*Article* **Detection of Circulating VZV-Glycoprotein E-Specific Antibodies by Chemiluminescent Immunoassay (CLIA) for Varicella–Zoster Diagnosis**

**Arnaud John Kombe Kombe 1, Jiajia Xie 1, Ayesha Zahid 2, Huan Ma 2, Guangtao Xu 2, Yiyu Deng 2, Fleury Augustin Nsole Biteghe 3,4, Ahmed Mohammed 2, Zhao Dan 2, Yunru Yang 2, Chen Feng 2, Weihong Zeng 2, Ruixue Chang 1, Keyuan Zhu 1, Siping Zhang 1,\* and Tengchuan Jin 1,2,5,\***


**Abstract:** Varicella and herpes zoster are mild symptoms-associated diseases caused by varicella–zoster virus (VZV). They often cause severe complications (disseminated zoster), leading to death when diagnoses and treatment are delayed. However, most commercial VZV diagnostic tests have low sensitivity, and the most sensitive tests are unevenly available worldwide. Here, we developed and validated a highly sensitive VZV diagnostic kit based on the chemiluminescent immunoassay (CLIA) approach. VZV-glycoprotein E (gE) was used to develop a CLIA diagnostic approach for detecting VZV-specific IgA, IgG, and IgM. The kit was tested with 62 blood samples from 29 VZV-patients classified by standard ELISA into true-positive and equivocal groups and 453 blood samples from VZV-negative individuals. The diagnostic accuracy of the CLIA kit was evaluated by receiveroperating characteristic (ROC) analysis. The relationships of immunoglobulin-isotype levels between the two groups and with patient age ranges were analyzed. Overall, the developed CLIA-based diagnostic kit demonstrated the detection of VZV-specific immunoglobulin titers depending on sample dilution. From the ELISA-based true-positive patient samples, the diagnostic approach showed sensitivities of 95.2%, 95.2%, and 97.6% and specificities of 98.0%, 100%, and 98.9% for the detection of VZV-gE-specific IgA, IgG, and IgM, respectively. Combining IgM to IgG and IgA detection improved diagnostic accuracy. Comparative analyses on diagnosing patients with equivocal results displaying very low immunoglobulin titers revealed that the CLIA-based diagnostic approach is overall more sensitive than ELISA. In the presence of typical VZV symptoms, CLIA-based detection of high titer of IgM and low titer of IgA/IgG suggested the equivocal patients experienced primary VZV infection. Furthermore, while no difference in IgA/IgG level was found regarding patient age, IgM level was significantly higher in young adults. The CLIA approach-based detection kit for diagnosing VZV-gE-specific IgA, IgG, and IgM is simple, suitable for high-throughput routine analysis situations, and provides enhanced specificity compared to ELISA.

**Keywords:** varicella–zoster virus (VZV); chemiluminescent immunoassay (CLIA); IgA; IgG; IgM; diagnostic test

**Citation:** Kombe Kombe, A.J.; Xie, J.; Zahid, A.; Ma, H.; Xu, G.; Deng, Y.; Nsole Biteghe, F.A.; Mohammed, A.; Dan, Z.; Yang, Y.; et al. Detection of Circulating VZV-Glycoprotein E-Specific Antibodies by Chemiluminescent Immunoassay (CLIA) for Varicella–Zoster Diagnosis. *Pathogens* **2022**, *11*, 66. https://doi.org/10.3390/ pathogens11010066

Academic Editors: Philipp A. Ilinykh, Kai Huang and Lawrence S. Young

Received: 11 October 2021 Accepted: 23 November 2021 Published: 5 January 2022

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

#### **1. Introduction**

Varicella–zoster virus (VZV), also known as human herpesvirus-3 (HHV-3), belongs to the α-herpesviridae subfamily [1]. It is responsible for varicella disease or chickenpox in children, adolescents, and young adults. The latent viral resurgence years later commonly occurs in older people and causes a secondary infection known as zoster or shingles [1,2].

Although considered among the mild-symptom diseases, VZV-related diseases are highly morbid. The most life-threatening complications include mental development deficit, meningoencephalitis, and post-infectious encephalopathy, in varicella cases. In herpes zoster cases, complications include vasculitis, zoster sine herpete, and post-herpetic neuralgia. A lack of early diagnosis results in treatment delays, which usually leads to fatalities, especially in newborns, elders, organ transplant recipients, and immunocompromised people experiencing disseminated herpes zoster [3–8].

Nowadays, VZV vaccination has led to a significant decrease in the incidence of varicella, particularly in countries where vaccination programs have been implemented and well followed [9–11]. Consequently, this has decreased hospitalization and remarkably reduced the routine biological diagnoses in laboratories [12,13]. In these countries, VZV immunodiagnostic tests assessing IgA, IgG, and/or IgM have been only recommended in pregnant women, critically ill patients before organ transplant surgery, and immunocompromised people, post-vaccinated people, and hospital practitioners [14,15]. However, many reports hypothesized that the implementation of varicella vaccines would be followed by an increase in post-vaccine varicella or herpes zoster cases [2,9,10,16–18], suggesting the need for using antibody detection tests in routine diagnoses for global epidemiologic surveillance, along with herpes zoster vaccination [17]. For instance, in many other countries, such as China [18–20] and Norway [21], where anti-VZV vaccines are not yet implemented or where VZV vaccination coverage is uneven, rapid case identifications are crucial [19,21,22]. Reporting varicella cases in these countries, especially in high-frequented public areas such as schools, institutions, healthcare centers, hospitals, etc., would prevent rapid infection spread to people at risk (pregnant women, immunocompromised). Consequently, this will prevent them from progressing toward infection complication stages and facilitate outbreak control, as diagnosis delays are often fatal.

While the diagnosis of VZV infection is needed in both countries with well-established and non-implemented VZV-vaccine programs, routine biological diagnostics has become challenging, as many currently available diagnostic tests have low sensitivity/specificity [9,13,23]. The few highly sensitive immune diagnostic tests are scarce in the market or not evenly available worldwide [13]. Practically, in the past decade, several biological diagnostic tests with variable sensitivities have been developed to detect VZV-specific IgA, IgG, or IgM, or polyclonal antibodies. Most of them, including direct fluorescent antibody (DFA) and Tzanck smear diagnostic kits based on immunofluorescent assay (IFA), yield in low to moderate antibody detection sensitivity, around 60–80%, and 42–90%, respectively [24–26]. The VZV detection using virus culture assays resulted in high toxicities and contaminations, biasing the diagnostic results, as yielded mainly in false-negative (46% of sensitivity) [25]. In addition, these immunodiagnostic approaches are generally labor-intensive and time consuming, requiring meticulous specimen collection and highly trained technicians [25]. Those with high detection sensitivities and specificities (around 97.8% and 96.8%, respectively), including glycoprotein-based enzyme-linked immunosorbent assay (gpELISA) or varicella zoster glycoprotein IgG enzyme immunoassay with a reference time-resolved fluorescence immunoassay (VZV TRFIA), VZV IgG glycoprotein assay (Merck gpEIA) for the detection of serum VZV IgG, are not evenly and/or commercially available worldwide [27].

Although molecular diagnostics, including viral isolation from vesicular fluid cultures and swab samples, and nucleic acid detection (by PCR), are the most sensitive in VZV diagnostic [24,28], antibody assessment is needed in VZV epidemiological surveillance [11,12] and vaccination effectiveness control [9]. For instance, vesicular rashes do not always appear during infection, compromising PCR results [29,30]. Moreover, the main diagnostic approach based on clinical presentation is not 100% reliable, since many

other herpetic diseases, including HSV, present similarly. Therefore, there is still a need for widely commercially distributed tests with high sensitivity and specificity for routine VZV diagnostics.

Several studies support that the chemiluminescent immunoassay (CLIA) approach for diagnosing serum/plasma viral antigen-specific IgA, IgG, or IgM has better diagnostic performance among the known immunoassays, including manual ELISA [31–33]. Manual ELISA is a labor-intensive multiple-wash-based assay; therefore, it is not suitable for high-throughput screening situations. Moreover, ELISA hardly detects weak antibody–antigen interactions and results in high background, compromising the test sensitivity. Interestingly, in the context of routine diagnostic of SARS-CoV-2, we and others [34,35] have developed CLIA-based diagnostic methods currently commercially available and with satisfying added values compared to ELISA [35] for diagnosing and monitoring COVID-19.

Therefore, we developed and validated a highly sensitive and specific diagnostic kit based on the CLIA approach for diagnosing VZV infections.

The developed CLIA-based VZV diagnostic approach demonstrated improved diagnostic accuracy, as it could detect very low IgA, IgG, and IgM titer in patients at the early stage of the VZV infection. Moreover, as the diagnosis process is automated, timesaving, and suitable for high-throughput situations, it can be used for routine diagnoses of VZV infections.
