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Reliable Serological Testing for the Diagnosis of Emerging Infectious Diseases

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Dengue and Zika: Control and Antiviral Treatment Strategies

Part of the book series: Advances in Experimental Medicine and Biology ((AEMB,volume 1062))

Abstract

Climate change, increased urbanization and international travel have facilitated the spread of mosquito vectors and the viral species they carry. Zika virus (ZIKV) is currently spreading in the Americas, while dengue virus (DENV) and chikungunya virus (CHIKV) have already become firmly established in most tropical and also many non-tropical regions. ZIKV, DENV and CHIKV overlap in their endemic areas and cause similar clinical symptoms, especially in the initial stages of infection. Infections with each of these viruses can lead to severe complications, and co-infections have been reported. Therefore, laboratory analyses play an important role in differential diagnostics. A timely and accurate diagnosis is crucial for patient management, prevention of unnecessary therapies, rapid adoption of vector control measures, and collection of epidemiological data.

There are two pillars to diagnosis: direct pathogen detection and the determination of specific antibodies. Serological tests provide a longer diagnostic window than direct methods, and are suitable for diagnosing acute and past infections, for disease surveillance and for vaccination monitoring. ELISA and indirect immunofluorescence test (IIFT) systems based on optimized antigens enable sensitive and specific detection of antibodies against ZIKV, DENV and CHIKV in patient serum or plasma. In recent years, Euroimmun (Lübeck, Germany) has developed numerous test systems for the serological diagnosis of (re-)emerging diseases, including a very sensitive and specific anti-ZIKV ELISA.

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Authors and Affiliations

  1. Institute for Experimental Immunology, Euroimmun AG, Lübeck, Germany

    Claudia Ohst, Sandra Saschenbrecker, Konstanze Stiba, Katja Steinhagen, Christian Probst, Christiane Radzimski, Erik Lattwein, Lars Komorowski, Winfried Stöcker & Wolfgang Schlumberger

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    Rolf Hilgenfeld

  2. Emerging Infectious Diseases Program, Duke-NUS Medical School Singapore, Singapore, Singapore

    Subhash G. Vasudevan

Discussion of Chapter 3 in Dengue and Zika: Control and Antiviral Treatment Strategies

Discussion of Chapter 3 in Dengue and Zika: Control and Antiviral Treatment Strategies

This discussion was held at the 2nd Advanced Study Week on Emerging Viral Diseases at Praia do Tofo, Mozambique.Transcribed by Hilgenfeld R and Vasudevan SG (Eds); approved by Dr. Claudia Ohst.

  • Aruna Sampath: Most of the tests that you have shown are based on serology. Do you have any tests that can specifically look at infectious virus particles?

  • Claudia Ohst: The only test that we have is the Dengue NS1 test, that shows the NS1 for all four types when the virus breaks down. We only have this test for Dengue virus and not for Zika virus or Chikungunya virus. We do not do any PCRs either.

  • Aruna Sampath: Ok, so for the Dengue, what sensitivity do you have? What is the sensitivity for the NS1 antigens?

  • Claudia Ohst: Our Dengue NS1 test looks into detecting the Dengue NS1 antigen, so it is not an antibody ELISA here, but an antigen test. Compared to the antibody ELISA, the correlation is quite good and even slightly more sensitive. This has been confirmed by serology where both, the antibody and the NS1 antigen test have been performed.

  • Subhash Vasudevan: And do you think you can find the serotype as well?

  • Claudia Ohst: No. This one is for all of the four, so you cannot distinguish the serotypes with this, it is just a screening test.

  • Kerstin Falk: But you have this mosaic slide with Dengue 1, 2, 3, and 4. But then you said you have to titrate it out. And how much work is that and how reliable is that?

  • Claudia Ohst: You see, in immunofluorescence you always have crossreactions between the different serotypes and all of them will come up positive in your screening dilution. That’s why you have to titrate. Obviously you can take bigger steps and just see where you end up. And then the one with the highest titer should be the virus in your serotype that you are dealing with. Maybe PCR is the better method, but at least there is an option to do this via serology.

  • Kerstin Falk: So what antigen are you using? So what part of the genome? For the Dengue 1, 2, 3 and 4?

  • Claudia Ohst: So we are using Dengue 2, I think. For the NS1 antigen ELISA we used antibodies from all four Dengue serotypes, for the antibody ELISA we used purified Dengue 2 virus particles (now purified particles from all four Dengue types).

  • Kerstin Falk: But when you have Dengue 1, 2, 3 and 4?

  • Claudia Ohst: Yes, but it does detect all of them.

  • Aravinda de Silva: But you have done this with IFA. IFA is immunofluorescence.

  • Claudia Ohst: Well, the IFA uses infected cells, there are four Biochips on each field of the Mosaic slide, each chip is coated with cells which were infected with only one of the four Dengue serotypes.

  • Aravinda de Silva: The Zika virus data looks very encouraging, much better than Dengue.

  • Claudia Ohst: We put a lot of work into it.

  • Aravinda de Silva: Have you looked at Zika virus in late convalescence, in other words, in people 6 months, 1 year after exposure and see how the NS1 antibodies change over the long term?

  • Claudia Ohst: I think, these data might just be on their way. You need to get the specimens for this. And everything is from the first half basically of 2016 and that is when we start to get the specimens. I cannot give an answer now.

  • Aravinda de Silva: The other question I have had is that you showed that in secondary cases the Zika IgM, you could not really detect, it was not very sensitive. Is it because there is no IgM or is it because the IgG antibodies are binding to your NS1 antigen, because you have very high IgG levels. And are the IgG antibodies binding the NS1 and preventing IgM from binding?

  • Claudia Ohst: No. There apparently is not very much IgM. What we do in our IgM tests, is to pre-absorb all the IgG, so they should not really interfere with the assay. We really only catch the IgM antibodies that are present in your specimen.

  • Aravinda de Silva: So even in the commercial assay, you pre-absorb the IgG?

  • Claudia Ohst: Yes. So our dilution solution provided in the test kit contains something that pre-absorbs the IgGs.

  • George Gao: I missed your NS1 antigen preparation. Did you use the mammalian-expressed protein or insect cells expressed proteins for your antigen, NS1 antigen? How do you prepare the antigen?

  • Claudia Ohst: Are you referring to the NS1 antigen for Dengue or for Zika?

  • George Gao: For both. Dengue 1–4 and the Zika. How do you prepare the antigen?

  • Claudia Ohst: For Dengue, it is an antigen capture test, so obviously we coat our ELISA plates with antibodies that catch the antigen. In the case of Zika, where we detect antibodies against the NS1 antigen, Zika NS1, we have NS1 protein coated on our plate. And this protein is prepared by our company, in our own molecular biology department that produces these recombinant antigens.

  • Paul Young: I am a little confused by the IgM and IgG antibody data against Zika. NS1. In Dengue, in primary infected patients at least, there is very little if any, and In many cases no, early IgG or IgM response to NS1. This comes along much later in convalescence. Very strong anti-E and sometimes prM, but very little NS1. Is it completely different in Zika? The patients actually mount a good antibody response to the NS1 protein?

  • Claudia Ohst: Well, we are using the NS1, because that is the one where we really get specific results. Otherwise it would not really help.

  • Paul Young: I understand completely, but it is not there in Dengue, so I am just surprised.

  • Claudia Ohst: I cannot tell you. You see, in our Dengue ELISAs, we do not even use the Dengue NS1. That might be the reason why.

  • Paul Young: Yes, that is why no one uses it. And I am just very surprised that Zika infection would actually induce such a high response, given that the viral load is lower in Zika virus. I suspect, no one has really done that analysis yet. And I know there is really quantitative data on the amount of NS1 in patients, but I suspect it is lower than in Dengue virus. So again it is still confusing.

  • Claudia Ohst: Maybe we were just lucky with our NS1.

  • Jonas Schmidt-Chanasit: I would support this comment, because I think it was neglected that the sensitivity especially for IgG is not that good. In combination with IgM, this Zika assay is okay, but if you only perform the IgG, you lose like 30–40% sometimes. Then, what is also important to mention is that the plaque titers do not correlate with the reactivity in the Zika virus ELISA. So you have sometimes very high viral titers and the ELISA is completely negative. So it is quite interesting and it might be related to what was just mentioned before. I just wanted to highlight that. High sensitivity is only achieved if you do this in combination, IgG and IgM together. We have a lot of samples and this was also very nicely illustrated by the data from Mozambique, that they were completely negative in IgG ELISA, but positive in the IFA. So this is due to a lack of sensitivity.

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Ohst, C. et al. (2018). Reliable Serological Testing for the Diagnosis of Emerging Infectious Diseases. In: Hilgenfeld, R., Vasudevan, S. (eds) Dengue and Zika: Control and Antiviral Treatment Strategies. Advances in Experimental Medicine and Biology, vol 1062. Springer, Singapore. https://doi.org/10.1007/978-981-10-8727-1_3

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