Confirmed case of Zika virus congenital infection , Spain , March 2016

S Perez 1 , R Tato 2 , JJ Cabrera 1 , A Lopez 3 , O Robles 4 , E Paz 3 , A Coira 5 , MP Sanchez-Seco 6 , A Vazquez 6 , R Carballo 1 , C Quintas 7 , A Pousa 8 1. Microbiology Department, University Hospital of Vigo, Spain 2. Microbiology Department, University Hospital of Pontevedra, Spain 3. Gynecology Department, Lucus Augusti Hospital, Lugo, Spain 4. Pathology Department, Lucus Augusti Hospital, Lugo, Spain 5. Microbiology Department, Lucus Augusti Hospital, Lugo, Spain 6. Arbovirus and Imported Viral Diseases, Virology Area, National Center of Microbiology (CNM), Carlos III Health Institute (ISCIII), Majadahonda, Madrid, Spain 7. Preventive Medicine Department, Lucus Augusti Hospital, Lugo, Spain 8. General Subdirection for Epidemiology and Information Systems, General Direction of Public Health, Consellería de Sanidade, Santiago de Compostela, Spain

Serum samples were collected upon hospital admission in March and recent ZIKV infection was diagnosed by serology and molecular biology.ZIKV IgG and IgM antibodies were detected by indirect immune fluorescence test (Euroimmun Arboviral Fever Mosaic 2, Luebeck, Germany).ZIKV IgG antibodies were confirmed by plaque reduction neutralization test (PRNT).NS2A protein gen of ZIKV was detected (2.4 x104 copies/mL) by real-time quantitative RT-PCR using Light Mix Modular ZikaVirus (Tib Molbiol, Berlin, Germany) and Light Cycler Multiplex RNA Virus Master (Roche Diagnostics, Mannheim, Germany) in a Cobas Z 480 analyser (Roche Diagnostics, Indianapolis, United States).Extraction of nucleic acids was performed with the MagNA Pure Compact Nucleic Acid Isolation Kit I (Roche Diagnostics, Indianapolis, United States).Ultrasound scan at 19 weeks of gestational age showed fetal hydrocephalus with dilation of both lateral ventricles (Figure 1), and stiffness and severe contractures of the four extremities compatible with arthrogryposis multiplex congenita (AMC) (Figure 2).Amniotic fluid was obtained by amniocentesis.Chromosomal abnormalities were discarded by array comparative genomic hybridisation and karyotype analysis.ZIKV RNA was detected in amniotic fluid (9.1 x104 copies/mL) by real-time quantitative RT-PCR.Toxoplasma gondii, rubella virus, cytomegalovirus, herpes virus, erythrovirus B19 and measles virus infections were discarded by PCR in amniotic fluid.
Due to severe malformations and brain disease, the neonatal health prognosis was poor.The patient asked for voluntary termination of the pregnancy, and the procedure was approved by national and hospital ethics committees.Medical termination of the pregnancy was performed at 21 weeks of gestation.Autopsy of the female fetus (295 g) showed no microcephaly (cranial, thoracic and abdominal circumference were 17 cm, 16 cm and 14 cm respectively, normal for sex and gestational age) but confirmed AMC with flexion contracture and deformity of joints of all four limbs, extreme flexion of hips and crossed femurs.Under-developed muscles with replacement of muscle by adipose tissue and fibrous proliferation in interarticularis spaces were also found.In addition, fetal hydrocephalus with dilation of both lateral ventricles, cerebral cortex 2.5 mm thick and multiple calcifications at cortical level and brainstem were diagnosed (Figure 3).
ZIKV real-time quantitative RT-PCR was performed in placenta, umbilical cord and brain tissue samples, as previously described.Before nucleic acid extraction, ca 10 mg of tissue were treated with 50 µL of proteinase K at 56 °C until sample digestion, and then heated at 95 °C for 10 min to inactivate proteinase K. ZIKV RNA was detected in umbilical cord (threshold cycle, Ct: 36.7) and brain tissue (Ct: 22.1), but it was not detected in the placenta sample.For phylogenetic analysis, amplification of NS5 gene (192 bp) was performed from amniotic fluid sample as previously described [1] with Mega 7 Software and it was deposited in GenBank (accession number: KX358623).The sequence clustered within the Asian lineage.

Background
ZIKV is an arbovirus (arthropod-borne virus) of the genus Flavivirus.It was isolated for the first time in 1947 from the blood of a Rhesus monkey in the Zika forest (Uganda), but the infection was relatively unknown until the recent outbreak in South America.Prior to 2015, minor ZIKV outbreaks were reported in areas of Africa, south-east Asia and the Pacific Islands [2].In May 2015, the Pan American Health Organization (PAHO) issued an alert regarding the first confirmed ZIKV infections in Brazil [3], which quickly spread all over the South America continent.

Discussion
Local mosquito transmission of ZIKV infection was reported in Venezuela in November 2015 [4].In January 2016, a total of 252 cases of Guillain-Barré syndrome with a spatiotemporal association to Zika virus were reported.Zika virus infection was confirmed in three of them [5].
Phylogenetic analysis based on the conserved region NS5 has revealed the existence of two major lineages: the African lineage that includes the African strains and the Asian lineage which has recently emerged in the Pacific and the Americas including the Asian and American strains [6].In this case, phylogenetic analysis showed that ZIKV strain isolated from this patient belonged to the Asian lineage.
The illness caused by ZIKV, commonly named Zika virus disease (ZVD), usually has a benign cause and presents with signs and symptoms similar to other arbovirus infections such as dengue virus (DENV) and chikungunya virus (CHKV): mild fever, exanthema, arthralgia and/or non-purulent conjunctivitis.Recently, ZIKV infection has been associated with neurological complications, such as Guillain-Barré syndrome, but encephalitis or meningitis have also been reported [7].The patient reported generalised skin rash compatible with ZIKV infection during her stay in Venezuela.
ZIKV is transmitted through mosquito bites of the genus Aedes, mainly Aedes aegypti, which is the most important vector in Asia and the Americas and in some regions in the Pacific.Other possible modes for human infection include sexual transmission and blood transfusion, and vertical transmission from mother to fetus can have serious consequences.If a pregnant woman acquires a ZIKV infection, the virus might cross placental barrier causing congenital infection.In this case, ZIKV RNA was detected in amniotic fluid, confirming that ZIKV crossed the placental barrier.When this happens, the fetus might develop brain damage including microcephaly and, less frequently, calcifications, ventriculomegaly or hydrocephalus, and other congenital malformations such as arthrogryposis [8].
Viraemia of ZIKV infection is relatively short, and viral RNA is usually detected in serum samples only around seven days after onset of symptoms.It is remarkable that in this case, ZIKV RNA was detected in the serum of the pregnant woman up to two months after the acute phase of the disease.Persistent viraemia was previously described in another case of congenital Zika transmission and it might be a consequence of high viral replication in the fetus [9].
The recent outbreaks in Brazil and French Polynesia indicate that the greatest risk of brain damage for the fetus is in the first trimester, often between seven and 13 weeks of gestation [10,11].A preliminary report from Brazil indicated that fetal abnormalities were present in almost 30% of women with ZIKV infection during pregnancy [12].In the case presented here, microcephaly was not present in the ultrasound scan at 19 weeks of gestation, probably due to the short gestational age, and this is in agreement with other reported cases in which microcephaly was not diagnosed until 27 to 35 weeks [12].However, other malformations, such as hydrocephalus and arthrogryposis, were detected in this case.Presence of ZIKV RNA was detected in the umbilical cord and brain tissue but not in placenta as recently reported in another congenital case [13].Another study showed that ZIKV is unlikely to access the fetal compartment by its direct replication in placental tissue.This might be explained due to the potent antiviral properties of type III interferons (IFNs), specifically IFNλ1, which protects the human placental trophoblasts from viral infections, suggesting that ZIKV may invade the intrauterine cavity by unknown mechanisms that are independent of direct placental infection [14].In this case, comparison of the Ct values between umbilical cord and brain tissue indicates a higher viral load (ca 10,000 times higher) in the central nervous system (CNS), showing a strong neurotropism of the virus, although the mechanism is not clear.A possible persistence of ZIKV in the fetal brain because of the immunologically secure milieu for the virus was suggested [15].Additionally, recent studies found that neural progenitor cells are more susceptible to ZIKV infection than mature cortical neurons, explaining microcephaly and other abnormalities in the developing brain [16][17][18].
In the Zika situation report of the World Health Organization (WHO) on 6 June 2016 [19], only eleven countries or territories have reported microcephaly and/or CNS malformation cases potentially associated with ZIKV infection.The total number of cases reached 1,520, and most of them are related to the recent outbreak in Brazil.In Spain, the Ministry of Health reported 141 cases of confirmed ZIKV infection on 6 June 2016, all of them imported from areas with active transmission [20].Nineteen cases were pregnant women, but no evidence of intrauterine transmission was detected, except in this case.The first case of imported ZIKV infection and two cases of ZIKV infection in pregnant women in Spain, have been published recently [21,22].Aside from this case, there has been only one other confirmed ZIKV congenital infection in Europe [15].
This case highlights the new challenge gynaecologists face when performing ultrasound in pregnant women originating from or having stayed in countries with risk of transmission of Zika.Nowadays, there is a considerable number of pregnant women who travel from South America; moreover, other pregnant women or their sexual partners may travel to affected areas.

Figure 1 Figure 2
Figure 1 Ultrasound scan on fetal skull showing bilateral hydrocephalus, case of vertical transmission of Zika virus infection, gestation week 19, imported to Spain, March 2016 This may have an impact on prenatal care in Europe.Positive and negative predictive values of screening for Zika virus in amniotic fluid or maternal serum have not yet been established.Accurate and quick detection of the presence of Zika virus in maternal samples in collaboration with expert gynaecologists follow-up might help the early diagnosis of congenital Zika infections.It is important to carefully communicate risks associated with ZVD to those possibly concerned, and to improve the evidence base to perform well-informed risk assessments.Microphotography of the entire cerebral cortex thickness with abundant microcalcifications, case of vertical transmission of Zika virus infection, gestation week 21, imported to Spain, April 2016 Arrows indicate microcalcifications. from the Microbiology Department of Lucus Augusti Hospital of Lugo, Spain, Ory F and all the staff from the 'Arbovirus and Imported Viral Diseases' laboratory of the National Center of Microbiology -Carlos III Health Institute, Majadahonda, Madrid, Spain.Authors specially thank the staff from the Microbiology Department of University Hospital of Vigo, Spain.