogenicity that lead to congenital malformation due to Zika virus infection. Articles www.thelancet.com/infection Vol 16 June 2016 655 viral metagenomics to detect and sequence the Zika virus genome in the amniotic fl uid samples of these two pregnant women with microcephalic fetuses. Methods Case histories The fi rst case in our study was of a 27-year-old woman in her fi rst pregnancy, from an inner city in the state of Paraíba, in the Northeast Region of Brazil (patient 1). Her prenatal care was uneventful until early September, 2015, when, at 18 weeks of gestation, the woman developed a cutaneous rash with itching of the hands and back. On the basis of her clinical status, she was diagnosed at an emergency service unit with allergic reaction, and was prescribed intravenous hydrocortisone. The next day, her symptoms worsened as she developed a fever and myalgia. She had a normal fetal ultrasound at 16 weeks. The patient had not travelled outside the state of Paraíba during the previous few years, and she had not had contact with any ill individuals. She had no immunodefi ciency or autoimmune diseases. At 21 weeks of gestation, a further ultrasound indicated a fetal microcephaly diagnosis with moderate ventriculomegaly and partial agenesis of the cerebellar vermis. A third ultrasound done at 27 weeks confi rmed the microcephaly diagnosis with relevant dilation of ventricles, asymmetry of hemispheres, and hypoplastic cerebellum with complete absence of the cerebellar vermis. The patient was healthy and stable during the ultrasound and amniocentesis procedures. Results of all laboratory examinations showed no diabetes and blood-pressure- related disorders. Additionally, the patient did not report taking any medication (other than hydrocortisone), recreational drug use, alcohol consumption, or smoking during the pregnancy. Patient 1 is still being monitored by the physicians in our group. At 40 weeks of gestation the fetus presented microcephaly with calcifi cation areas and head circumference of 29 cm assessed by ultrasonography before birth. The baby was born at 40 weeks of gestation and had an actual head circumference of 30 cm. The second case in our study was of a 35-year-old woman in her fi rst pregnancy, also from the state of Paraíba (patient 2). The patient, with no relevant past medical history, sought care when she developed mild Zika virus disease-like symptoms at 10 weeks of gestation. She was prescribed symptomatic treatment. A morph- ological ultrasound at 22 weeks of gestation revealed mild hypoplasia of the cerebellar vermis. The fetal head circumference on the 22nd week of gestation was below the 10th percentile. A second ultrasound done at 25 weeks of gestation revealed more severe hypoplasia of the cerebellar vermis, enlargement of the posterior fossa, and microcephaly, yielding a head circumference below the third percentile. The brain parenchyma was normal. The patient was healthy and stable during the ultrasound and amniocentesis procedures. All the laboratory examinations showed no evidence of diabetes or blood-pressure-related disorders. Additionally, she did not report taking any medication, recreational drug use, alcohol consumption, or smoking during the pregnancy. Patient 2 is still being monitored by the physicians in our group. She delivered on Feb 3, 2016, and the neonate presented severe ventriculomegaly, microphthalmia, cataract, and severe arthrogryposis in the legs and arms. Sample collection Following Brazilian health public recommendations, amniocentesis was done at gestational week 28 in both women to investigate the cause of microcephaly. Ultrasound-guided transabdominal amniocentesis was done and about 5 mL of amniotic fl uid was aspirated and immediately stored at –80°C. Viral metagenomics and sequence analysis A 0·5 mL sample of the amniotic fl uid from each patient was fi ltered through 0·45 μm fi lters to remove residual host cells. The samples were then centrifuged at 21 130 × g and 15 000 rpm (rotor FA-45–24–11, Eppendorf, Hamburg, Germany) for 90 min at 4°C to concentrate virus particles. Pelleted virus particles were treated with deoxy ribonuclease and ribonuclease A at 37°C for 90 min according to previously reported protocols.19 RNA was isolated using the QIAamp MinElute Virus Spin Kit (Qiagen, Hilden, Germany), omitting carrier RNA. Double-stranded cDNA libraries were prepared using the TruSeq Stranded Total RNA LT Sample Preparation Kit (Illumina, San Diego, CA, USA). Library size distribution was assessed using the 2100 Bioanalyzer (Agilent, Santa Clara, CA, USA) and the High Sensitivity DNA Kit (Agilent). Accurate quanti fi cation of the libraries was accomplished with the 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) and the KAPA Library Quantifi cation Kit (Kapa Biosystems, Wilmington, MA, USA). Paired-end sequencing (2 × 210 bp) was done using a MiSeq sequencing system (Illumina). The sequences obtained were preprocessed using the PRINSEQ software to remove reads smaller than 35 bp and sequences with scores of lower quality than a Phred quality score of 20. Fast length adjustment of short reads (FLASH) software was used to merge and extend the paired-end Illumina reads using the default parameters, with a maximum overlap of 400 bp. The extended reads were analysed with basic local alignment search tool (BLAST), against the Human Transcriptome Database (RefSeq, Annotation Release 107; 162 916 sequences), with e-value cutoff of 1e-5, to remove human RNA sequences. Non-human reads were analysed against all GenBank viral genomes (65 052 sequences), and reads that were similar to the Zika virus were collected and used for genomic assembly. The Zika virus genome (Brazil strain) was assembled de novo using the CAP3 assembly software, Articles 656 www.thelancet.com/infection Vol 16 June 2016 using the parameters overlap length cutoff (-o) of 16, and overlap percent identity cutoff (-p) of 85. The Atlas genome was constructed using BRIG (BLAST Ring Image Generator) software. We used the Zika virus genome sequence H/PF/2013 (KJ776791.1) as the reference. This strain was isolated in French Polynesia, and we compared it with a strain from Uganda, MR 766 (accession: NC_012532.1), another strain isolated in Senegal, ArD157995 (accession: KF383118), and our assembled Zika virus genome. Phylogenetic analysis Phylogenetic reconstruction was completed using both maximum likelihood and Bayesian inference methods on alignments of the envelope and NS5 regions of the polyprotein coding sequence. The best choice of substitution model used in the maximum likelihood and Bayesian inference analyses was determined with the likelihood-ratio test, implemented using HyPhy software. The generalised time-reversible (GTR) model with gamma-distributed evolutionary rates (G) and invariable sites (I), GTR + G + I, was chosen. We undertook maximum likelihood analysis with PhyML 3.0 phylogeny software, using the approximate likelihood-ratio test as a means of assigning statistical signifi cance to internal branches. Bayesian inference was run on MrBayes 3.2 software with default Markov chain Monte Carlo (MCMC) algorithm settings—ie, two independent runs with four chains each were sampled every 500th generation until 1 000 000 samples were obtained. 25% of the MCMC samples were discarded as a burn-in step. Chain convergence was measured by the Gelman-Rubin statistic, using the potential scale reduction factor, or PSRF, which was close to 1 for all parameters. Maximum likelihood and Bayesian inference topologies were identical. We therefore report the results from the maximum likelihood analysis. To investigate recombination breakpoints along the Zika virus genome, a sliding window strategy was implemented using an in-house script. By building a stand-alone BLAST database containing all reference fl avivirus genomes, we scanned the Zika virus genome every 50 bp regions and registered their BLAST hits using a cutoff e-value of 0·0001. We did genome-wide multiple alignments using the Multi-LAGAN algorithm as implemented in the VISTA database. Phylogeny of whole genomes was also inferred by maximum likelihood and Bayesian inference methods. Role of the funding source The funder of the study had no role in study design, data collection, data analysis, data interpretation, or writing of the report. The corresponding author had full access to all the data in the study and had fi nal responsibility for the decision to submit for publication. Results Serum, urine, and amniotic fl uid samples from both patients (all taken at week 28 of gestation) were tested for dengue virus, chikungunya virus, and Zika virus. The RT- qPCR for dengue virus20 and the RT-qPCR for chikungunya virus21 were negative in all samples. The RT-qPCRs for Zika virus22 confi rmed Zika virus infection in the amniotic fl uids of patients 1 and 2, but were negative in urine and serum samples in both patients. Serology tests of serum, urine, and amniotic fl uid samples using anti-dengue- virus IgM, anti-dengue-virus IgG, anti-chikungunya-virus IgM, and anti-chikungunya-virus IgG yielded negative results by ELISA. However, ELISA for Zika virus was positive for anti-Zika-virus IgM in amniotic fl uid, and negative in serum and urine in both patients 1 and 2. TORCH (toxoplasmosis, HIV, syphilis, measles, rubella, cytomegalovirus, and herpes simplex) panels of both women were also negative, as well as specifi c HIV, syphilis, cyto megalovirus, and parvovirus B19 screens. Virus particles were fi ltrated and concentrated from the amniotic fl uid samples. After cellular contaminants and human sequences were eliminated, 288 904 sequences Figure 1: Comparative genome BLAST Atlas diagram of Zika virus The green outer circle corresponds to the complete Brazilian Zika virus genome isolated from the amniotic fl uid of patient 1. 10 793 bases were sequenced. The red circle corresponds to the Senegal (KF383118.1) strain of Zika virus and the blue circle corresponds to the Uganda strain (NC_012532.1). The percentage deviation in GC content between the Brazilian Zika virus and the reference Zika virus is represented along the Zika virus genome by the varying heights of the black bars. The innermost (black) circle corresponds to the reference genome (French Polynesia, KJ776791.1). Genome shared identity between each strain and the reference genome are shown as percentages. BLAST=basic local alignment search tool. Zika virus Uganda 87–90% similarity Zika virus Senegal 87–90% similarity Zika virus Brazil 97–100% similarity Shared identity with reference genome Reference: Zika virus French Polynesia 1 kbp 2 kbp 3 kbp 4 kbp 5 kbp 6 kbp 7 kbp 8 kbp 9 kbp 10 kbp 10617 bp Capside Membrane Envelope NS1 NS2A NS2B NS3 NS4A NS4B NS5 Articles www.thelancet.com/infection Vol 16 June 2016 657 showed similarity with virus sequences through BLAST analysis of the GenBank viral genome database. 683 sequences matched the Zika virus, comprising 167 143 bp, used to assemble the genome. Two diff erent fragments corresponding to Zika virus genome positions 1641–1763 and 6466–6566 were sequenced from samples of patient 2. Metagenomic analysis of samples of patient 1 covered 10 793 bases of the Zika virus genome with 19 × coverage. The complete sequence (10 793 nucleotides) was deposited at the GenBank database (accession number ID: KU497555). Figure 1 shows the whole Zika virus genome isolated from the amniotic fl uid of patient 1 with viral gene annotation. The Brazilian Zika virus shares 97–100% of Figure 2: Maximum likelihood topologies of envelope genomic region from Brazilian Zika virus Brazilian Zika virus (in red text) isolated from the amniotic fl uid of patient 1, whose fetus was diagnosed with microcephaly, was compared with previously released sequence data. Approximate likelihood-ratio test support values greater than 0·5 are shown at nodes. Zika virus Brazil shares the same origin as those of Asian, Pacifi c, and American lineages (red branches). For most strains, the country of isolation is shown, in some cases followed by the date of isolation. Burkina=Burkina Faso. Central=Central African Republic. Cook=Cook Islands. KF383015_Senegal_2001 KF383018_Senegal_2000 KF383016_Senegal_2001 KF383017_Senegal_2001 KF382020_Côte_d’Ivoire KF383019_Senegal_1998 KF383021_Senegal_1998 KF383044_Côte_d’Ivoire KF383043_Côte_d’Ivoire KF383045_Côte_d’Ivoire KF383041_Côte_d’Ivoire KF383042_Côte_d’Ivoire KF383040_Côte_d’Ivoire KF383039_Senegal_1991 KF383029_Senegal_2002 KF383030_Burkina KF383028_Senegal_2002 KF383031_Senegal_1969 KF383116 HQ234501_Senegal_1984 KF383033_Senegal_1979 KF383032_Senegal_1979 KF383034_Senegal_1979 HQ234500_Nigeria_1968 HQ234499_Malaysia_1966 EU545988_Micronesia_Jun-2007 JN860885_Cambodia_2010 KF993678_Canada_19-Feb-2013 KJ634273_Cook KJ776791_French Polynesia Zika virus Brazil KR815990_Brazil_2015 KR815989_Brazil_2015 KR816336_Brazil_May-2015 KR816334_Brazil_May-2015 KR816333_Brazil_May-2015 KR816335_Brazil_May-2015 DQ859059_Uganda KF383035_Uganda_1963 AF372422 KF383115 KF268949_Central KF268948_Central KF268950_Central AY632535_Uganda NC_012532_Uganda HQ234498_Uganda_1947 LC002520_Uganda KF383118 KF383119 KF383121 KF383037_Côte_d’Ivoire KF383036_Côte_d’Ivoire KF383046_Côte_d’Ivoire KF383038_Côte_d’Ivoire KF383025_Senegal_1997 KF383024_Senegal_1997 KF383027_Senegal_1997 KF383026_Senegal_1997 KF383022_Senegal_1997 KF383023_Senegal_1997 KF383117 0·91 0·8 0·83 0·9 0·75 0·88 0·94 0·7 0·52 0·77 0·78 0·71 1 0·97 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0·83 0·89 0·870·84 0·85 0·97 0·62 0·63 0·73 0·68 0·66 Articles 658 www.thelancet.com/infection Vol 16 June 2016 its genomic identity with the Zika virus sequence KJ776791.1 isolated in French Polynesia. The comparison with African strains such as NC_012532.1 (Zika virus Uganda) and KF383118.1 (Zika virus Senegal) yielded 87–90% identity. The proportion of GC content in the Brazilian Zika virus was 51·2% (fi gure 1). We compared the viral sequences from patient 1 with previously released sequence data from Zika virus outbreaks in Asia and Africa and other fl aviviruses, including dengue virus serotypes 1–4, West Nile virus, and yellow fever virus. Phylogenetic analyses were done using the coding region for the envelope (fi gure 2) and NS5 genes (fi gure 3). The geographical origin of the Brazilian Zika virus strain could not be determined because of sampling limitations, but Brazilian Zika virus is probably more closely related to French Polynesia sequences than to African strains. Maximum likelihood and Bayesian inference methods applied to the alignment of the envelope and NS5 regions of the polyprotein coding sequence yielded identical estimates of phylogenetic topologies. In both envelope (fi gure 2) and NS5 (fi gure 3) genomic regions, the new genome Figure 3: Maximum likelihood topologies of NS5 genomic region from Brazilian Zika virus Brazilian Zika virus (in red text) isolated from the amniotic fl uid of patient 1, whose fetus was diagnosed with microcephaly, was compared with previously released sequence data. Approximate likelihood-ratio test support values greater than 0·5 are shown at nodes. Zika virus Brazil shares the same origin as those of Asian, Pacifi c, and American lineages (red branches). For most sequences, the country of isolation is shown, in some cases followed by the date of isolation. JX041632_India_Madras_state_1955 GQ851604_India_1957 AF013406 DQ859064_South_Africa HQ234500_Nigeria_1968 KF383107_Côte_d’Ivoire_1990 KF383106_Côte_d’Ivoire_1990 KF383117 KF383089_Senegal_2002 KF383113_Côte_d’Ivoire_1980 KF383101_Senegal_1997 KF383098_Senegal_1997 KF383099_Senegal_1997 KF383097_Senegal_1997 HQ234501_Senegal_1984 KF383085_Senegal_1969 KF383116 KF383088_Senegal_1979 KF383087_Senegal_1979 KF383114_Senegal_1979 KF383115 KF268949_Central_African_Republic KF268950_Central_African_Republic KF268948_Central_African_Republic_1976 KF383121 KF383119 KF383118 KF383091_Senegal_2001 KF383092_Senegal_2001 KF383093_Senegal_2001 KF383086_Côte_d’Ivoire_1999 AY632535_Uganda LC002520_Uganda HQ234498_Uganda_1947 AF013415 KF383104_Côte_d’Ivoire_1999 KF383103_Côte_d’Ivoire_1999 DQ859059_Uganda HQ234499_Malaysia_1966 KM851038_Philippines_09-May-2012 EU545988_Micronesia_Jun-2007 KF258813_Indonesia_2012 JN860885_Cambodia_2010 KF993678_Canada_19-Feb-2013 KM851039_Thailand_19-Jul-2014 KJ873160_New_Caledonia_02-Apr-2014 KM078936_Chile_Easter_Island_01-Mar-2014 KJ873160_New_Caledonia_03-Apr-2014 KM078933_Chile_Easter_Island_17-Feb-2014 KM078961_Chile_Easter_Island_24-Apr-2014 KM078970_Chile_Easter_Island_20-Apr-2014 KM078971_Chile_Easter_Island_21-Apr-2014 KM078930_Chile_Easter_Island_04_Apr-2014 KM078929_Chile_Easter_Island_21-Mar-2014 KJ776791_French_Polynesia_28-Nov-2013 Zika virus Brazil Ultrasound Obstet Gynecol 2016; 47: 6 – 7 Published online in Wiley Online Library (wileyonlinelibrary.com). Physician Alert Zika virus intrauterine infection causes fetal brain abnormality and microcephaly: tip of the iceberg? An unexpected upsurge in diagnosis of fetal and pediatric microcephaly has been reported in the Brazilian press recently. Cases have been diagnosed in nine Brazilian states so far. By 28 November 2015, 646 cases had been reported in Pernambuco state alone. Although reports have circulated regarding the declaration of a state of national health emergency, there is no information on the imaging and clinical findings of affected cases. Authorities are considering different theories behind the ‘microcephaly outbreak’, including a possible association with the emergence of Zika virus disease within the region, the first case of which was detected in May 20151. Zika virus is a mosquito-borne disease closely related to yellow fever, dengue, West Nile and Japanese encephalitis viruses2. It was first identified in 1947 in the Zika Valley in Uganda and causes a mild disease with fever, erythema and arthralgia. Interestingly, vertical transmission to the fetus has not been reported previously, although two cases of perinatal transmission, occurring around the time of delivery and causing mild disease in the newborns, have been described3. We have examined recently two pregnant women from the state of Paraiba who were diagnosed with fetal microcephaly and were considered part of the ‘microcephaly cluster’ as both women suffered from symptoms related to Zika virus infection. Although both patients had negative blood results for Zika virus, amniocentesis and subsequent quantitative real-time polymerase chain reaction4, performed after ultrasound diagnosis of fetal microcephaly and analyzed at the Oswaldo Cruz Foundation, Rio de Janeiro, Brazil, was positive for Zika virus in both patients, most likely representing the first diagnoses of intrauterine transmission of the virus. The sequencing analysis identified in both cases a genotype of Asian origin. In Case 1, fetal ultrasound examination was performed at 30.1 weeks’ gestation. Head circumference (HC) was 246 mm (2.6 SD below expected value) and weight was estimated as 1179 g (21st percentile). Abdominal circumference (AC), femur length (FL) and transcranial Doppler were normal for gestational age as was the width of the lateral ventricles. Anomalies were limited to the brain and included brain atrophy with coarse calcifications involving the white matter of the frontal lobes, including the caudate, lentostriatal vessels and cerebellum. Corpus callosal and vermian dysgenesis and enlarged cisterna magna were observed (Figure 1). In Case 2, fetal ultrasound examination was performed at 29.2 weeks’ gestation. HC was 229 mm (3.1 SD below Figure 1 Case 1: (a) Transabdominal axial ultrasound image shows cerebral calcifications with failure of visualization of a normal vermis (large arrow). Calcifications are also present in the brain parenchyma (small arrow). (b) Transvaginal sagittal image shows dysgenesis of the corpus callosum (small arrow) and vermis (large arrow). (c) Coronal plane shows a wide interhemispheric fissure (large arrow) due to brain atrophy and bilateral parenchymatic coarse calcifications (small arrows). (d) Calcifications are visible in this more posterior coronal view and can be seen to involve the caudate (arrows). expected value) and estimated fetal weight was 1018 g (19th percentile). AC was below the 3rd percentile but FL was normal. The cerebral hemispheres were markedly asymmetric with severe unilateral ventriculomegaly, displacement of the midline, thinning of the parenchyma on the dilated side, failure to visualize the corpus callosum and almost complete disappearance or failure to develop the thalami. The pons and brainstem were thin and continuous with a non-homogeneous small mass at the position of the basal ganglia. Brain calcifications were more subtle than in Case 1 and located around the lateral ventricles and fourth ventricle. Both eyes had cataracts and intraocular calcifications, and one eye was smaller than the other (Figure 2). In the meantime, in Paraiba state, six children diagnosed with Zika virus were born to mothers who were apparently symptomatic during pregnancy, all of them with neonatal HC below the 10th percentile. Fetal neurosonograms showed two cases with cerebellar involvement and three with brain calcifications. One had severe arthrogryposis. Intrauterine infections affecting the brain are relatively rare; cytomegalovirus (CMV), toxoplasmosis, herpes virus, syphilis and rubella are well known vectors of fetal disease. Among the Flaviviruses there have been only isolated reports linking West Nile encephalitis virus to fetal brain insults5. Copyright © 2015 ISUOG. Published by John Wiley & Sons Ltd. P H Y S I C I A N A L E R T Physician Alert 7 Figure 2 Case 2: (a) Anterior coronal view shows severe asymmetric ventriculomegaly with cystic formation (arrow). (b) Posterior horn of the lateral ventricle (LV) in coronal view is dilated. Note calcifications in the fourth ventricle (arrows). (c) The thalamus is absent (arrow) and the brainstem and pons are thin and difficult to visualize (sagittal view). (d) Axial view shows calcifications in both eyes (arrows). Note that the proximal eye is very small and lacks normal anatomic landmarks. The presence of calcifications was suggestive of an intrauterine infection but severe damage of the cerebel- lum, brainstem and thalami is rarely associated with intrauterine infection. Both cases showed some similari- ties to CMV cases but with a more severe and destructive pattern and they lacked the nodules characteristic of toxoplasmosis. Interestingly, the reported case of fetal West Nile virus infection has similar characteristics5. It is difficult to explain why there have been no fetal cases of Zika virus infection reported until now but this may be due to the underreporting of cases, possible early acquisition of immunity in endemic areas or due to the rarity of the disease until now. As genomic changes in the virus have been reported6, the possibility of a new, more virulent, strain needs to be considered. Until more cases are diagnosed and histopathological proof is obtained, the possibility of other etiologies cannot be ruled out. As with other intrauterine infections, it is possible that the reported cases of microcephaly represent only the more severely affected children and that newborns with less severe disease, affecting not only the brain but also other organs, have not yet been diagnosed. If patients diagnosed in other states are found to be seropositive for Zika virus, this represents a severe health threat that needs to be controlled expeditiously. The Brazilian authorities reacted rapidly by declaring a state of national health emergency. As there is no known medical treatment for this disease, a serious attempt will be needed to eradicate the mosquito and prevent the spread of the disease to other Brazilian states and across the border7. A. S. Oliveira Melo†, G. Malinger*‡, R. Ximenes§, P. O. Szejnfeld¶, S. Alves Sampaio** and A. M. Bispo de Filippis** †Instituto de Pesquisa Professor Joaquim Amorim Neto (IPESQ), Instituto de Saúde Elpidio de Almeida (ISEA), Campina Grande, Brazil; ‡Division of Ultrasound in Obstetrics & Gynecology, Lis Maternity Hospital, Tel Aviv Sourasky Medical Center, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel; §Fetal Medicine Foundation Latinamerica – FMFLA, Centrus – Fetal Medicine, Campinas, Brazil; ¶FIDI - Fundação Instituto de Ensino e Pesquisa em Diagnóstico por Imagem, Departamento de Diagnóstico por Imagem -DDI- UNIFESP, Escola Paulista de Medicina, Universidade Federal de São Paulo, São Paulo, Brazil; **Laboratório de Flavivı́rus, Instituto Oswaldo Cruz – FIOCRUZ, Rio de Janeiro, Brazil *Correspondence. (e-mail: [email protected]) DOI: 10.1002/uog.15831 References 1. Campos GS, Bandeira AC, Sardi SI. Zika Virus Outbreak, Bahia, Brazil. Emerg Infect Dis 2015; 21: 1885 – 1886. 2. Ioos S, Mallet HP, Leparc Goffart I, Gauthier V, Cardoso T, Herida M. Current Zika virus epidemiology and recent epidemics. Medecine et maladies infectieuses 2014; 44: 302 – 307. 3. Besnard M, Lastere S, Teissier A, Cao-Lormeau V, Musso D. Evidence of perinatal transmission of Zika virus, French Polynesia, December 2013 and February 2014. Euro Surveill 2014; 19. 4. Lanciotti RS, Kosoy OL, Laven JJ, Velez JO, Lambert AJ, Johnson AJ, Stanfield SM, Duffy MR. Genetic and serologic properties of Zika virus associated with an epidemic, Yap State, Micronesia, 2007. Emerg Infect Dis 2008; 8: 1232 – 1239. 5. Centers for Disease Control and Prevention (CDC). Intrauterine West Nile virus infection--New York, 2002. MMWR Morb Mortal Wkly Rep 2002; 51: 1135 – 1136. 6. Faye O, Freire CC, Iamarino A, Faye O, de Oliveira JV, Diallo M, Zanotto PM, Sall AA. Molecular evolution of Zika virus during its emergence in the 20(th) century. PLoS Negl Trop Dis 2014; 8: e2636. 7. Goenaga S, Kenney JL, Duggal NK, Delorey M, Ebel GD, Zhang B, Levis SC, Enria DA, Brault AC. Potential for Co-Infection of a Mosquito-Specific Flavivirus, Nhumirim Virus, to Block West Nile Virus Transmission in Mosquitoes. Viruses 2015; 7: 5801 – 5812. Copyright © 2015 ISUOG. Published by John Wiley & Sons Ltd. Ultrasound Obstet Gynecol 2016; 47: 6 – 7. Infants & Young Children Vol. 30, No. 1, pp. 17–27 Copyright C⃝ 2017 Wolters Kluwer Health, Inc. All rights reserved. Infants With Congenital Zika Virus Infection A New Challenge for Early Intervention Professionals Sallie Porter, DNP, PhD, APN; Nancy Mimm, MSN, APHN-BC, RN-BC Zika virus infection-associated microcephaly has generated public health and media concern. Un- settling images emerging from Brazil of infants with abnormally small heads have raised concern among women of childbearing age, international travelers, government officials, and health care professionals. The World Health Organization declared the most recent, ongoing Zika virus in- fection outbreak a “public health emergency of international concern.” The Centers for Disease Control and Prevention is working to understand the impact of Zika virus infection in the United States and elsewhere. Zika virus is a mosquito-transmitted Flavivirus that can also be transmit- ted through sexual contact. Congenital Zika virus infection is a cause of microcephaly and other serious neurological harm to the fetus. The early intervention professional should understand Zika virus infection including the geographical risk, etiology, epidemiology, and potential devel- opmental impact. Still evolving clinical, policy, and research implications for early intervention professionals need to be based on the context of emerging scientific information. It is important for early intervention professionals to remain attentive, as scientific knowledge concerning the impact of congenital Zika virus infection in infants and families will be evolving for years to come. Key words: brain calcification, congenital infection, early intervention, microcephaly, Zika virus Z IKA VIRUS (ZIKV) infection-associatedmicrocephaly has created public health and media concern. Unsettling images emerg- ing from Brazil of young infants with abnor- mally small heads have raised concern among Author Affiliations: Division of Advanced Nursing Practice, Rutgers School of Nursing, Newark, New Jersey (Dr Porter); and Division of Family Health Services, Reproductive and Perinatal Health Services, New Jersey Department of Health, Trenton (Ms Mimm). Ms Mimm is a Doctor of Nursing Practice student at Rutgers. The authors thank Margaret P. Disston for her editing assistance. The authors report no conflicts of interest. Correspondence: Sallie Porter, DNP, PhD, APN, Di- vision of Advanced Nursing Practice, Rutgers School of Nursing, 180 University Ave, Newark, NJ 07102 ([email protected]). DOI: 10.1097/IYC.0000000000000084 women of childbearing age, international trav- elers, government officials, and health profes- sionals. Before 2015, health experts believed ZIKV infection to be relatively harmless, of- ten with those infected showing no symptoms (Bell, Boyle, & Petersen, 2016; Costello et al., 2016). The rapid spread of ZIKV infection, along with realization by health care profes- sionals that for some individuals, there are se- rious ZIKV infection-related health outcomes, has captured the interest of federal and state government officials in the United States and elsewhere (Miner & Diamond, 2016). On February 1, 2016, the World Health Organization ([WHO], 2016a) declared the most recent, ongoing ZIKV infection outbreak a “public health emergency of international concern.” The Centers for Disease Control and Prevention (CDC) is working to better understand the incidence and impact of ZIKV infection in the United States and elsewhere. Copyright © 2017 Wolters Kluwer Health, Inc. Unauthorized reproduction of this article is prohibited. 17 18 INFANTS & YOUNG CHILDREN/JANUARY–MARCH 2017 States are working to develop and implement plans to address the potential spread as well as health ramifications more locally. ZIKV infection is a mosquito-transmitted Flavivirus that can also be transmitted through sexual contact. Congenital ZIKV infection is a cause of microcephaly and other serious neurological damage in the fetus and the infant (Cauchemez et al., 2016; Johansson, Mier-y-Teran-Romero, Reefhuis, Gilboa, & Hills, 2016; Rasmussen, Jamieson, Honein, & Petersen, 2016). Early intervention professionals may be called upon to care for infants and young children and families affected by ZIKV infection. Early intervention professionals potentially have a significant role in documenting and elu- cidating health and developmental outcomes for infants and young children with congeni- tal ZIKV infection. This important role will be complicated by the evolving knowledge base and the unprecedented nature of the ZIKV outbreak. Early intervention professionals will need to remain alert to epidemiology and etio- logical knowledge advances, especially as data may change frequently. GEOGRAPHY The ZIKV was first described in humans in 1952 in Uganda (WHO, 2015). Prior to hu- man identification, the ZIKV was identified in a rhesus monkey in 1947 also in Uganda. Since 2007, concerning outbreaks of ZIKV infection have been noted in a number of countries in- cluding Micronesia, French Polynesia, Central America, South America, and Cape Verde off the coast of Africa. As of August 10, 2016, mosquito-borne ZIKV is present in at least 69 countries and territories (WHO, 2016b). Since February 11, 2016, a total of 11 countries have reported person-to-person transmission of ZIKV infec- tion (WHO, 2016a). Weekly updates to the WHO Zika situation report may be found on their website (http://www.who.int/ emergencies/zika-virus/situation-report/en). The ZIKV infection spread in the Americas is especially concerning, as the area population has little immunity to ZIKV infection as compared with populations in Africa and Asia. Brazil is considered the epicenter of the cur- rent outbreak in the Americas. As of October 2015, there have been as many as 4,000 cases of microcephaly and other fetal neurological malformations that may be ZIKV infection- related in Brazil (Berkrot, 2016; Melo et al., 2016; Zavis, 2016). The count accuracy of the 4,000 cases of microcephaly is controversial, as case definitions vary among sources and the window for laboratory confirmation of ZIKV infection is time sensitive. In addition, limited health resources in Brazil have delayed and hindered the robust investigation of all pre- sumed congenital ZIKV infection cases. As of August 10, 2016, likely congenital ZIKV infection-associated microcephaly and other anomalies have been reported in 15 countries (WHO, 2016b). Thirty states encompass habitat friendly to the Aedes mosquito (CDC, 2016a). Mosquitoes that may transmit ZIKV have been identified in almost all 50 states (Malo, 2016). People residing in the southern United States are at higher risk for ZIKV infection, as this geographic area has a large mosquito popu- lation. As of mid-August 2016, the ZIKV in- fection spread by local mosquitoes has been detected in the United States in the state of Florida (CDC, 2016a). ETIOLOGY ZIKV infection has multiple routes of trans- mission to humans. The vector is primarily the Aedes aegypti mosquito, but the Aedes albopictus and Asian tiger mosquitoes are also a source of ZIKV transmission (Malo, 2016). ZIKV infection can also be spread by sexual contact and laboratory exposure. Transmission through blood transfusion, or- gan or tissue transplant, and fertility treatment has been theorized (Fleming-Dutra et al., 2016). More information about transmission specifics via sexual, perinatal, transplacental, blood transfusion, saliva, urine, and breast Copyright © 2017 Wolters Kluwer Health, Inc. Unauthorized reproduction of this article is prohibited. Infants With Congenital Zika Virus Infection 19 milk is needed (dos Santos & Goldenberg, 2016). Congenital ZIKV infection is most concern- ing, as serious health outcomes to the fe- tus and the infant have been noted. ZIKV outbreaks preface microcephaly outbreaks (Johansson et al., 2016). It is not fully un- derstood whether there is mother-to-infant transmission of ZIKV infection during labor and delivery. ZIKV RNA has been found in breast milk, but at this time, breast-feeding by ZIKV-infected mothers is not contraindicated (Fleming-Dutra et al., 2016). EPIDEMIOLOGY U.S. health officials state that thousands of individuals may have been infected with the ZIKV while traveling internationally and have now arrived back in the United States. Be- cause 80% of people with ZIKV infection have no symptoms, it is likely that many people do not know they are infected (Tavernise, 2016). All these individuals who both know and do not know they have ZIKV infection are po- tentially able to start an outbreak through the local mosquito population. World Health Organization believes that potentially many thousands of infants with congenital ZIKV infection, worldwide, will exhibit neurological abnormalities (Berkrot, 2016). These neurological defects may range in severity from moderate to severe. As of August, 31, 2016, the CDC aggregated reports of 1,595 pregnant women with lab- oratory evidence of possible ZIKV infection including 624 pregnant women residing in the United States and District of Columbia and 971 living in U.S. territories. As of September 1, 2016, a total of 16 infants with laboratory-confirmed congenital ZIKV infection and anomalies have been live-born in the United States (WHO, 2016b). CDC Arborviral Disease Branch updates to the current number of cases may be found at http: //www.cdc.gov/zika/geo/united-states.html. The number of infants born with congenital ZIKV infection in the United States and its territories will very likely rise over time. Experts believe that the ZIKV could infect 25% of the population of Puerto Rico by the end of 2016, with the possibility that hun- dreds of infants could be affected by micro- cephaly and other concerning neurological issues (McKay, 2016). Puerto Rico’s budget difficulties are hindering ZIKV infection pre- vention efforts (McKay, 2016). Again, these numbers will very likely change and increase over time. ZIKV INFECTION IN PREGNANT WOMEN Four of five individuals infected with the ZIKV have no symptoms (Rathore, 2016). The incubation period for ZIKV infection is 2– 14 days (Rathore, 2016). Symptoms are gener- ally mild and last for a few days to a week and include fever, maculopapular rash, arthral- gia, conjunctivitis, myalgia, and headache. The rash is often pruritic and maculopapular (Fleming-Dutra et al., 2016). ZIKV infection has also been linked to neurological disorders in adults including Guillian–Barré syndrome and paralysis-causing myelitis (WHO, 2016a). In pregnant women, ZIKV infection presents most often with a pruritic descend- ing maculopapular rash, arthralgia, fever, and conjunctivitis (Simeone et al., 2016). World Health Organization (2016a) is advis- ing people in ZIKV infection transmission geographical areas to delay pregnancy. ZIKV infection may damage the developing fetus re- gardless of whether the pregnant women had symptoms of infection or not. Asymptomatic pregnant women with laboratory evidence of ZIKV infection have delivered infants with microcephaly and other serious brain anoma- lies (Jamieson & Honein, 2016). Monitoring of at-risk pregnant women will assist in the early identification of some affected infants and adequate mobilization of resources. MATERNAL-TO-FETAL TRANSMISSION OF ZIKV INFECTION Information is accumulating to improve sci- entific understanding of the precise mecha- nism of transmission from mother to fetus. Copyright © 2017 Wolters Kluwer Health, Inc. Unauthorized reproduction of this article is prohibited. 20 INFANTS & YOUNG CHILDREN/JANUARY–MARCH 2017 The ZIKV has been found in the placenta, amniotic fluid, and fetal brain tissue (Zavis, 2016). Animal models show the ZIKV is neu- rotropic. Intrapartum transmission has been documented, but a better scientific under- standing is needed. There are no reports of transmission through breast-feeding. Absolute certainty about all transmission particulars cannot be concluded at the present. The evidence is clear that prenatal expo- sure to the ZIKV especially in the first and second trimesters is associated with severe microcephaly. There is a 1%–13% risk for mi- crocephaly when a mother is infected with the ZIKV during the first trimester (Johansson et al., 2016). Although first-trimester ZIKV in- fection is a risk factor for microcephaly, an increase in central nervous system abnormal- ities has been identified that are not gesta- tional age specific (Johansson et al., 2016). ZIKV infection in later pregnancy has been associated with infant’s poor growth and pos- sible fetal death (Rasmussen et al., 2016). Pre- liminary research results out of Colombia sug- gests that structural defects are not linked to third-trimester ZIKV infection (Pacheco et al., 2016). Evidence is still unclear of the full spec- trum of defects that is caused by ZIKV infec- tion. It is important, therefore, for early inter- vention professionals to maintain awareness and keep abreast of new developments and findings. In at least one set of twins, born to a woman infected with the ZIKV, only one infant has overt ZIKV infection manifestations (Doce & Garcia, 2016). The health and developmen- tal impact on the apparently unaffected twin is not known. The permeability of the pla- centa, the resistance of neurons, and genetic predisposition may have an impact on which infants are seriously damaged by ZIKV infec- tion and which infants have no effects or less overt problems from congenital ZIKV expo- sure (Doce & Garcia, 2016). CLINICAL FINDINGS Congenital ZIKV infection of the human fe- tus is a cause of microcephaly (WHO, 2016a). Microcephaly, however, is not the only serious outcome exhibited by infants with congenital ZIKV infection. Other serious out- comes in infants with congenital ZIKV in- fection include spasticity, seizures, irritabil- ity, feeding difficulties, visual impairment, and documentation of several brain anoma- lies (Berkrot, 2016; Costello et al., 2016). The full phenotype spectrum of congenital ZIKV infection in infants is not known (Fleming- Dutra et al., 2016; Rasmussen et al., 2016). Much of what is reported concerning clinical features of congenital ZIKV infection comes from small studies that may not be general- izable. As with all components of ZIKV in- fection knowledge, the science is still evolv- ing and what is known may change and expand. IMPLICATIONS FOR EARLY INTERVENTION PROFESSIONALS There is not much known about the health and developmental outcomes for infants with congenital ZIKV infection including those born to symptomatic and asymptomatic preg- nant women (Simeone et al., 2016). What does seem clear is that early intervention pro- fessionals need to plan and prepare for the potential impact of congenital ZIKV-related adverse pregnancy and birth events and the resultant increased demand for services (de Barros Miranda-Filho et al., 2016; Johansson et al., 2016). The cohort of congenitally ZIKV- infected infants may exhibit more intense health and developmental needs than the typ- ical children enrolled in early intervention ser- vices. The potential severity and complexity of infants with congenital ZIKV infection may prove challenging for families, professionals, and programs, especially in the midst of evolv- ing scientific knowledge and resource con- straints including less than optimal funding and therapist availability. CLINICAL IMPLICATIONS There is a range of clinical findings in infants with congenital ZIKV infection (Table 1). Copyright © 2017 Wolters Kluwer Health, Inc. Unauthorized reproduction of this article is prohibited. Infants With Congenital Zika Virus Infection 21 Table 1. Potential Clinical Findings in Infants With Congenital Zika Virus Infection Arthrogryposis Clubfoot Eye anomalies Hearing loss Hyperreflexia Hypertonia Hypotonia Irritability Microcephaly and other serious brain anomalies Seizures Spasticity Sucking impairment Swallowing dysfunction Note. From Leal et al. (2016); Russell et al. (2016); van der Linden et al. (2016). The range of clinical findings is likely to ex- pand as more is learned about the condition and its impact on infants (Costello et al., 2016). Therefore, it behooves early interven- tion professionals to remain vigilant look- ing for new scientific knowledge concern- ing the health and development implications of congenital ZIKV infection. Development- enhancing interventions will need to evolve right along with scientific knowledge. The American Academy of Pediatrics, CDC, state health departments, and other vetted sites are good sources for up-to-date information for professionals as well as resources designed for parents (Table 2). A checklist for early intervention services and support for infants and their families with congenital ZIKV expo- sure care is available (the Figure). For children with complex health care challenges and their families, a checklist may help organize care and ensure that more basic care needs are not missed. As with all early intervention infants and families, using a family-centered approach to evaluation and assessment, development of the individualized family services plan, and service provision is essential. Infants with con- genital ZIKV infection likely require multi- ple medical consultation appointments and follow-up visits, so flexibility as to assess- ment and planning meetings may be espe- cially important. Considering the locales cur- rently most associated with ZIKV infection, Portuguese or Spanish-speaking program staff may be helpful in providing family-centered care. Early intervention professionals will need to also pay special attention to social determi- nants of health when arranging for evaluation, meetings, and services (Baptista, Quaghebeur, & Alarcon, 2016). Access to care, neighbor- hood conditions, and socioeconomic status likely all have an impact on outcomes for infants with ZIKV infection and their fami- lies. Low-income and uninsured people are less likely to get ZIKV testing (Santora, 2016). Prior research on sociodemographic and clin- ical characteristics that influence early inter- vention enrollment found that infants with more severe disabilities, as may often be the case for infants with congenital ZIKV infec- tion, were less likely to participate fully in early intervention services, so outreach and follow-up may need even closer attention than the typical enrollee family (Litt & Perrin, 2014). Table 2. Zika Virus Information Resources Centro de Recursos sobre Virus Zika (http://zika-virus-resource-center.elsevier.com.br) El virus del Zika: Lo que los padres deben saber (https://www.healthychildren.org/Spanish/ages- stages/prenatal/Paginas/zika-virus.aspx) Facts about Microcephaly (http://www.cdc.gov/ncbddd/birthdefects/microcephaly.html) MMWR Zika Reports (http://www.cdc.gov/mmwr/zika reports.html) United States Zika Pregnancy Registry (http://www.cdc.gov/zika/hc-providers/registry.html) Zika Resources for Hispanic Communities (http://espanol.cdc.gov/enes/zika/index.html) Copyright © 2017 Wolters Kluwer Health, Inc. Unauthorized reproduction of this article is prohibited. 22 INFANTS & YOUNG CHILDREN/JANUARY–MARCH 2017 Figure. Checklist for early intervention services and support for the family with an infant congenitally exposed to ZIKV infection (September 2016). Note. ABR = audio brainstem response; ZIKV = Zika virus. Because of Aedes mosquito behavior, pop- ulation density and poverty may place people at a greater risk for ZIKV infection. This is especially concerning in Puerto Rico where about half of the population lives in poverty. ZIKV infection appears to flourish in areas that are impoverished. Inadequate housing, lack of air-conditioning, screen-less doors and win- dows, standing water found in areas with poor drainage, and inadequate trash removal may contribute to conditions that support a habi- tat for mosquito proliferation. Transportation issues and out-of-pocket expenses may hinder optimal early intervention participation. The emotional health of infants and fam- ilies affected by congenital ZIKV infection needs to be a special emphasis both during interviews and treatment. The novel circum- stances of infants with congenital ZIKV in- fection have made some mothers in Brazil leery of attention even from health care pro- fessionals (Zavis, 2016). Media interest, so- cial media, and an undercurrent of still evolv- ing scientific information may make parents especially vulnerable to unwanted attention, stigma, and anxiety. Privacy and confiden- tiality may be challenging. Adequate psy- chosocial services to address the parent’s Copyright © 2017 Wolters Kluwer Health, Inc. Unauthorized reproduction of this article is prohibited. Infants With Congenital Zika Virus Infection 23 emotional status as a parent of an infant with complex health and developmental needs are essential. The CDC offers Interim Guidance for the Evaluation and Management of Infants With Possible Congenital Zika Virus Infec- tion (Russell et al., 2016). As this guidance is likely to change as more is learned about con- genital ZIKV infection, it is important for early intervention professionals to confirm they are using the most current evidence-based guide- lines. Pediatric health care clinicians are en- couraged to refer affected infants and fami- lies to early intervention services as soon as possible. The initial evaluation of infants prior to hos- pital discharge and recommended outpatient management varies depending upon maternal laboratory evidence of ZIKV infection and infant clinical examination results (Russell et al., 2016). These recommendations include comprehensive physical examination, with special attention to precise occipital-frontal head circumference, weight, length, neuro- logical examination, assessment for dysmor- phic features, hearing screen, postnatal head ultrasound study, and ZIKV infection testing for all infants with possible congenital ZIKV infection prior to hospital discharge (Russell et al., 2016). Infants with abnormalities consis- tent with congenital ZIKV infection should re- ceive all recommended evaluation previously noted plus additional subspecialty care consis- tent with clinical findings, laboratory testing, ophthalmology examination, audio brainstem response (ABR) test, and consideration of advanced neuroimaging (Russell et al., 2016). Action on many of these recommendations will likely take place prior to early interven- tion evaluation, but early intervention pro- fessionals should document and follow-up as necessary. Overall, ongoing monitoring of growth parameters, neurological status, nutri- tion and feeding, vision, and hearing is im- portant throughout at least the infant’s first year of life. Consultation reports should be obtained. Parents should be assisted in under- standing the consultant’s findings and recom- mendations. Early intervention professionals with experience serving children with other congenital infection sequelae will likely recog- nize the importance of such documentation, follow-up, and explanation. However, this may be especially important for infants with congenital ZIKV infection due to so many un- certainties about longer term outcomes. Hearing screening prior to hospital dis- charge and an ABR at approximately 2 weeks of age, when not done prior to hospital dis- charge and infant has laboratory evidence of ZIKV infection, are recommended (Russell et al., 2016). A repeat ABR at 4–6 months of age is also recommended (Russell et al., 2016). Retrospective evidence suggests that sensorineural hearing loss occurs at a preva- lence of 5.8% in infants with congenital ZIKV infection similar to prevalence rates found with other congenital viral infections (Leal et al., 2016). Ophthalmology examination prior to hospi- tal discharge or at approximately 2 weeks of age is recommended, as is a repeat ophthal- mology examination at 3 months of age for in- fants with abnormalities consistent with con- genital ZIKV infection (Russell et al., 2016). Ideally, both initial hearing and vision eval- uations will take place prior to hospital dis- charge. Involvement with hearing and vision specialists in evaluation, service plan develop- ment, and treatment is essential. Because many infants with congenital ZIKV infection may be small for their age and ex- hibit poor feeding skills, optimal nutrition in- take and management are vital to their over- all health and development. Dieticians and those with feeding therapy expertise will be essential as part of the interprofessional team. Nutrition consultation and feeding therapy in- terventions should not be delayed, as this is a critical period for brain development and overall growth for all infants. At this time, breast-feeding is not contraindicated for this population and early consultation with lacta- tion specialists should be considered along with ongoing encouragement and support for breast-feeding (Russell et al., 2016). Irritability as a behavioral concern should be addressed. Irritability may affect the Copyright © 2017 Wolters Kluwer Health, Inc. Unauthorized reproduction of this article is prohibited. 24 INFANTS & YOUNG CHILDREN/JANUARY–MARCH 2017 infant’s sleep patterns as well contribute to parent’s poor sleep and emotional health. Nonpharmacological interventions to address infant irritability should be shared, keeping in mind safe sleep and other safety con- siderations. Parents may need considerable emotional support and opportunities for self-care and respite. Arthrogryposis has been found in infants with congenital ZIKV infection involving arms and/or legs (van der Linden et al., 2016). Some infants evidence hip dislocation and knee sub- luxation. Handling and positioning informa- tion should be provided early on to family members. Adaptive equipment may be use- ful, but again keeping in mind safe sleep and other safety considerations. POLICY IMPLICATIONS Early intervention policy makers will need to determine whether health and develop- mental needs for families with an infant with congenital ZIKV infection-associated micro- cephaly or another severe outcome have been assessed and planned for and that an appro- priate system is in place with the capacity and resources to adequately address those needs (CDC, 2016b). Differing and evolving case definitions for how congenital ZIKV infection is defined may have an impact on inclusion criteria for research studies including efforts to quantify risk, incidence, and prevalence. As the case definition is refined and case numbers change, it may have significant ramifications for policy makers and early intervention program planning. With life span costs of care for children with micro- cephaly estimated at $4,000,000, even small incidence differences may have substantial economic impact (Ellis, 2016). To gain further knowledge for future treat- ment and prevention, the CDC has created the Zika Pregnancy Registry. This registry is a de- identified registry that contains vital informa- tion regarding a pregnant woman’s exposure and infants born to ZIKV-exposed women. By collecting and analyzing data, researchers can examine pregnancy outcomes and possible fu- ture treatment needs. This information should prove helpful for early intervention program planning and advocacy staff to plan and lobby for adequate services for these children and their families. As the actual need will likely precede the re- search results, early intervention profession- als will need to prepare for a potential influx of infants and young children who will likely need intensive and expensive care from a va- riety of professionals (de Barros Miranda-Filho et al., 2016). Early intervention professionals as well as policy makers will need to con- sider the impact of a cohort of infants with congenital ZIKV infection and their families who present all at the same time first for early intervention services and then to the preschool education system at 3 years of age. System functioning improvements coupled with additional funding to create expanded community-based programs including addi- tional therapists are likely needed, as some states already struggle to meet federally man- dated evaluation timelines (Sun, 2016). In a way, this may present an opportunity for early intervention systems to improve organization and communication among providers (Ruble, 2016). Advocacy and careful planning to ade- quately address the enrollment demands, vari- ety of services, and intensity of services such a cohort will need are vital. In addition, addi- tional personnel to handle a potential influx of developmental evaluation requests and repeat developmental screening may be needed. Planning for transition issues including longer term health and developmental needs will need consideration. Infants born with congenital ZIKV infection-associated micro- cephaly will likely have intensive health and developmental needs over their life span (Baptista et al., 2016). This necessitates plan- ning not only for the birth to 3 years, but for care transitions, school services, legal, fi- nancial, recreational, and adulthood require- ments. Because some individuals with con- genital ZIKV infection have a shortened life span, palliative and hospice care may be nec- essary and early intervention professionals may need to adapt to that model of care. Copyright © 2017 Wolters Kluwer Health, Inc. Unauthorized reproduction of this article is prohibited. Infants With Congenital Zika Virus Infection 25 On a more …