Performance Evaluation of the CareGENETM Zika Virus Reverse Transcription-PCR Kit for Urine Specimen

ABSTRACTZika virus (ZIKV) is a mosquito-borne flavivirus that is responsible for recent explosive epidemics in the Americas. Notably, ZIKV infection during pregnancy has been found to cause congenital birth defects, including microcephaly, and ZIKV has been associated with Guillain-Barré syndrome in adults. Diagnosis and surveillance of Zika in the Americas have been challenging due to similar clinical manifestations and extensive antibody cross-reactivity with endemic flaviviral diseases, such as dengue. We evaluated four serological and two reverse transcription-PCR (RT-PCR) methods in acute-phase (mean day, 1.8), early-convalescent-phase (mean day, 16.7), and late-convalescent-phase (mean, ~7 months) samples from the same individuals in a long-term pediatric cohort study in Nicaragua. Well-characterized samples from 301 cases of Zika, dengue, or non-Zika, nondengue febrile illnesses were tested. Compared to a composite reference, an in-house IgM antibody capture enzyme-linked immunosorbent assay (MAC-ELISA) and the NIAID-Biodefense and Emerging Infections (BEI) MAC-ELISA measuring IgM yielded sensitivities of 94.5% and 70.1% and specificities of 85.6% and 82.8%, respectively. The NS1 blockade-of-binding ELISA measuring anti-ZIKV NS1 antibody levels yielded sensitivities of 85.0% and 96.5% and specificities of 91.4% and 92.6% at early and late convalescence, respectively. An inhibition ELISA detecting total anti-ZIKV antibodies had sensitivity and specificity values of 68.3% and 58.3% for diagnosis and 94.0% and 98.6% for measuring annual infection incidence. Finally, the ZCD and Trioplex real-time RT-PCR assays detecting Zika, chikungunya, and dengue viruses both yielded a sensitivity of 96.1% and specificity of 100%. Together, these assays resolve the urgent need for diagnostic and surveillance tools for countries affected by Zika virus infections.

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Vertical transmission of zika virus in Aedes albopictus

PLoS Neglected Tropical Diseases ◽

10.1371/journal.pntd.0008776 ◽

2020 ◽

Vol 14 (10) ◽

pp. e0008776

Author(s):

Zetian Lai ◽

Tengfei Zhou ◽

Jiayong Zhou ◽

Shuang Liu ◽

Ye Xu ◽

...

Keyword(s):

Vertical Transmission ◽

Aedes Albopictus ◽

Reverse Transcription ◽

Zika Virus ◽

Transmission Rate ◽

Horizontal Transmission ◽

Infection Rates ◽

Transmission Rates ◽

Suckling Mice ◽

Reverse Transcription Pcr

Background Zika virus (ZIKV) is an arthropod-borne flavivirus transmitted by Aedes mosquitoes. Aedes albopictus is an important vector of ZIKV worldwide. To date, most experiments have focused on the vertical transmission of ZIKV in Ae. aegypti, while studies on Ae. albopictus are very limited. To explore vertical transmission in Ae. albopictus, a series of laboratory studies were carried out. Methodology/Principal findings In this study, Ae. albopictus were blood-fed with ZIKV-infectious blood, and the ovaries and offspring viral infection rates were analyzed by reverse transcription PCR (RT-PCR), real-time reverse transcription PCR (RT-qPCR) and immunohistochemistry (IHC). ZIKV was detected in the ovaries and oviposited eggs in two gonotrophic cycles. The minimum filial egg infection rates in two gonotrophic cycles were 2.06% and 0.69%, and the effective population transmission rate was 1.87%. The hatching, pupation, and emergence rates of infected offspring were not significantly different from those of uninfected offspring, indicating that ZIKV did not prevent the offspring from completing the growth and development process. ZIKV was detected in three of thirteen C57BL/6 suckling mice bitten by ZIKV-positive F1 females, and the viremia persisted for at least seven days. Conclusions/Significance ZIKV can be vertically transmitted in Ae. albopictus via transovarial transmission. The vertical transmission rates in F1 eggs and adults were 2.06% and 1.87%, respectively. Even though the vertical transmission rates were low, the female mosquitoes infected via the congenital route horizontally transmitted ZIKV to suckling mice through bloodsucking. This is the first experimental evidence of offspring with vertically transmitted ZIKV initiating new horizontal transmission. The present study deepens the understanding of the vertical transmission of flaviviruses in Aedes mosquitoes and sheds light on the prevention and control of mosquito-borne diseases.

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Zika Virus Testing Considerations: Lessons Learned from the First 80 Real-Time Reverse Transcription-PCR-Positive Cases Diagnosed in New York State

Journal of Clinical Microbiology ◽

10.1128/jcm.01232-16 ◽

2016 ◽

Vol 55 (2) ◽

pp. 535-544 ◽

Cited By ~ 20

Author(s):

Kirsten St. George ◽

Inderbir S. Sohi ◽

Elizabeth M. Dufort ◽

Amy B. Dean ◽

Jennifer L. White ◽

...

Keyword(s):

New York ◽

Pregnant Women ◽

Real Time ◽

Molecular Diagnosis ◽

Reverse Transcription ◽

Zika Virus ◽

New York State ◽

Lessons Learned ◽

York State ◽

Reverse Transcription Pcr

ABSTRACTThe performance and interpretation of laboratory tests for Zika virus (ZKV) continue to be evaluated. Serology is cross-reactive, laborious, and frequently difficult to interpret, and serum was initially solely recommended for molecular diagnosis. ZKV testing was initiated in January 2016 in New York State for symptomatic patients, pregnant women, their infants, and patients with Guillain-Barré syndrome who had traveled to areas with ZKV transmission. Subsequently, eligibility was expanded to pregnant women with sexual partners with similar travel histories. Serum and urine collected within 4 weeks of symptom onset or within 6 weeks of travel were tested with real-time reverse transcription-PCR (RT-PCR) assays targeting the ZKV envelope and NS2B genes. In this review of lessons learned from the first 80 positive cases in NYS, ZKV RNA was detected in urine only in 50 patients, in serum only in 19 patients, and in both samples concurrently in 11 patients, with average viral loads in urine a log higher than those in serum. Among 93 positive samples from the 80 patients, 41 were positive on both gene assays, 52 were positive on the envelope only, and none were positive on the NS2B only. Of the 80 infected patients, test results for 74 (93%) would have defined their infection status as not detected or equivocal if the requirement for positive results from two assay targets (two-target-positive requirement) in the initial federal guidance to public health laboratories was enforced, if urine was not tested, or if the extended eligibility time for molecular testing was not implemented. These changes facilitated more extensive molecular diagnosis of ZKV, reducing reliance on time-consuming and potentially inconclusive serology.

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Development and Performance Evaluation of a Quantitative Reverse Transcription PCR Kit for the Determination of prohibitin Gene Expression

Laboratory Medicine Online ◽

10.3343/lmo.2020.10.3.214 ◽

2020 ◽

Vol 10 (3) ◽

pp. 214-220

Author(s):

Bo-Ram Na ◽

Young-Eun Lee ◽

Min-Gu Kang ◽

Yonggwan Won ◽

Hye-Ran Kim ◽

...

Keyword(s):

Gene Expression ◽

Performance Evaluation ◽

Reverse Transcription ◽

Quantitative Reverse Transcription Pcr ◽

Reverse Transcription Pcr ◽

And Performance

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SARS-CoV-2 Diagnostic Tests: Algorithm and Field Evaluation From the Near Patient Testing to the Automated Diagnostic Platform

Frontiers in Medicine ◽

10.3389/fmed.2021.650581 ◽

2021 ◽

Vol 8 ◽

Author(s):

Nicolas Yin ◽

Cyril Debuysschere ◽

Marc Decroly ◽

Fatima-Zohra Bouazza ◽

Vincent Collot ◽

...

Keyword(s):

Performance Evaluation ◽

Reverse Transcription ◽

Diagnostic Tests ◽

Point Of Care ◽

Diagnostic Algorithm ◽

Molecular Techniques ◽

Diagnostic Tools ◽

Target Populations ◽

Reverse Transcription Pcr ◽

Rapid Tests

Introduction: Since the first wave of COVID-19 in Europe, new diagnostic tools using antigen detection and rapid molecular techniques have been developed. Our objective was to elaborate a diagnostic algorithm combining antigen rapid diagnostic tests, automated antigen dosing and rapid molecular tests and to assess its performance under routine conditions.Methods: An analytical performance evaluation of four antigen rapid tests, one automated antigen dosing and one molecular point-of-care test was performed on samples sent to our laboratory for a SARS-CoV-2 reverse transcription PCR. We then established a diagnostic algorithm by approaching median viral loads in target populations and evaluated the limit of detection of each test using the PCR cycle threshold values. A field performance evaluation including a clinical validation and a user-friendliness assessment was then conducted on the antigen rapid tests in point-of-care settings (general practitioners and emergency rooms) for outpatients who were symptomatic for <7 days. Automated antigen dosing was trialed for the screening of asymptomatic inpatients.Results: Our diagnostic algorithm proposed to test recently symptomatic patients using rapid antigen tests, asymptomatic patients using automated tests, and patients requiring immediate admission using molecular point-of-care tests. Accordingly, the conventional reverse transcription PCR was kept as a second line tool. In this setting, antigen rapid tests yielded an overall sensitivity of 83.3% (not significantly different between the four assays) while the use of automated antigen dosing would have spared 93.5% of asymptomatic inpatient screening PCRs.Conclusion: Using tests not considered the “gold standard” for COVID-19 diagnosis on well-defined target populations allowed for the optimization of their intrinsic performances, widening the scale of our testing arsenal while sparing molecular resources for more seriously ill patients.

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Detection of Zika Virus in Desiccated Mosquitoes by Real-Time Reverse Transcription PCR and Plaque Assay

Emerging Infectious Diseases ◽

10.3201/eid2304.161772 ◽

2017 ◽

Vol 23 (4) ◽

pp. 680-681 ◽

Cited By ~ 5

Author(s):

Kristen L. Burkhalter ◽

Harry M. Savage

Keyword(s):

Real Time ◽

Reverse Transcription ◽

Zika Virus ◽

Plaque Assay ◽

Reverse Transcription Pcr

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Evaluation of Altona Diagnostics RealStar Zika Virus Reverse Transcription-PCR Test Kit for Zika Virus PCR Testing

Journal of Clinical Microbiology ◽

10.1128/jcm.02153-16 ◽

2017 ◽

Vol 55 (5) ◽

pp. 1576-1584 ◽

Cited By ~ 21

Author(s):

Arnaud G. L'Huillier ◽

Ernesto Lombos ◽

Elaine Tang ◽

Stephen Perusini ◽

Alireza Eshaghi ◽

...

Keyword(s):

Reverse Transcription ◽

Zika Virus ◽

Sanger Sequencing ◽

Diagnostic Testing ◽

Large Set ◽

Reverse Transcription Pcr ◽

Test Kit ◽

The U.S ◽

Pcr Test ◽

Dual Target

ABSTRACT With the emerging Zika virus (ZIKV) epidemic, accessible real-time reverse transcription-PCR (rRT-PCR) assays are needed to streamline testing. The commercial Altona Diagnostics RealStar ZIKV rRT-PCR test kit (Altona PCR) has been approved for emergency use authorization by the U.S. FDA. Our aim was to verify the Altona PCR by comparing it to the CDC-designed dual-target ZIKV rRT-PCR reference assay (reference PCR) and describe the demographics of patients tested for ZIKV by rRT-PCR in Ontario, Canada. A large set of clinical specimens was tested for ZIKV by the Altona PCR and the reference PCR. Positive or equivocal specimens underwent PCR and Sanger sequencing targeting the ZIKV NS5 gene. A total of 671 serum specimens were tested by the reference PCR: 58 (8.6%) were positive, 193 (28.8%) were equivocal, and 420 (62.6%) were negative. Ninety percent of the reference PCR-positive patients were tested in the first 5 days after symptom onset. The Altona PCR was performed on 284/671 specimens tested by the reference PCR. The Altona PCR was positive for 53/58 (91%) reference PCR-positive specimens and 16/193 (8%) reference PCR-equivocal specimens; the ZIKV NS5 PCR was positive for all 68 Altona PCR-positive specimens and negative for all 181 Altona PCR-negative specimens that underwent the NS5 PCR. The Altona PCR has very good sensitivity (91%) and specificity (97%) compared to the reference PCR. The Altona PCR can be used for ZIKV diagnostic testing and has less extensive verification requirements than a laboratory-developed test.

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