Capítulo 6. Descripción de la empresa y análisis e interpretación de
6.2. Relación del control interno con la función de auditoría
3.4.4.1 The IgM and IgG ZIKV NS1 ELISA assays
The IgM and IgG NS1 anti-ZIKV ELISAs from Euroimmun (Lubeck, Germany) use recombinant Zika non-structural protein 1 (NS1) as the ZIKV antigen. This is an indirect, colourimetric ELISA that detects IgM human antibodies generated against ZIKV infection. I performed the procedures following the manufacturer’s instructions as described in Chapter 2.
3.4.4.2 IgM ZIKV µ-capture ELISA.
The Novagnost® Zika Virus IgM µ-capture ELISA (NovaTec Immunodiagnostica GmbH, Germany) is based on the ELISA µ-capture technique. It uses ZIKV NS1 as antigen. Protocol described in further detail in Chapter 2.
3.4.4.3 IgM CDC MAC ELISA
The FDA CDC-designed IgM antibody capture ELISA also known as the ZIKV MAC ELISA was performed as previously described by using the US Centers for Disease Control and
Evaluation of diagnostic assays for Zika virus Chapter 3
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Prevention (CDC) emergency use authorization protocol (CDC Fort Collins, CO, USA)[115].
Results were reported as recommended by the CDC protocol as positive if 𝑃
𝑁 ≥3; equivocal if f 𝑃
𝑁 ≥2 but 𝑃
𝑁 <3 and negative if f 𝑃
𝑁 <2.
3.4.4.4 DENV IgM capture ELISA
For the qualitative detection of DENV IgM antibodies Panbio dengue IgM capture ELISA (Cat.
No. E-DEN01M, Alere, UK) was performed following the manufacturer’s manual. Further details are shown in Chapter 2..
3.4.4.5 IgG Indirect DENV ELISA
For detection of DENV IgG antibodies, the IgG DENV indirect ELISA (Cat. No. E-DEN02G) (Panbio, Alere, United Kingdom) was performed. The manufacturers indicate that the assay is able to determine levels of IgG antibodies against the four dengue serotypes (DENV-1 to 4). The assay was performed according to the manufacturer’s instructions and a further detailed protocol is shown in Chapter 2.
3.4.4.6 BOB Assay
The Blockade-of-binding (BOB) assay is an NS1-based competition ELISA using a Zika specific monoclonal antibody developed as described in [116]. This assay is based on a competitive principle. Thus, when no anti-ZIKV NS1 antibodies are present in the sample, the enzyme labelled Mab will bind and there will be a chromogenic reaction. A percentage of inhibition of 50% was determined to be the best threshold for this assay. Patients exhibiting higher percentages of inhibition than 50% are considered positive for IgG ZIKV antibodies. More details are described in Chapter 2.
3.4.4.7 Green BOB Assay
The Green BOB assay is a Zika competitive enzyme-linked immunoabsorbent assay that detects antibodies specifically to the NS1 region of the ZIKV. It has been developed by collaborators in Public Health England as a modification of the Blockade-of-binding (BOB) assay. The Green BOB assay is based on an ELISA format and it also uses the same ZIKA35 mAb as described above. The main differences between BOB and Green BOB assays are the following:
1- Plates are pre-coated with NS1 ZIKV Antigen.
2- Serum concentration is modified to reduce the amount of serum required.
3- The concentrations of the reagents had been adjusted
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4- No previous serum lysis step or serum dilution step is needed, aiming to reduce the time of the assay.
5- No incubation step of the serum in the plate before adding the Anti-ZKA35 mAb.
6- The assay can be performed in only one day and it can be completed in less than 4 hours.
7- The Anti-ZKA35 Monoclonal antibody is conjugated to HorseRadish Peroxidase (HRP).
8- The substrate used is 3,3',5,5'-Tetramethylbenzidine (TMB) instead of p-Nitrophenyl Phosphate (pNPP).
The Green BOB is based on a one-step incubation competitive protocol. If the Anti-ZIKV antibodies are present in the sample, they will compete with the monoclonal anti-ZIKV antibodies that are conjugated to HRP for the fixed amount of recombinant ZIKV NS1- antigen that is already pre-coated in the plate wells. For a graphical visualization of the assay, and further details on the protocol, see Chapter 2.
3.4.4.8 Plaque Reduction Neutralization Tests (PRNTs)
The protocol I used was adapted from protocol used for DENV and JEV PRNT in the Liverpool group, based on the previously described protocol [118]. Testing was conducted both in Liverpool and in Brazil. Further details on the protocol can be found in Chapter 2.
3.4.4.9 Single-use point-of-care (POC) assays for ZIKV diagnosis (DPP® Zika IgM/IgG) A Rapid Diagnostic Test (RDT) for ZIKV diagnosis for use with an automatic reader. The assay was designed to support the diagnosis of ZIKV in primary care centres in order to reduce the delay in the response to patients suspected to have ZIKV.
3.4.4.10 Indirect immunofluorescence test for Zika Virus detection
The indirect immunofluorescence test (IIFT) based on virus-infected cells offers an alternative sensitive screening assay for ZIKV antibodies. I evaluated the first commercial immunofluorescence test for detection of antibodies against Zika virus. It is based in a BIOCHIP combination, which aims to differentiate Zika, dengue and chikungunya antibodies in serum samples. It is produced by Euroimmun (Lubeck, Germany) and their evaluations report excellent specificity and sensitivity (>90%). The two assays evaluated are:
3- IIFT Arbovirus Fever Mosaic 2. Designed for specific detection of 6 pathogens, including ZIKV, DENV (serotypes 1, 2, 3 and 4) and CHIKV.
Evaluation of diagnostic assays for Zika virus Chapter 3
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4- IIFT Arbovirus Profile Mosaic 3. Designed for specific detection of 10 pathogens, including the 6 above mentioned in the IIFT Mosaic 2 [ZIKV, DENV (serotypes 1, 2, 3 and 4) and CHIKV] but also Tick-Borne Encephalitis Virus (TBEV), Yellow Fever Virus (YFV), West Nile Virus (WNV) and Japanese Encephalitis Virus (JEV).
The detailed protocol for both assays is shown in Chapter 2.
Molecular Diagnostic Assays
Molecular detection of pathogen RNA was performed for ZIKV and DENV though RT-PCR.
3.4.5.1 RNA extraction
The individuals in the ZIKV panel with clinical presentation indicative of recent ZIKV infection (during 2015 and 2016) were tested for detection of ZIKV nucleic acid material. RNA was extracted using the QIAamp Mini Elute (Qiagen, Brazil) following the manufacturer’s instructions as described in Chapter 2.
3.4.5.2 RT-PCR
RT-PCR reactions were conducted for detection of specific RNA from ZIKV, DENV1, DENV2, DENV3 and DENV4. The primers and probes used for all the RT-PCRs and the RT-PCR thermomixer protocols are described in Chapter 2.
Calculation of ROC Curves, sensitivity and specificity
Diagnostic performance of the ELISAs was assessed by calculating the probability of a true positive (sensitivity), a true negative result (specificity) and by generating receiver operating characteristic (ROC) curves as described in Chapter 2 [123].
The optimal cut-off values for the ELISAs were investigated to maximize the combination of sensitivity and specificity. ROC curves provide graphical visualization of the sensitivity and specificity at all the cut-off values possible for a particular test. The larger the area under the curve, the better the assay performance. The sensitivity, specificity and accuracy of the assays was determined using MedCalc Statistical Software, version 16.2.0 [MedCalc Software, Ostend, Belgium; https://www.medcalc.org; 2016]). ROC curves were generated using PRISM GraphPad v7 (GraphPad Software, La Jolla, CA).
Samples with borderline antibody results were re-tested (when enough reagents and sample volume were available). If samples were still borderline, they were considered negative for calculation of sensitivity, specificity and ROC curves.
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In this chapter, I will also be using the kappa statistic also known as Cohen’s Kappa. Kappa is a tool that controls for random agreement factor. The Kappa statistic varies from 0 to 1, where:
0 = agreement equivalent to chance.
0.1 – 0.20 = slight agreement.
0.21 – 0.40 = fair agreement.
0.41 – 0.60 = moderate agreement.
0.61 – 0.80 = substantial agreement.
0.81 – 0.99 = near perfect agreement 1 = perfect agreement.
Kappa is described in further detail in Chapter 2. The Coefficient of Variation (CV) for the Green BOB assay was calculated as follows:
Coefficient of Variation (CV) = 𝑆𝑡𝑎𝑛𝑑𝑎𝑟𝑑 𝐷𝑒𝑣𝑖𝑎𝑡𝑖𝑜𝑛 (𝑆𝐷)
𝑀𝑒𝑎𝑛 𝑜𝑓 𝑡ℎ𝑒 5 𝐺𝑟𝑒𝑒𝑛 𝐵𝑜𝑏 𝑅𝑎𝑡𝑖𝑜 𝑟𝑒𝑠𝑢𝑙𝑡𝑠 𝑥 100
Statistical methods and graphical representations
Statistics and figures were generated using PRISM GraphPad. A one way ANOVA with repeated measures was used to assess the differences among more than two groups. Non-parametric Mann–Whitney U tests were used to determine differences among two groups.