Realstar zika virus rt pcr kit 1

Fourteen days post-infection, mosquitoes were screened for infection rate (IR), transmission rate (TR) and transmission efficiency (TE), as described previously [32 (link)]. Briefly, the IR is defined as the number of virus-positive mosquito bodies per number of fed females, TR as the number of virus-positive saliva per the number of virus-positive bodies and TE is calculated as the number of virus-positive saliva per the total number of fed and analyzed specimens. Detection and quantification of RNA was done by quantitative real-time PCR assay (RealStar Chikungunya RT-PCR Kit 2.0; RealStar Zika Virus RT-PCR Kit 1.0; RealStar WNV RT-PCR Kit 1.0; altona Diagnostics, Hamburg, Germany). A salivation assay was performed as previously described, detecting viable virus particles in the saliva by incubation of saliva solution on Vero cells (Chlorocebus sabaeus; CVCL_0059, obtained from ATCC, Cat# CCL-81) [35 (link)]. The R program [36 ] was used for all data analysis and visualizations, including the readxl [37 ], stringr [38 ], dplyr [39 ], plyr [40 (link)] and ggplot2 [41 ] packages.

ZIKV nucleic acid extraction was conducted in a fully automated manner according to the manufacturers instruction (NucliSense® easyMAG®, Biomerieux France). 200 μl EDTA-plasma was eluted into 110μl of purified RNA. For the amplification process, we used the recommended volume of 10μl of RNA extract with 20μl primer-enzyme-mix provided by the test kit to make a final reaction volume of 30 μl (RealStar® Zika Virus RT-PCR Kit 1.0 Altona Diagnostics, Germany) [16 ]. The test is based on real-time technology with reverse transcription of target RNA (NS1 gene region) to cDNA followed by amplification of target cDNA with simultaneous detection of amplified product using fluorescence labelled probes. The analytical sensitivity of the test l0.61 copies/μl of nucleic acid extracts 95% CI: (0.39–1.27 copies/μl). The kit is equipped with a system that controls for the quality of extracted sample (internal control) as well as positive and negative controls. The sample is considered negative, if there is no fluorescence signal detected up to the fortieth amplification cycle.

Pregnant women were eligible for recruitment when transferred to referral hospitals for the reasons specified above. At recruitment, we collected demographic data, including maternal birth date, birth country, ZIKV exposure country, presence of symptoms, and departure and return dates from ZIKV endemic area. Likewise, blood and/or urine ZIKV samples were collected and analyzed via RT-PCR commercial assay (RealStar® Zika Virus RT-PCR kit 1.0, Altona Diagnostics), ZIKV-IgG, and ZIKV-IgM (IIFT Arboviral fever Mosaic IgG and IgM, Euroimmun, Germany from 2016 to December 2017; and ELISA Virus Zika IgG and IgM, Euroimmun, Germany, from December 2017 to the end of study) (Table 1). If ZIKV RT-PCR was negative but ZIKV-IgG tested positive, specimens were sent to the Instituto de Salud Carlos III (ISCIII, Madrid, Spain) for ZIKV-PRNT. However, ISCIII was not able to perform specific PRNT for DENV; consequently, positive or equivocal ZIKV-PRNT titers were not compared with DENV-PRNT results. Neutralization titer ≥1/32 was considered indicative of the presence of ZIKV neutralizing antibodies. The date of ZIKV test was considered to be the date of diagnosis for ZIKV infection.

In utero ZIKV exposure was determined by RT-PCR or serological evidence of ZIKV infection in serum and/or urine samples from the mother collected during pregnancy. A toddler was considered exposed to ZIKV if the mother had RT-PCR (RealStar Zika Virus RT-PCR Kit 1.0, Altona Diagnostics) positive result for ZIKV in blood, urine, or both at any stage during her pregnancy; if the toddler had anti-ZIKV IgM (EuroImmun ELISA or in-house MAC-ELISA in French Guiana [16 (link)]) in the cord blood or blood taken within the first 10 days of life; or if the toddler had anti-ZIKV IgG (EuroImmun ELISA or in-house MAC-ELISA in French Guiana [16 (link)]) in the blood beyond 12 months of age and the date of birth was posterior to the end of the ZIKV epidemic: 11 September 2016 in French Guiana, 25 September 2016 in Guadeloupe, and 16 October 2016 in Martinique.
Anti-ZIKV kinetic studies have shown that anti-ZIKV IgG antibodies appear rapidly after infection and remain detectable up to 6 months with 100% sensitivity [17 (link)–19 (link)]. Therefore, a negative serology for anti-ZIKV IgG in mothers at the time of delivery has a 100% negative predictive value for ZIKV infection in the mother during pregnancy and was used to define toddlers as unexposed to ZIKV in utero.

All the pregnant women testing positive for ZIKV-IgG or ZIKV-IgM and (i) travelling to endemic areas during the pregnancy or the two previous months, or (ii) who had sexual partners that had visited endemic areas in the last 6 months were eligible for recruitment.
We collected demographic data, blood, and/or urine samples to test for ZIKV, and the samples were collected and analyzed with reverse transcriptase polymerase chain reaction (RT-PCR) (RealStar^®Zika Virus RT-PCR kit 1.0, Altona Diagnostics), ZIKV-IgG, and ZIKV-IgM (IIFT Arboviral fever Mosaic IgG and IgM, Euroimmun, Germany from 2016 to December 2017; and ELISA Virus Zika IgG and IgM, Euroimmun, Germany, from December 2017 to the end of study) (Table 1). In cases where ZIKV RT-PCR tested negative but ZIKV-IgG was positive, samples were shipped to the national reference laboratory Instituto de Salud Carlos III (ISCIII) located in Madrid. They processed the samples using a specific plaque reduction neutralization test for ZIKV (ZIKV-PRNT). Unfortunately, ISCIII did not perform PRNT for other flaviviruses; therefore, positive ZIKV-PRNT titers were not able to be compared with other flaviviruses. A neutralization titer ≥1/32 was considered positive, detecting the presence of ZIKV-neutralizing antibodies. The ZIKV test date was considered to be the diagnosis date for ZIKV infection [12 (link)].