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Immunoglobulin M seroneutralization for improved confirmation of Japanese encephalitis virus infection in a flavivirus-endemic area

Immunoglobulin M seroneutralization for improved confirmation of Japanese encephalitis virus... Trans R Soc Trop Med Hyg 2022; 116 : 1032–1042 https://doi.org/10.1093/trstmh/trac036 Advance Access publication 18 May 2022 Immunoglobulin M seroneutralization for improved confirmation of Japanese encephalitis virus infection in a flavivirus-endemic area a , b , ∗ c c b b Tehmina Bharucha , Nazli Ayhan , Boris Pastorino , Sayaphet Rattanavong , Manivanh Vongsouvath , b , d , e b b b Mayfong Mayxay , Anisone Changthongthip , Onanong Sengvilaipaseuth , Ooyanong Phonemixay , f , g , h , i j a b , e c Jean-David Pommier , Christopher Gorman , Nicole Zitzmann , Paul N. Newton , Xavier de Lamballerie , b , c , e and Audrey Dubot-Pérès a b Department of Biochemistry, University of Oxford, Oxford, UK ; Lao-Oxford-Mahosot Hospital-Wellcome Trust-Research Unit, Microbiology Laboratory, Mahosot Hospital, Vientiane, Lao PDR ; Unité des Virus Émergents, Aix-Marseille Univ-IRD 190-Inserm 1207, Marseille, France ; Institute of Research and Education Development, University of Health Sciences, Ministry of Health, Vientiane, Lao e f PDR ; Centre for Tropical Medicine and Global Health, Nuffield Department of Medicine, University of Oxford, Oxford, UK; Epidemiology and Public Health Unit, Institut Pasteur du Cambodge, Phnom Penh, Cambodia; Institut Pasteur, Biology of Infection Unit, Paris, h i j France; Inserm U1117, Paris, France; Intensive Care Department, University Hospital of Guadeloupe, France; Virology Unit, Institut Pasteur du Cambodge, Phnom Penh, Cambodia Corresponding author: Tel: + 01865275723; E-mail: t.bharucha@doctors.org.uk Received 26 November 2021; revised 21 January 2022; editorial decision 21 February 2022; accepted 28 March 2022 Background: The mainstay of diagnostic confirmation of acute Japanese encephalitis (JE) involves detection of anti-JE virus (JEV) immunoglobulin M (IgM) by enzyme-linked immunosorbent assay (ELISA). Limitations in the specificity of this test are increasingly apparent with the introduction of JEV vaccinations and the endemicity of other cross-reactive flaviviruses. Virus neutralization testing (VNT) is considered the gold standard, but it is challenging to implement and interpret. We performed a pilot study to assess IgG depletion prior to VNT for detection of anti-JEV IgM neutralizing antibodies (IgM-VNT) as compared with standard VNT. Methods: We evaluated IgM-VNT in paired sera from anti-JEV IgM ELISA-positive patients (JE n = 35) and negative controls of healthy flavivirus-naïve (n = 10) as well as confirmed dengue (n = 12) and Zika virus (n = 4) patient sera. IgM-VNT was subsequently performed on single sera from additional JE patients (n = 76). Results: Anti-JEV IgG was detectable in admission serum of 58% of JE patients. The positive, negative and over- all percentage agreement of IgM-VNT as compared with standard VNT was 100%. A total of 12/14 (86%) patient samples were unclassified by VNT and, with sufficient sample available for IgG depletion and IgG ELISA confirm- ing depletion, were classified by IgM-VNT. IgM-VNT enabled JE case classification in 72/76 (95%) patients for whom only a single sample was available. Conclusions: The novel approach has been readily adapted for high-throughput testing of single patient samples and it holds promise for incorporation into algorithms for use in reference centres. Keywords: diagnostics, flavivirus, Laos, neglected tropical disease, neurological infection, seroneutralization is brief and low in humans and JEV RNA is rarely detected. Introduction Correspondingly, serological methods are the mainstay of Progress has been made in the implementation of vaccination diagnostic confirmation. The World Health Organization (WHO)- programmes for Japanese encephalitis virus (JEV) in endemic recommended test is the anti-JEV immunoglobulin M (IgM) 1 –3 areas. Nonetheless, gaps remain in understanding the epi- capture enzyme-linked immunosorbent assay (JEV MAC-ELISA) 2 , 4 demiology of the disease. Incorporation of JEV immunization to be performed and interpreted alongside an anti-dengue virus in routine schedules and coverage remain suboptimal and there 11 (DENV) MAC-ELISA. The availability of commercial kits has facil- is inadequate surveillance to identify vaccine failure and JEV geo- itated widespread use of the JEV MAC-ELISA as the standard test. 2 , 5 –8 graphical expansion. However, in line with other flaviviruses, there are increasingly Detection of JEV nucleic acid is highly specific and pro- 12 –15 recognized problems with specificity. For this reason, the 7 , 9 vides additional molecular information. However, viraemia © The Author(s) 2022. Published by Oxford University Press on behalf of Royal Society of Tropical Medicine and Hygiene. This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( https:// creativecommons.org/ licenses/ by/ 4.0/ ), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. ORIGINAL ARTICLE Transactions of the Royal Society of Tropical Medicine and Hygiene all samples) Figure 1. Summary of the suspected JE patient samples tested. Centers for Disease Control and Prevention (CDC) recommends test (PRNT), however, laboratories are increasingly adopting that positive results obtained through JEV MAC-ELISA undergo high-throughput 96-well formats with comparable results. The confirmation by neutralizing antibody (NAb) testing. high VNT requirements limit implementation: testing involves Gold-standard serological confirmation of JEV infection relatively large ( > 150 µL) sample volumes, the need for paired involves assessment of NAb titres using a virus neutralization samples, biosafety 3 category laboratories, reference virus and 13 , 17 test (VNT). This is more specific than the JEV MAC-ELISA. Con- cell strains and technical expertise. Indeed, interpreting VNT ventional VNT methods involve a plaque reduction neutralisation results is challenging due to cross-reactivity that is attributable 1033 T. Bharucha et al. to anamnestic responses related to immunological reactions the section on suspected JE patients above), confirmed by IgM against a previously encountered flavivirus. As there are specific and/or NS1 ELISA and negative for anti-JEV IgM. All procedures major overlaps in the distribution of JEV and other flaviviruses, relating to the conduct, evaluation and documentation of contemporaneous VNT for other endemic flaviviruses is required. the study have been conceived in agreement with the good In Asia, this involves testing for DENV serotypes 1–4, Zika virus clinical practices and ethical principles of the Helsinki Dec- (ZIKV) and, in some areas, West Nile virus (WNV). All of these laration. Written informed consent was obtained from all viruses can manifest as neurological complications. subjects included in the study. All data and samples were Multiple methods have been attempted to mitigate cross- anonymised. reactivity and anamnestic response interference in serological 22 –31 testing for non-JEV flaviviruses. These include analysis of IgA, 25 22 , 32 –35 Anti-JEV IgG ELISA IgG subclasses, antibody avidity, incorporation of block- 34 , 36 ing agents and production of specific monoclonal antibodies Anti-JEV IgG was detected using the Euroimmun ELISA kit 37 –41 for identification of specific viral epitopes. A modification of (Lübeck, Germany) according to manufacturer’s instructions. A VNT, involving prior depletion of IgG, has been successfully per- standard curve using three calibration samples was used to cal- 19 42 , 43 formed for ZIKV and DENV infections. The underlying prin- culate the concentration of antibodies in relative units (RU)/mL ciple is that long-lasting IgG responses from vaccination and pre- for each sample using optical density results; < 16 RU/mL was vious infection are major contributors to non-specific VNT results. negative, ≥16–< 22 RU/mL was equivocal and ≥22 RU/mL was IgG removal results in detection of specific neutralizing IgM anti- positive. bodies, which are markers of acute infection. We performed a pilot study to evaluate the utility of IgG deple- tion prior to VNT (IgM-VNT) to detect anti-JEV IgM neutralizing IgG depletion antibody for confirming acute JEV infection. IgG depletion was performed using Protein G HP SpinTrap/Ab Spin Trap columns (28-4083-47; Cytiva, Marlborough, MA, USA). These contain recombinant protein G, a protein present in group G Strep- tococcus with high affinity for IgG. An in-house method devel- Methods oped by the French National Centre for Arboviruses was used, Patient samples substituting commercial binding buffer by phosphate-buffered saline (PBS). Two IgG depletion columns were used for 100– A prospective study of central nervous system (CNS) infec- 150 µL sample serum. Columns were inverted three times and tions has been conducted at Mahosot Hospital, Vientiane, Laos, briefly vortexed. Each column was inserted in a 2-mL tube and since 2003. Methods and results from 2003 to 2011 have been 44 centrifuged. All centrifugation steps were performed at 500 g described. Patients from 2014 to 2017 were included in the 45 for 2 min. The subsequent eluate was discarded, 600 µL of PBS Southeast Asia Encephalitis Project. The laboratory also receives added to each column and centrifuged again. Columns were samples from patients from other hospitals around Vientiane transferred to clean 2-mL tubes and 100–150 µL of sample was City (i.e. Friendship, Children’s and Setthathirat Hospitals). Writ- added to one column and incubated at room temperature for ten informed consent was obtained from patients or responsi- 4 min before centrifugation. The eluate was transferred to the ble guardians. Anti-JEV and anti-DENV IgM were detected by the second column, incubated at room temperature for 4 min and Japanese encephalitis/dengue IgM combo ELISA (Panbio, Bris- centrifuged again. The final eluate was stored at −20°C until the bane, QLD, Australia; now Alere) until July 2014, for which result VNT. interpretation included a ratio between DENV and JEV. After August 2014, as per WHO recommendations, the JEV IgM ELISA (Inbios, Seattle, WA, USA) was utilized. All samples used were VNT aliquoted and stored at −80°C. This pilot study involved a con- venience sample of consecutive patients with available speci- Two-fold dilutions from 1/20 to 1/2560 of each serum sam- mens to be tested; hence a sample size calculation was not ple were tested in duplicate by VNT for JEV, DENV1–4, ZIKV performed. and WNV. Serum dilutions from 1/10 to 1/1280 were pre- Suspected JE patients included in this study had anti-JEV IgM pared and mixed in a 1:1 ratio with 100 TCID50 viral sus- detected by MAC-ELISA in cerebrospinal fluid (CSF) or serocon- pension (Table 1 ) using epMotion 5075 (Eppendorf, Hamburg, version between acute and follow-up serum, no other pathogen Germany) in a 96-well microplate (Figure S1). Negative con- detected in any body fluid and a sufficient volume of acute trols containing minimum essential medium (MEM), with or and/or follow-up serum for VNT. Patients with DENV and JEV RNA without serum, were included in each microplate. Plates were or DENV non-structural protein 1 (NS1) in serum or CSF were incubated at 37°C for 2 h. A 100-µL suspension of Vero cells excluded. (ATCC CCL-81) containing approximately 2 ×10 cells/mL, was Negative controls included samples from three groups: added to each well using the epMotion 5070 (Eppendorf) and healthy flavivirus-naïve blood donors living in Puy-de-Dôme, in incubated at 37°C in a 5% carbon dioxide incubator. After 5– central France; ZIKV VNT-confirmed sera collected in Peru in 7 d, microplates were read under an inverted microscope. Two the framework of a seroprevalence study ; and DENV infec- investigators read the results for each replicate to identify the tion patients from the Laos CNS study (study details reported in end dilution at which there was no cytopathic effect, with a 1034 Transactions of the Royal Society of Tropical Medicine and Hygiene Table 1. Virus strain used in VNTs Virus Strain Country of isolation GenBank number EVAg number Titre (TCID /mL) Day read JEV Laos 2009 Laos KC196115 001V-02217 2 ×10 5 WNV UVE/WNV/2008/US/R94224 USA – 001V-02224 2.1 ×10 5 ZIKV ZIKV strain H/PF/2013 French French Polynesia KJ776791 – 3.7 ×10 5 Polynesia DENV-1 DENV1 2012 Saint Vincent and VC16692 001V-02335 3.1 ×10 7 the Grenadines DENV-2 UVE/DENV-2/1998/MQ/703 Martinique AF208496 – 6.7 ×10 5 DENV-3 UVE/DENV-3/2001/MQ/2023 Martinique AH011666 – 4.5 ×10 6 DENV-4 UVE/DENV-4/1998/ID/814 Indonesia – – 3 ×10 6 EVAg: European Virus Archive – GLOBAL; TCID : 50% tissue culture infective dose. third investigator to resolve disagreement. For duplicates, the compatible; and 10 (29%) as unknown (Table 2 and Table S2). geometric mean of end dilutions was calculated and reported as Eighteen of these patients had sufficient serum available for IgM- 47 , 48 an NAb titre and ≥40 was considered as positive. Suspected VNT in at least one sample. The results enabled reclassification JE patients were categorized as acute JE positive, confirmed or through the removal of cross-reactive IgG to other viruses and compatible, JE negative and unknown, according to the criteria in the specific detection of anti-JEV IgM, such that 17 (94%) were Figure 2 . classified as JE positive, 16 (89%) confirmed, 1 (6%) compatible; and 1 (6%) as JE negative. Five patients classified as unknown by VNT did not have sufficient acute and/or follow-up sample to perform IgG depletion and/or anti-JEV IgG ELISA testing. Results For the subset of 32 patients classified as JE positive, con- firmed or compatible (before or after depletion), the median From 2003 to March 2021, 264 patients with suspected CNS infec- duration of onset of illness was 5 d (IQR 4–7) and the median tion were positive for anti-JEV IgM (in CSF or with seroconversion) duration between paired serum samples was 14 d (IQR 11–24). and negative for other screened aetiologies (see Figure 1 ). A total of 17/24 (71%) of these patients had detectable anti-JEV Paired serum samples (admission and follow-up) were avail- IgG in the admission serum before IgG depletion and 23/24 [96%] able for 35 patients and a single acute sample for 98 patients. had detectable anti-JEV IgG in the follow-up sample. Among these 133 included patients, 130 (98%) had anti-JEV IgM detected in CSF and 3 (2%) demonstrated IgM seroconversion Negative control sera only (no anti-JEV IgM in CSF) in paired sera. The median age of the patients was 11 y (interquartile range [IQR] 6–20) and IgM-VNT was performed on three other groups of negative con- 32% (43/133) were female. The median duration of illness on trol sera to assess the specificity of the novel method. JEV NAb admission was 5 d (IQR 4–6) and the median time between was not detected by IgM-VNT or VNT in the healthy flavivirus- admission and follow-up serum collection was 14 d (IQR 10–25). naïve blood donors (n = 10) or ZIKV infection sera (n = 4) (see Table S3). In the DENV patient sera, 2/12 (17%) did not have detectable JEV NAb, and for both of these patients, IgM-VNT IgG depletion was performed and was also negative. In the 10/12 (83%) A total of 102/156 (65%) serum samples, including 72/125 (58%) patients with DENV infection with JEV NAb detected by VNT, admission sera and 30/31 (97%) follow-up sera, were anti-JEV 8/10 (80%) did not have detectable JEV NAb after IgG deple- IgG positive by ELISA before IgG depletion. Seventy samples had tion. For the remaining two, one did not have a result for IgM- sufficient volumes to be tested for anti-JEV IgG by ELISA after VNT and the other showed negative JEV VNT for admission IgG depletion. Fifty-nine (84%) were negative or equivocal after serum and a low JEV NAb titre of 40 in follow-up serum. There IgG depletion. Six samples were equivocal before IgG depletion were not sufficient sample volumes available to perform DENV and all of these were negative after IgG depletion. Samples that VNT. remained positive after IgG depletion demonstrated decrease in the titre, however, the starting anti-JEV IgG result in these cases was high, all > 125 RU/mL (positive > 22 RU). Positive, negative and overall percentage agreement The IgM-VNT was compared with the reference standard VNT. This was based on results for patients classified as JE positive VNT for the patients with paired serum samples or negative by standard VNT and with sufficient sera to com- VNT results prior to IgG depletion enabled classification of 25/35 plete IgM-VNT, i.e. VNT performed after IgG depletion and IgG (71%) patients as JE positive, 7 (20%) confirmed, 18 (51%) ELISA to confirm IgG depletion. This included 14 JE-positive and 1035 T. Bharucha et al. Figure 2. Criteria for interpretation of the results and patient categorisation for JE status. 1036 Transactions of the Royal Society of Tropical Medicine and Hygiene Table 2. VNT antibody titre in acute and follow-up serum samples for patients with positive anti-JEV IgM capture ELISA Before IgG depletion (standard VNT) After IgG depletion (IgM-VNT) NAb titre NAb titre Patient Sample Days of number type illness Class JEV IgG JEV D1 D2 D3 D4 ZIK WN Class JEV IgG JEV D1 D2 D3 D4 ZIK WN a b b b b b b 1597 Adm 5 Conf − 1280 Neg Neg Neg Neg Conf − 160 Neg Neg Neg Neg Neg b b b b b FU 59 + 2560 14 14 Neg 20 640 Neg Neg Neg Neg b b b b b b b 1704 Adm 5 Conf + 640 20 Neg 20 28 Conf − 640 Neg Neg Neg Neg Neg Neg b b b b b b b FU 13 + 2560 40 Neg 28 80 2560 20 Neg Neg Neg Neg Neg b b b b b b b 829 Adm 4 Conf − 1280 Neg Neg Neg Neg Neg 14 Conf − 640 Neg Neg Neg Neg Neg Neg FU 21 + 2560 Neg Neg Neg 14 Neg 908 Adm 4 Conf + 40 160 160 40 113 Neg Conf b b b b b FU 14 + 2560 20 Neg Neg 14 − 2560 Neg Neg Neg Neg Neg b b b b b b b 928 Adm 5 Conf − 1810 20 Neg Neg Neg Neg 56 Conf − 2560 Neg Neg Neg Neg Neg Neg b b b b b b FU 44 + 2560 Neg Neg Neg 14 − 2560 Neg Neg Neg Neg Neg Neg 2078 Adm 7 Conf Eq 160 20 Neg Neg Neg Conf − FU 17 + 2560 Neg Neg 20 20 − ≥2560 Neg Neg Neg Neg 101 Adm 4 Conf − 2560 Neg Neg Neg Neg Conf − 453 Neg Neg Neg Neg FU 6 − 2560 Neg Neg 40 Neg − ≥2560 Neg Neg Neg Neg b b b b b b b 1610 Adm 6 Comp + 2560 14 Neg Neg 20 Conf − 1280 Neg Neg Neg Neg Neg Neg FU 40 + 2560 160 20 Neg 113 2560 Neg Neg Neg Neg Neg Neg b b b b b b b 483 Adm 7 Comp + 2560 Neg Neg Neg Neg Conf − 2560 Neg Neg Neg Neg Neg Neg b b b b b b b FU 21 + 2560 20 20 Neg 20 Neg Neg 2560 Neg Neg Neg Neg Neg Neg b b b b b b b 884 Adm 5 Comp + 2560 40 Neg Neg 40 Neg 40 Conf − 1280 Neg Neg Neg Neg Neg Neg b b b b b b b FU 19 + 2560 Neg Neg Neg 20 − 2560 Neg Neg Neg Neg Neg Neg 1074 Adm 6 Comp Eq 452 Neg Neg Neg Conf b b b b b b FU 84 + 1810 40 40 Neg 80 − 1280 Neg Neg Neg Neg Neg b b b b b b 1180 Adm 7 Comp + 1280 452 640 452 160 Neg Conf − 1280 Neg Neg Neg Neg Neg FU 13 + 2560 226 226 160 160 20 80 2053 Adm 5 Comp + 2560 20 Neg Neg Neg Conf − 2560 Neg Neg Neg Neg FU 18 + 1280 80 Neg 40 Neg − 775 Adm 3 Comp − 320 Neg Neg Neg Comp − 113 Neg Neg Neg FU 12 + 640 Neg Neg 40 640 Neg Neg Neg b b b b b 5149 Adm 1 Unkn + 2560 2560 2560 2560 2560 Conf 1280 20 20 20 Neg b b b b b b FU 28 + 2560 2560 2560 2560 2560 − 2560 40 20 40 20 Neg b b b b b b 1056 Adm 3 Unkn + 320 1810 320 320 320 28 40 Conf − 640 20 Neg Neg Neg Neg FU 28 + 2560 1917 Adm 7 Unkn + 2560 2560 2560 Neg 453 Conf − 2560 Neg Neg Neg Neg FU 12 + 1280 2560 2560 Neg 320 − 1280 Neg Neg Neg Neg b b b b b b 1036 Adm 4 Unkn + 80 2560 226 320 226 Neg 20 Neg − Neg 80 Neg 20 Neg Neg FU 16 + 640 2560 1280 2560 Adm: serum on admission; FU: serum at follow-up; NAb: NAb assessed by VNT, geometric mean calculated from duplicate results, = indeter- minate, NAb titre underlined to indicate the maximum dilution tested, neg: no NAb detected in duplicate samples (observation of cytopathic effect) for all serum dilutions tested (lowest = 20); NAb titre ≥40 considered as positive; D1–4: dengue virus 1–4; ZIK: Zika virus; WN: West Nile virus; class: classification for JE status according to criteria in Table 2 ; Conf: confirmed; Comp: compatible; Unkn: unknown; JEV IgG: anti-JEG IgG detection by ELISA (Euroimmun); + : positive; Eq: equivocal; −: negative. JEV IgG negative before depletion. Only one replicate tested or interpretable, the other samples were tested in duplicate. 16 JE-negative patients. Positive, negative and overall percentage Results allowed classification for 72/76 (95%) patients: 70 (92%) agreements (PPA, NPA and OPA, respectively) were all 100% (see JE, 63 (83%) confirmed and 3 (4%) compatible, and 6 (8%) neg- Table 3 ). ative. Four (5%) were unknown (Table 4 ). VNT after IgG depletion for patients with single acute Discussion serum A total of 76/98 (78%) patient samples had sufficient volumes This pilot study included a large set of well-characterized patients for IgG depletion, confirmatory IgG ELISA testing and IgM-VNT. recruited prospectively in clinical studies, with extensive VNT for 1037 T. Bharucha et al. gradient centrifugation of whole serum and 2-mercaptoethanol Table 3. 2 ×2 table of the results of IgM-VNT as compared with treatment. The improved discrimination of evidence for acute JE standard VNT in patient samples gave rise to further work developing the widely 50 , 51 used anti-JEV IgM ELISA. However, with evidence suggesting suboptimal performance of MAC-ELISA, the increasing use of Reference test (standard VNT) the JEV vaccine, as well as hyperendemicity of DENV serotypes, a a IgM-VNT JE positive , n JE negative , n Total, n the requirement for accurate diagnostic confirmation becomes even more pertinent. Although the performance of contempora- JE positive 14 0 14 neous anti-DENV IgM ELISA and calculation of a JEV:DENV IgM JE negative 0 16 16 ratio has improved specificity, the combination of VNT and IgG Total 14 16 30 depletion (IgM-VNT) permits IgM detection with higher specificity The classification of patients followed the criteria set out in than by using MAC-ELISA alone. Figure 2 . Calvert et al. showed that IgG depletion prior to neutral- ization testing considerably improved (15% before to 77% after IgG depletion) the differentiation of acute Zika from dengue JEV, DENV 1–4, ZIKV and WNV. We show that the implementa- viral infections. This has also been demonstrated for DENV infec- 42 , 43 tion of IgG depletion prior to VNT performed on par with stan- tions. It is notable that as JE is predominantly a neurological dard VNT (100% PPA, NPA and OPA) and also resulted in a sig- infection, and the natural history of the immunological response nificantly higher proportion, compared with standard VNT, of is different to flavivirus infections presenting as acute febrile syn- patients being classified. Of the patients with paired sera tested to dromes, by the time of clinical presentation, anti-JEV IgM and IgG confirm acute JEV infection, 74% (26/35) were classified without is detectable in a larger proportion of patients. Therefore use of an IgG depletion step, in contrast to 100% when IgG depletion the IgM-VNT method for JE confirmation is a logical approach. was included. Furthermore, IgG depletion improved the diagnos- The humoral responses to JEV infection are directed mainly tic confidence of patients classed as JE positive, from 7/26 (27%) against antigenic epitopes on the viral envelope protein. There is confirmed as opposed to 19/26 (73%) compatible with standard major cross-reactivity with other endemic circulating flaviviruses 52 , 53 VNT to 16/17 (94%) confirmed as opposed to 1/17 (6%) compat- and therefore it was crucial to test for all DENV serotypes, 54 44 ible with IgM-VNT. Depleting IgG also enabled a diagnosis of JE ZIKV and WNV where they are sympatric. Likewise, IgG deple- in 95% of patients for whom only a single sample was available, tion and seroneutralization might play a role in the diagnosis of allowing for specific neutralization of the IgM remaining in the DENV neurological infections for which there is considerable diag- sample. nostic uncertainty. The high proportion of patients presenting with detectable We acknowledge that a diagnostic accuracy study should anti-JEV IgG before depletion and a reduction in DENV neutral- ideally be performed with an a priori sample size calculation, ization titres after depletion strengthen the underlying premise of prospectively testing consecutive patients with suspected neu- this study, that IgG complicates discrimination by VNT, especially rological infection by the reference standard VNT to ascertain in areas with high endemicity of other flaviviruses and increasing JE-positive and negative patient samples. However, we were utilization of JEV vaccination. unable to conduct this in this pilot study and flavivirus-naïve A limitation is that there were not sufficient sample volumes patients from France were included as an additional category available to perform standard and IgM-VNT in all samples. How- of negative controls. That patients already had anti-JEV IgM ever, the testing was retrospectively performed on a relatively detected in CSF or experienced JEV seroconversion reflects the large number of very precious samples. It would be realistic in role of VNT within reference centres. Further limitations include clinical practice to secure the serum volume (400 μL) needed for missing data due to limited sample volumes and that dilutions prospective IgM-VNT testing. This is one of the advantages of the were 1/20 to 1/2560 for the sera. Ideally serum should be tested new technique, that it relies on a single serum sample rather than to the end point of dilution. IgM-VNT is a diagnostic test suited for paired sera or CSF. The efficiency of the IgG depletion was eval- reference centres and optimization will be required to adapt the uated using anti-JEV IgG ELISA. We found that 84% of the anti- technique to be high throughput, using protein G slurry and an JEV IgG ELISA-positive sera became negative after IgG depletion. automatized format for VNT testing of 1/20 to 1/5120. Addition- All samples with an anti-JEV IgG ELISA result < 125 RU/mL were ally, not all the virus strains used were sourced from the countries negative after IgG depletion, suggesting IgG depletion was prob- where the samples were derived; the DENV strains isolated from ably incomplete in samples with high titres. Further optimiza- Laos did not provide a sufficient cytopathic effect for the assay tion is required to ensure that depletion is fully effective, perhaps and neither ZIKV nor WNV have been isolated from patients in with alternative methods depending on the initial anti-JEV IgG Laos. result, such as the use of three rather than two IgG depletion In conclusion, measurement of anti-JEV IgG and the per- columns. formance of IgM-VNT significantly improved performance and The principle of removing IgG and the use of IgM as a allowed the use of a single serum sample instead of paired sera biomarker for confirming acute infection is by no means novel. In for JE confirmation. This innovation holds promise for wider incor- 1973, Edelman and Pariyanonda reported a modified haemag- poration into testing algorithms in the reference confirmation of glutination inhibition involving depletion of IgG by sucrose density JE and DENV neurological infections. 1038 Transactions of the Royal Society of Tropical Medicine and Hygiene Table 4. VNT antibody titre for patients with only a single acute serum sample After IgG depletion (IgM-VNT) NAb titre Days of Before IgG Patient number illness Class depletion, JEV IgG JEV IgG JEV D1 D2 D3 D4 ZIK WN 34 Conf − − 160 Neg Neg Neg Neg Neg Neg 37 3 Conf − − 640 Neg Neg Neg Neg Neg Neg 38 2 Conf − − 57 Neg Neg Neg Neg Neg 40 14 Conf − − 80 Neg Neg Neg Neg Neg Neg 44 4 Conf − − 57 Neg Neg Neg Neg Neg Neg 47 4 Conf − − 320 Neg Neg Neg Neg Neg Neg 52 4 Conf − − 80 Neg Neg Neg Neg Neg Neg 53 4 Conf − − 57 Neg Neg Neg Neg Neg Neg 59 4 Conf − − 320 Neg Neg Neg Neg Neg Neg 60 1 Conf − − 160 Neg Neg Neg Neg Neg Neg 64 3 Conf − − 80 Neg Neg Neg Neg Neg Neg 57 6 Conf − − 640 Neg Neg Neg Neg Neg Neg 66 8 Conf − − 40 Neg Neg Neg Neg Neg Neg 73 5 Conf − − 1810 Neg Neg Neg Neg Neg Neg 76 5 Conf − − 320 Neg Neg Neg Neg Neg Neg 87 4 Conf − − 57 Neg Neg Neg Neg Neg Neg 88 6 Conf − − 160 Neg Neg Neg Neg Neg Neg 92 3 Conf − − 905 Neg Neg Neg Neg Neg Neg 98 Conf − − 320 Neg Neg 14 Neg Neg Neg 101 Conf − − 1280 Neg Neg Neg Neg Neg Neg 102 Conf − − 640 Neg Neg 28 Neg Neg Neg 103 Conf − − 320 Neg Neg Neg Neg Neg Neg 104 Conf − − 226 Neg Neg Neg Neg Neg Neg 105 Conf − − 160 Neg Neg Neg 14 Neg Neg 111 Conf − − 452 Neg Neg Neg Neg Neg Neg 112 Conf − − 160 Neg Neg Neg Neg Neg Neg 127 Conf − − 640 Neg Neg Neg Neg Neg 14 118 Conf − − 640 Neg Neg Neg 14 Neg Neg 89 3 Conf − − 160 Neg Neg Neg Neg Neg Neg 97 Conf − − 640 Neg Neg Neg Neg Neg Neg 94 3 Conf − − 640 Neg Neg Neg Neg Neg Neg 110 Conf − − 160 Neg Neg Neg Neg Neg Neg 121 Conf − − 160 14 Neg Neg Neg 54 3 Conf − 57 Neg Neg Neg Neg Neg Neg 128 Conf Eq − 640 Neg Neg Neg Neg Neg Neg 51 5 Conf Eq − 905 Neg Neg Neg Neg Neg Neg 33 Conf Eq − 452 Neg Neg Neg Neg Neg Neg 58 5 Conf Eq − 2560 Neg Neg Neg Neg Neg Neg 62 6 Conf Eq − 226 20 Neg Neg Neg Neg Neg 35 4 Conf + − 160 Neg Neg Neg Neg Neg Neg 36 Conf + − 40 Neg Neg Neg Neg Neg Neg 41 4 Conf + − 320 Neg Neg Neg Neg Neg Neg 65 13 Conf + − 80 14 Neg 14 Neg Neg Neg 67 4 Conf + − 1280 Neg Neg Neg Neg Neg 28 68 6 Conf + − 2560 Neg Neg Neg Neg Neg Neg 69 5 Conf + − 452 Neg Neg Neg Neg Neg Neg 70 6 Conf + − 640 Neg Neg Neg Neg Neg Neg 71 8 Conf + − 640 Neg Neg Neg Neg Neg 14 74 4 Conf + − 226 Neg Neg Neg Neg Neg Neg 1039 T. Bharucha et al. Table 4. Continued After IgG depletion (IgM-VNT) NAb titre Days of Before IgG Patient number illness Class depletion, JEV IgG JEV IgG JEV D1 D2 D3 D4 ZIK WN 75 14 Conf − 905 Neg Neg Neg Neg Neg Neg 79 5 Conf + − 640 Neg Neg Neg Neg Neg Neg 80 7 Conf + − 226 Neg Neg Neg Neg Neg Neg 81 6 Conf + − 640 Neg Neg Neg Neg Neg Neg 82 6 Conf + − 905 Neg Neg Neg Neg Neg Neg 86 Conf + − 2560 Neg Neg Neg Neg Neg 20 91 7 Conf + − 1280 Neg Neg Neg Neg Neg Neg 95 4 Conf + − 320 Neg Neg Neg Neg Neg 14 99 Conf + − 640 40 Neg Neg 20 Neg Neg 108 Conf + − 320 Neg Neg Neg 40 Neg Neg 109 Conf + − 113 Neg Neg Neg Neg Neg Neg 116 Conf + − 113 Neg Neg Neg Neg Neg Neg 122 Conf + − 1280 Neg Neg Neg Neg Neg Neg 125 Conf + − 160 Neg Neg 14 Neg Neg Neg 31 Comp − − 226 Neg Neg Neg 32 Comp − − 226 Neg Neg Neg 45 10 Comp − − 80 Neg Neg Neg 56 6 Unkn + − 80 Neg 40 20 Neg Neg Neg 77 6 Unkn + − 80 Neg 28 Neg Neg Neg Neg 85 Unkn + − 320 98 160 57 20 Neg Neg 78 4 Unkn + − 160 Neg Neg Neg Neg 80 Neg 39 3 Neg − − Neg Neg Neg Neg Neg Neg Neg 83 5 Neg − − Neg Neg Neg Neg Neg Neg Neg 48 3 Neg + − Neg 20 Neg Neg Neg Neg Neg 61 14 Neg + − Neg 14 14 Neg 14 Neg Neg 49 10 Neg + − Neg 14 Neg Neg Neg 120 Neg + − 20 40 Neg 20 14 Neg Neg NAb titre: NAb assessed by VNT, geometric mean calculated from duplicate results. neg: no NAb detected in duplicate samples (observation of cytopathic effect) for all serum dilutions tested (lowest = 20); NAb titre ≥40 considered as positive; D1–4: dengue virus 1–4; ZIK: Zika virus; WN: West Nile virus; class: classification for JE status according to the criteria set out in Figure 2 ; Conf: confirmed; Comp: compatible; Unkn: unknown; JEV IgG: = anti-JEG IgG detection by ELISA (Euroimmun); + : positive; Eq: equivocal; −: negative. Only one replicate tested or interpretable, the other samples were tested in duplicate. JEV IgG negative before depletion. Maximum dilution tested. Test not performed. provided the clinical samples. TB, NA and BP performed the experimental Supplementary data work. TB, NA, ADP, BP, NZ and XDL analysed and interpreted the data. Supplementary data are available at Transactions online. TB wrote the manuscript. All the authors edited successive drafts and approved the final version. Acknowledgements: We are very grateful to the patients and to Boun- Authors’ contributions: TB, ADP and XDL conceived the study. TB, NA, thaphany Bounxouei, the former Director of Mahosot Hospital, the late ADP, BP, XDL and NZ developed the methodology. SR, MV, MM, AC, OS, Rattanaphone Phetsouvanh, Director of the Microbiology Laboratory, and OP, ADP, JDP, CG and PNN designed and conducted the clinical study and the staff of the wards and Microbiology Laboratory of Mahosot Hospi- 1040 Transactions of the Royal Society of Tropical Medicine and Hygiene tal. We also thank Bounnak Saysanasongkham, the former Director of References the Department of Healthcare and Rehabilitation, Ministry of Health, and 1 World Health Organization. Japanese encephalitis vaccines: Bounkong Syhavong, Minister of Health, Lao PDR for their very kind help WHO position paper, February 2015–recommendations. Vaccine. and support. We thank the stakeholders of the SEAe project, members 2016;34(3):302–3. of the Unité des Virus Émergents (Christine Isnard and Camille Placidi) 2 Heffelfinger JD, Li X, Batmunkh N, et al. Japanese encephalitis surveil- and the CNR des Arbovirus (Patrick Gravier, Gilda Grard, Isabelle Leparc- lance and immunization –Asia and Western Pacific regions, 2016. Goffart and Mathilde Galla). We also thank Rodrigo Cachay, Eduardo Got- MMWR Morb Mortal Wkly Rep. 2017;66(22):579–83. tuzo and Humberto Guerra (Instituto de Medicina Tropical Alexander von Humboldt, Universidad Peruana Cayetano Heredia) for providing the Zika 3 Hills SL, Walter EB, Atmar RL, et al. Japanese encephalitis vaccine: virus patient samples. recommendations of the Advisory Committee on Immunization Prac- tices. MMWR Recomm Rep. 2019;68(2):1–33. Collaborators: We are grateful to all the SEAe study researchers, includ- 4 Pearce JC, Learoyd TP, Langendorf BJ, et al. Japanese encephali- ing Philippe Buchy, Em Bunnakea, Julien Cappelle, Mey Channa, Veronique tis: the vectors, ecology and potential for expansion. J Travel Med. 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New strains of Japanese encephali- manam, Viengmon Davong, Patrice Debré, Jean-François Delfraissy, Chris- tis virus circulating in Shanghai, China after a ten-year hiatus in local tian Devaux, Anousone Douangnouvong, Veasna Duong, Benoit Durand, mosquito surveillance. Parasites Vectors. 2019;12:14. Chanreaksmey Eng, Catherine Ferrant, Didier Fontenille, Lukas Hafner, Le 8 Ojha JK, Samantaray K, Mohanty S. Assess the coverage rate of Thanh Hai, Do Thu Huong, Marc Jouan, May July, Magali Lago, Jean- Japanese encephalitis vaccination and factors of non-compliance as Paul Moatti, Bernadette Murgue, Khin Yi Oo, MengHeng Oum, Khansouda- reported by parents of selected areas of Khurdha. Eur J Mol Clin Med. phone Phakhounthong, Anh Tuan Pham, Do Quyen, Malee Seephonelee, 2021;7(11):5049–60. Maud Seguy, Bountoy Sibounheunang, Kanarith Sim, Luong Minh Tan, Cho 9 Do LP, Bui TM, Hasebe F, et al. 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Evidence and rationale for the Aviesan Sud, Institut national de la santé et de la recherche médicale World Health Organization recommended standards for Japanese (Inserm), and innovation programme EVAg (grant agreement 653316). encephalitis surveillance. BMC Infect Dis. 2009;9:214. The Zika virus patient samples were provided by the EC-funded project 12 Dubot-Peres A, Sengvilaipaseuth O, Chanthongthip A, et al. How ZIKAlliance, Grant agreement no. 734548. many patients with anti-JEV IgM in cerebrospinal fluid really have Japanese encephalitis? Lancet Infect Dis. 2015;15(12): Competing interests: None declared. 1376–7. 13 Maeki T, Tajima S, Ikeda M, et al. 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Natl Acad Sci USA. 2017;114(31):8384–9. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Transactions of The Royal Society of Tropical Medicine and Hygiene Oxford University Press

Immunoglobulin M seroneutralization for improved confirmation of Japanese encephalitis virus infection in a flavivirus-endemic area

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Abstract

Trans R Soc Trop Med Hyg 2022; 116 : 1032–1042 https://doi.org/10.1093/trstmh/trac036 Advance Access publication 18 May 2022 Immunoglobulin M seroneutralization for improved confirmation of Japanese encephalitis virus infection in a flavivirus-endemic area a , b , ∗ c c b b Tehmina Bharucha , Nazli Ayhan , Boris Pastorino , Sayaphet Rattanavong , Manivanh Vongsouvath , b , d , e b b b Mayfong Mayxay , Anisone Changthongthip , Onanong Sengvilaipaseuth , Ooyanong Phonemixay , f , g , h , i j a b , e c Jean-David Pommier , Christopher Gorman , Nicole Zitzmann , Paul N. Newton , Xavier de Lamballerie , b , c , e and Audrey Dubot-Pérès a b Department of Biochemistry, University of Oxford, Oxford, UK ; Lao-Oxford-Mahosot Hospital-Wellcome Trust-Research Unit, Microbiology Laboratory, Mahosot Hospital, Vientiane, Lao PDR ; Unité des Virus Émergents, Aix-Marseille Univ-IRD 190-Inserm 1207, Marseille, France ; Institute of Research and Education Development, University of Health Sciences, Ministry of Health, Vientiane, Lao e f PDR ; Centre for Tropical Medicine and Global Health, Nuffield Department of Medicine, University of Oxford, Oxford, UK; Epidemiology and Public Health Unit, Institut Pasteur du Cambodge, Phnom Penh, Cambodia; Institut Pasteur, Biology of Infection Unit, Paris, h i j France; Inserm U1117, Paris, France; Intensive Care Department, University Hospital of Guadeloupe, France; Virology Unit, Institut Pasteur du Cambodge, Phnom Penh, Cambodia Corresponding author: Tel: + 01865275723; E-mail: t.bharucha@doctors.org.uk Received 26 November 2021; revised 21 January 2022; editorial decision 21 February 2022; accepted 28 March 2022 Background: The mainstay of diagnostic confirmation of acute Japanese encephalitis (JE) involves detection of anti-JE virus (JEV) immunoglobulin M (IgM) by enzyme-linked immunosorbent assay (ELISA). Limitations in the specificity of this test are increasingly apparent with the introduction of JEV vaccinations and the endemicity of other cross-reactive flaviviruses. Virus neutralization testing (VNT) is considered the gold standard, but it is challenging to implement and interpret. We performed a pilot study to assess IgG depletion prior to VNT for detection of anti-JEV IgM neutralizing antibodies (IgM-VNT) as compared with standard VNT. Methods: We evaluated IgM-VNT in paired sera from anti-JEV IgM ELISA-positive patients (JE n = 35) and negative controls of healthy flavivirus-naïve (n = 10) as well as confirmed dengue (n = 12) and Zika virus (n = 4) patient sera. IgM-VNT was subsequently performed on single sera from additional JE patients (n = 76). Results: Anti-JEV IgG was detectable in admission serum of 58% of JE patients. The positive, negative and over- all percentage agreement of IgM-VNT as compared with standard VNT was 100%. A total of 12/14 (86%) patient samples were unclassified by VNT and, with sufficient sample available for IgG depletion and IgG ELISA confirm- ing depletion, were classified by IgM-VNT. IgM-VNT enabled JE case classification in 72/76 (95%) patients for whom only a single sample was available. Conclusions: The novel approach has been readily adapted for high-throughput testing of single patient samples and it holds promise for incorporation into algorithms for use in reference centres. Keywords: diagnostics, flavivirus, Laos, neglected tropical disease, neurological infection, seroneutralization is brief and low in humans and JEV RNA is rarely detected. Introduction Correspondingly, serological methods are the mainstay of Progress has been made in the implementation of vaccination diagnostic confirmation. The World Health Organization (WHO)- programmes for Japanese encephalitis virus (JEV) in endemic recommended test is the anti-JEV immunoglobulin M (IgM) 1 –3 areas. Nonetheless, gaps remain in understanding the epi- capture enzyme-linked immunosorbent assay (JEV MAC-ELISA) 2 , 4 demiology of the disease. Incorporation of JEV immunization to be performed and interpreted alongside an anti-dengue virus in routine schedules and coverage remain suboptimal and there 11 (DENV) MAC-ELISA. The availability of commercial kits has facil- is inadequate surveillance to identify vaccine failure and JEV geo- itated widespread use of the JEV MAC-ELISA as the standard test. 2 , 5 –8 graphical expansion. However, in line with other flaviviruses, there are increasingly Detection of JEV nucleic acid is highly specific and pro- 12 –15 recognized problems with specificity. For this reason, the 7 , 9 vides additional molecular information. However, viraemia © The Author(s) 2022. Published by Oxford University Press on behalf of Royal Society of Tropical Medicine and Hygiene. This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( https:// creativecommons.org/ licenses/ by/ 4.0/ ), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. ORIGINAL ARTICLE Transactions of the Royal Society of Tropical Medicine and Hygiene all samples) Figure 1. Summary of the suspected JE patient samples tested. Centers for Disease Control and Prevention (CDC) recommends test (PRNT), however, laboratories are increasingly adopting that positive results obtained through JEV MAC-ELISA undergo high-throughput 96-well formats with comparable results. The confirmation by neutralizing antibody (NAb) testing. high VNT requirements limit implementation: testing involves Gold-standard serological confirmation of JEV infection relatively large ( > 150 µL) sample volumes, the need for paired involves assessment of NAb titres using a virus neutralization samples, biosafety 3 category laboratories, reference virus and 13 , 17 test (VNT). This is more specific than the JEV MAC-ELISA. Con- cell strains and technical expertise. Indeed, interpreting VNT ventional VNT methods involve a plaque reduction neutralisation results is challenging due to cross-reactivity that is attributable 1033 T. Bharucha et al. to anamnestic responses related to immunological reactions the section on suspected JE patients above), confirmed by IgM against a previously encountered flavivirus. As there are specific and/or NS1 ELISA and negative for anti-JEV IgM. All procedures major overlaps in the distribution of JEV and other flaviviruses, relating to the conduct, evaluation and documentation of contemporaneous VNT for other endemic flaviviruses is required. the study have been conceived in agreement with the good In Asia, this involves testing for DENV serotypes 1–4, Zika virus clinical practices and ethical principles of the Helsinki Dec- (ZIKV) and, in some areas, West Nile virus (WNV). All of these laration. Written informed consent was obtained from all viruses can manifest as neurological complications. subjects included in the study. All data and samples were Multiple methods have been attempted to mitigate cross- anonymised. reactivity and anamnestic response interference in serological 22 –31 testing for non-JEV flaviviruses. These include analysis of IgA, 25 22 , 32 –35 Anti-JEV IgG ELISA IgG subclasses, antibody avidity, incorporation of block- 34 , 36 ing agents and production of specific monoclonal antibodies Anti-JEV IgG was detected using the Euroimmun ELISA kit 37 –41 for identification of specific viral epitopes. A modification of (Lübeck, Germany) according to manufacturer’s instructions. A VNT, involving prior depletion of IgG, has been successfully per- standard curve using three calibration samples was used to cal- 19 42 , 43 formed for ZIKV and DENV infections. The underlying prin- culate the concentration of antibodies in relative units (RU)/mL ciple is that long-lasting IgG responses from vaccination and pre- for each sample using optical density results; < 16 RU/mL was vious infection are major contributors to non-specific VNT results. negative, ≥16–< 22 RU/mL was equivocal and ≥22 RU/mL was IgG removal results in detection of specific neutralizing IgM anti- positive. bodies, which are markers of acute infection. We performed a pilot study to evaluate the utility of IgG deple- tion prior to VNT (IgM-VNT) to detect anti-JEV IgM neutralizing IgG depletion antibody for confirming acute JEV infection. IgG depletion was performed using Protein G HP SpinTrap/Ab Spin Trap columns (28-4083-47; Cytiva, Marlborough, MA, USA). These contain recombinant protein G, a protein present in group G Strep- tococcus with high affinity for IgG. An in-house method devel- Methods oped by the French National Centre for Arboviruses was used, Patient samples substituting commercial binding buffer by phosphate-buffered saline (PBS). Two IgG depletion columns were used for 100– A prospective study of central nervous system (CNS) infec- 150 µL sample serum. Columns were inverted three times and tions has been conducted at Mahosot Hospital, Vientiane, Laos, briefly vortexed. Each column was inserted in a 2-mL tube and since 2003. Methods and results from 2003 to 2011 have been 44 centrifuged. All centrifugation steps were performed at 500 g described. Patients from 2014 to 2017 were included in the 45 for 2 min. The subsequent eluate was discarded, 600 µL of PBS Southeast Asia Encephalitis Project. The laboratory also receives added to each column and centrifuged again. Columns were samples from patients from other hospitals around Vientiane transferred to clean 2-mL tubes and 100–150 µL of sample was City (i.e. Friendship, Children’s and Setthathirat Hospitals). Writ- added to one column and incubated at room temperature for ten informed consent was obtained from patients or responsi- 4 min before centrifugation. The eluate was transferred to the ble guardians. Anti-JEV and anti-DENV IgM were detected by the second column, incubated at room temperature for 4 min and Japanese encephalitis/dengue IgM combo ELISA (Panbio, Bris- centrifuged again. The final eluate was stored at −20°C until the bane, QLD, Australia; now Alere) until July 2014, for which result VNT. interpretation included a ratio between DENV and JEV. After August 2014, as per WHO recommendations, the JEV IgM ELISA (Inbios, Seattle, WA, USA) was utilized. All samples used were VNT aliquoted and stored at −80°C. This pilot study involved a con- venience sample of consecutive patients with available speci- Two-fold dilutions from 1/20 to 1/2560 of each serum sam- mens to be tested; hence a sample size calculation was not ple were tested in duplicate by VNT for JEV, DENV1–4, ZIKV performed. and WNV. Serum dilutions from 1/10 to 1/1280 were pre- Suspected JE patients included in this study had anti-JEV IgM pared and mixed in a 1:1 ratio with 100 TCID50 viral sus- detected by MAC-ELISA in cerebrospinal fluid (CSF) or serocon- pension (Table 1 ) using epMotion 5075 (Eppendorf, Hamburg, version between acute and follow-up serum, no other pathogen Germany) in a 96-well microplate (Figure S1). Negative con- detected in any body fluid and a sufficient volume of acute trols containing minimum essential medium (MEM), with or and/or follow-up serum for VNT. Patients with DENV and JEV RNA without serum, were included in each microplate. Plates were or DENV non-structural protein 1 (NS1) in serum or CSF were incubated at 37°C for 2 h. A 100-µL suspension of Vero cells excluded. (ATCC CCL-81) containing approximately 2 ×10 cells/mL, was Negative controls included samples from three groups: added to each well using the epMotion 5070 (Eppendorf) and healthy flavivirus-naïve blood donors living in Puy-de-Dôme, in incubated at 37°C in a 5% carbon dioxide incubator. After 5– central France; ZIKV VNT-confirmed sera collected in Peru in 7 d, microplates were read under an inverted microscope. Two the framework of a seroprevalence study ; and DENV infec- investigators read the results for each replicate to identify the tion patients from the Laos CNS study (study details reported in end dilution at which there was no cytopathic effect, with a 1034 Transactions of the Royal Society of Tropical Medicine and Hygiene Table 1. Virus strain used in VNTs Virus Strain Country of isolation GenBank number EVAg number Titre (TCID /mL) Day read JEV Laos 2009 Laos KC196115 001V-02217 2 ×10 5 WNV UVE/WNV/2008/US/R94224 USA – 001V-02224 2.1 ×10 5 ZIKV ZIKV strain H/PF/2013 French French Polynesia KJ776791 – 3.7 ×10 5 Polynesia DENV-1 DENV1 2012 Saint Vincent and VC16692 001V-02335 3.1 ×10 7 the Grenadines DENV-2 UVE/DENV-2/1998/MQ/703 Martinique AF208496 – 6.7 ×10 5 DENV-3 UVE/DENV-3/2001/MQ/2023 Martinique AH011666 – 4.5 ×10 6 DENV-4 UVE/DENV-4/1998/ID/814 Indonesia – – 3 ×10 6 EVAg: European Virus Archive – GLOBAL; TCID : 50% tissue culture infective dose. third investigator to resolve disagreement. For duplicates, the compatible; and 10 (29%) as unknown (Table 2 and Table S2). geometric mean of end dilutions was calculated and reported as Eighteen of these patients had sufficient serum available for IgM- 47 , 48 an NAb titre and ≥40 was considered as positive. Suspected VNT in at least one sample. The results enabled reclassification JE patients were categorized as acute JE positive, confirmed or through the removal of cross-reactive IgG to other viruses and compatible, JE negative and unknown, according to the criteria in the specific detection of anti-JEV IgM, such that 17 (94%) were Figure 2 . classified as JE positive, 16 (89%) confirmed, 1 (6%) compatible; and 1 (6%) as JE negative. Five patients classified as unknown by VNT did not have sufficient acute and/or follow-up sample to perform IgG depletion and/or anti-JEV IgG ELISA testing. Results For the subset of 32 patients classified as JE positive, con- firmed or compatible (before or after depletion), the median From 2003 to March 2021, 264 patients with suspected CNS infec- duration of onset of illness was 5 d (IQR 4–7) and the median tion were positive for anti-JEV IgM (in CSF or with seroconversion) duration between paired serum samples was 14 d (IQR 11–24). and negative for other screened aetiologies (see Figure 1 ). A total of 17/24 (71%) of these patients had detectable anti-JEV Paired serum samples (admission and follow-up) were avail- IgG in the admission serum before IgG depletion and 23/24 [96%] able for 35 patients and a single acute sample for 98 patients. had detectable anti-JEV IgG in the follow-up sample. Among these 133 included patients, 130 (98%) had anti-JEV IgM detected in CSF and 3 (2%) demonstrated IgM seroconversion Negative control sera only (no anti-JEV IgM in CSF) in paired sera. The median age of the patients was 11 y (interquartile range [IQR] 6–20) and IgM-VNT was performed on three other groups of negative con- 32% (43/133) were female. The median duration of illness on trol sera to assess the specificity of the novel method. JEV NAb admission was 5 d (IQR 4–6) and the median time between was not detected by IgM-VNT or VNT in the healthy flavivirus- admission and follow-up serum collection was 14 d (IQR 10–25). naïve blood donors (n = 10) or ZIKV infection sera (n = 4) (see Table S3). In the DENV patient sera, 2/12 (17%) did not have detectable JEV NAb, and for both of these patients, IgM-VNT IgG depletion was performed and was also negative. In the 10/12 (83%) A total of 102/156 (65%) serum samples, including 72/125 (58%) patients with DENV infection with JEV NAb detected by VNT, admission sera and 30/31 (97%) follow-up sera, were anti-JEV 8/10 (80%) did not have detectable JEV NAb after IgG deple- IgG positive by ELISA before IgG depletion. Seventy samples had tion. For the remaining two, one did not have a result for IgM- sufficient volumes to be tested for anti-JEV IgG by ELISA after VNT and the other showed negative JEV VNT for admission IgG depletion. Fifty-nine (84%) were negative or equivocal after serum and a low JEV NAb titre of 40 in follow-up serum. There IgG depletion. Six samples were equivocal before IgG depletion were not sufficient sample volumes available to perform DENV and all of these were negative after IgG depletion. Samples that VNT. remained positive after IgG depletion demonstrated decrease in the titre, however, the starting anti-JEV IgG result in these cases was high, all > 125 RU/mL (positive > 22 RU). Positive, negative and overall percentage agreement The IgM-VNT was compared with the reference standard VNT. This was based on results for patients classified as JE positive VNT for the patients with paired serum samples or negative by standard VNT and with sufficient sera to com- VNT results prior to IgG depletion enabled classification of 25/35 plete IgM-VNT, i.e. VNT performed after IgG depletion and IgG (71%) patients as JE positive, 7 (20%) confirmed, 18 (51%) ELISA to confirm IgG depletion. This included 14 JE-positive and 1035 T. Bharucha et al. Figure 2. Criteria for interpretation of the results and patient categorisation for JE status. 1036 Transactions of the Royal Society of Tropical Medicine and Hygiene Table 2. VNT antibody titre in acute and follow-up serum samples for patients with positive anti-JEV IgM capture ELISA Before IgG depletion (standard VNT) After IgG depletion (IgM-VNT) NAb titre NAb titre Patient Sample Days of number type illness Class JEV IgG JEV D1 D2 D3 D4 ZIK WN Class JEV IgG JEV D1 D2 D3 D4 ZIK WN a b b b b b b 1597 Adm 5 Conf − 1280 Neg Neg Neg Neg Conf − 160 Neg Neg Neg Neg Neg b b b b b FU 59 + 2560 14 14 Neg 20 640 Neg Neg Neg Neg b b b b b b b 1704 Adm 5 Conf + 640 20 Neg 20 28 Conf − 640 Neg Neg Neg Neg Neg Neg b b b b b b b FU 13 + 2560 40 Neg 28 80 2560 20 Neg Neg Neg Neg Neg b b b b b b b 829 Adm 4 Conf − 1280 Neg Neg Neg Neg Neg 14 Conf − 640 Neg Neg Neg Neg Neg Neg FU 21 + 2560 Neg Neg Neg 14 Neg 908 Adm 4 Conf + 40 160 160 40 113 Neg Conf b b b b b FU 14 + 2560 20 Neg Neg 14 − 2560 Neg Neg Neg Neg Neg b b b b b b b 928 Adm 5 Conf − 1810 20 Neg Neg Neg Neg 56 Conf − 2560 Neg Neg Neg Neg Neg Neg b b b b b b FU 44 + 2560 Neg Neg Neg 14 − 2560 Neg Neg Neg Neg Neg Neg 2078 Adm 7 Conf Eq 160 20 Neg Neg Neg Conf − FU 17 + 2560 Neg Neg 20 20 − ≥2560 Neg Neg Neg Neg 101 Adm 4 Conf − 2560 Neg Neg Neg Neg Conf − 453 Neg Neg Neg Neg FU 6 − 2560 Neg Neg 40 Neg − ≥2560 Neg Neg Neg Neg b b b b b b b 1610 Adm 6 Comp + 2560 14 Neg Neg 20 Conf − 1280 Neg Neg Neg Neg Neg Neg FU 40 + 2560 160 20 Neg 113 2560 Neg Neg Neg Neg Neg Neg b b b b b b b 483 Adm 7 Comp + 2560 Neg Neg Neg Neg Conf − 2560 Neg Neg Neg Neg Neg Neg b b b b b b b FU 21 + 2560 20 20 Neg 20 Neg Neg 2560 Neg Neg Neg Neg Neg Neg b b b b b b b 884 Adm 5 Comp + 2560 40 Neg Neg 40 Neg 40 Conf − 1280 Neg Neg Neg Neg Neg Neg b b b b b b b FU 19 + 2560 Neg Neg Neg 20 − 2560 Neg Neg Neg Neg Neg Neg 1074 Adm 6 Comp Eq 452 Neg Neg Neg Conf b b b b b b FU 84 + 1810 40 40 Neg 80 − 1280 Neg Neg Neg Neg Neg b b b b b b 1180 Adm 7 Comp + 1280 452 640 452 160 Neg Conf − 1280 Neg Neg Neg Neg Neg FU 13 + 2560 226 226 160 160 20 80 2053 Adm 5 Comp + 2560 20 Neg Neg Neg Conf − 2560 Neg Neg Neg Neg FU 18 + 1280 80 Neg 40 Neg − 775 Adm 3 Comp − 320 Neg Neg Neg Comp − 113 Neg Neg Neg FU 12 + 640 Neg Neg 40 640 Neg Neg Neg b b b b b 5149 Adm 1 Unkn + 2560 2560 2560 2560 2560 Conf 1280 20 20 20 Neg b b b b b b FU 28 + 2560 2560 2560 2560 2560 − 2560 40 20 40 20 Neg b b b b b b 1056 Adm 3 Unkn + 320 1810 320 320 320 28 40 Conf − 640 20 Neg Neg Neg Neg FU 28 + 2560 1917 Adm 7 Unkn + 2560 2560 2560 Neg 453 Conf − 2560 Neg Neg Neg Neg FU 12 + 1280 2560 2560 Neg 320 − 1280 Neg Neg Neg Neg b b b b b b 1036 Adm 4 Unkn + 80 2560 226 320 226 Neg 20 Neg − Neg 80 Neg 20 Neg Neg FU 16 + 640 2560 1280 2560 Adm: serum on admission; FU: serum at follow-up; NAb: NAb assessed by VNT, geometric mean calculated from duplicate results, = indeter- minate, NAb titre underlined to indicate the maximum dilution tested, neg: no NAb detected in duplicate samples (observation of cytopathic effect) for all serum dilutions tested (lowest = 20); NAb titre ≥40 considered as positive; D1–4: dengue virus 1–4; ZIK: Zika virus; WN: West Nile virus; class: classification for JE status according to criteria in Table 2 ; Conf: confirmed; Comp: compatible; Unkn: unknown; JEV IgG: anti-JEG IgG detection by ELISA (Euroimmun); + : positive; Eq: equivocal; −: negative. JEV IgG negative before depletion. Only one replicate tested or interpretable, the other samples were tested in duplicate. 16 JE-negative patients. Positive, negative and overall percentage Results allowed classification for 72/76 (95%) patients: 70 (92%) agreements (PPA, NPA and OPA, respectively) were all 100% (see JE, 63 (83%) confirmed and 3 (4%) compatible, and 6 (8%) neg- Table 3 ). ative. Four (5%) were unknown (Table 4 ). VNT after IgG depletion for patients with single acute Discussion serum A total of 76/98 (78%) patient samples had sufficient volumes This pilot study included a large set of well-characterized patients for IgG depletion, confirmatory IgG ELISA testing and IgM-VNT. recruited prospectively in clinical studies, with extensive VNT for 1037 T. Bharucha et al. gradient centrifugation of whole serum and 2-mercaptoethanol Table 3. 2 ×2 table of the results of IgM-VNT as compared with treatment. The improved discrimination of evidence for acute JE standard VNT in patient samples gave rise to further work developing the widely 50 , 51 used anti-JEV IgM ELISA. However, with evidence suggesting suboptimal performance of MAC-ELISA, the increasing use of Reference test (standard VNT) the JEV vaccine, as well as hyperendemicity of DENV serotypes, a a IgM-VNT JE positive , n JE negative , n Total, n the requirement for accurate diagnostic confirmation becomes even more pertinent. Although the performance of contempora- JE positive 14 0 14 neous anti-DENV IgM ELISA and calculation of a JEV:DENV IgM JE negative 0 16 16 ratio has improved specificity, the combination of VNT and IgG Total 14 16 30 depletion (IgM-VNT) permits IgM detection with higher specificity The classification of patients followed the criteria set out in than by using MAC-ELISA alone. Figure 2 . Calvert et al. showed that IgG depletion prior to neutral- ization testing considerably improved (15% before to 77% after IgG depletion) the differentiation of acute Zika from dengue JEV, DENV 1–4, ZIKV and WNV. We show that the implementa- viral infections. This has also been demonstrated for DENV infec- 42 , 43 tion of IgG depletion prior to VNT performed on par with stan- tions. It is notable that as JE is predominantly a neurological dard VNT (100% PPA, NPA and OPA) and also resulted in a sig- infection, and the natural history of the immunological response nificantly higher proportion, compared with standard VNT, of is different to flavivirus infections presenting as acute febrile syn- patients being classified. Of the patients with paired sera tested to dromes, by the time of clinical presentation, anti-JEV IgM and IgG confirm acute JEV infection, 74% (26/35) were classified without is detectable in a larger proportion of patients. Therefore use of an IgG depletion step, in contrast to 100% when IgG depletion the IgM-VNT method for JE confirmation is a logical approach. was included. Furthermore, IgG depletion improved the diagnos- The humoral responses to JEV infection are directed mainly tic confidence of patients classed as JE positive, from 7/26 (27%) against antigenic epitopes on the viral envelope protein. There is confirmed as opposed to 19/26 (73%) compatible with standard major cross-reactivity with other endemic circulating flaviviruses 52 , 53 VNT to 16/17 (94%) confirmed as opposed to 1/17 (6%) compat- and therefore it was crucial to test for all DENV serotypes, 54 44 ible with IgM-VNT. Depleting IgG also enabled a diagnosis of JE ZIKV and WNV where they are sympatric. Likewise, IgG deple- in 95% of patients for whom only a single sample was available, tion and seroneutralization might play a role in the diagnosis of allowing for specific neutralization of the IgM remaining in the DENV neurological infections for which there is considerable diag- sample. nostic uncertainty. The high proportion of patients presenting with detectable We acknowledge that a diagnostic accuracy study should anti-JEV IgG before depletion and a reduction in DENV neutral- ideally be performed with an a priori sample size calculation, ization titres after depletion strengthen the underlying premise of prospectively testing consecutive patients with suspected neu- this study, that IgG complicates discrimination by VNT, especially rological infection by the reference standard VNT to ascertain in areas with high endemicity of other flaviviruses and increasing JE-positive and negative patient samples. However, we were utilization of JEV vaccination. unable to conduct this in this pilot study and flavivirus-naïve A limitation is that there were not sufficient sample volumes patients from France were included as an additional category available to perform standard and IgM-VNT in all samples. How- of negative controls. That patients already had anti-JEV IgM ever, the testing was retrospectively performed on a relatively detected in CSF or experienced JEV seroconversion reflects the large number of very precious samples. It would be realistic in role of VNT within reference centres. Further limitations include clinical practice to secure the serum volume (400 μL) needed for missing data due to limited sample volumes and that dilutions prospective IgM-VNT testing. This is one of the advantages of the were 1/20 to 1/2560 for the sera. Ideally serum should be tested new technique, that it relies on a single serum sample rather than to the end point of dilution. IgM-VNT is a diagnostic test suited for paired sera or CSF. The efficiency of the IgG depletion was eval- reference centres and optimization will be required to adapt the uated using anti-JEV IgG ELISA. We found that 84% of the anti- technique to be high throughput, using protein G slurry and an JEV IgG ELISA-positive sera became negative after IgG depletion. automatized format for VNT testing of 1/20 to 1/5120. Addition- All samples with an anti-JEV IgG ELISA result < 125 RU/mL were ally, not all the virus strains used were sourced from the countries negative after IgG depletion, suggesting IgG depletion was prob- where the samples were derived; the DENV strains isolated from ably incomplete in samples with high titres. Further optimiza- Laos did not provide a sufficient cytopathic effect for the assay tion is required to ensure that depletion is fully effective, perhaps and neither ZIKV nor WNV have been isolated from patients in with alternative methods depending on the initial anti-JEV IgG Laos. result, such as the use of three rather than two IgG depletion In conclusion, measurement of anti-JEV IgG and the per- columns. formance of IgM-VNT significantly improved performance and The principle of removing IgG and the use of IgM as a allowed the use of a single serum sample instead of paired sera biomarker for confirming acute infection is by no means novel. In for JE confirmation. This innovation holds promise for wider incor- 1973, Edelman and Pariyanonda reported a modified haemag- poration into testing algorithms in the reference confirmation of glutination inhibition involving depletion of IgG by sucrose density JE and DENV neurological infections. 1038 Transactions of the Royal Society of Tropical Medicine and Hygiene Table 4. VNT antibody titre for patients with only a single acute serum sample After IgG depletion (IgM-VNT) NAb titre Days of Before IgG Patient number illness Class depletion, JEV IgG JEV IgG JEV D1 D2 D3 D4 ZIK WN 34 Conf − − 160 Neg Neg Neg Neg Neg Neg 37 3 Conf − − 640 Neg Neg Neg Neg Neg Neg 38 2 Conf − − 57 Neg Neg Neg Neg Neg 40 14 Conf − − 80 Neg Neg Neg Neg Neg Neg 44 4 Conf − − 57 Neg Neg Neg Neg Neg Neg 47 4 Conf − − 320 Neg Neg Neg Neg Neg Neg 52 4 Conf − − 80 Neg Neg Neg Neg Neg Neg 53 4 Conf − − 57 Neg Neg Neg Neg Neg Neg 59 4 Conf − − 320 Neg Neg Neg Neg Neg Neg 60 1 Conf − − 160 Neg Neg Neg Neg Neg Neg 64 3 Conf − − 80 Neg Neg Neg Neg Neg Neg 57 6 Conf − − 640 Neg Neg Neg Neg Neg Neg 66 8 Conf − − 40 Neg Neg Neg Neg Neg Neg 73 5 Conf − − 1810 Neg Neg Neg Neg Neg Neg 76 5 Conf − − 320 Neg Neg Neg Neg Neg Neg 87 4 Conf − − 57 Neg Neg Neg Neg Neg Neg 88 6 Conf − − 160 Neg Neg Neg Neg Neg Neg 92 3 Conf − − 905 Neg Neg Neg Neg Neg Neg 98 Conf − − 320 Neg Neg 14 Neg Neg Neg 101 Conf − − 1280 Neg Neg Neg Neg Neg Neg 102 Conf − − 640 Neg Neg 28 Neg Neg Neg 103 Conf − − 320 Neg Neg Neg Neg Neg Neg 104 Conf − − 226 Neg Neg Neg Neg Neg Neg 105 Conf − − 160 Neg Neg Neg 14 Neg Neg 111 Conf − − 452 Neg Neg Neg Neg Neg Neg 112 Conf − − 160 Neg Neg Neg Neg Neg Neg 127 Conf − − 640 Neg Neg Neg Neg Neg 14 118 Conf − − 640 Neg Neg Neg 14 Neg Neg 89 3 Conf − − 160 Neg Neg Neg Neg Neg Neg 97 Conf − − 640 Neg Neg Neg Neg Neg Neg 94 3 Conf − − 640 Neg Neg Neg Neg Neg Neg 110 Conf − − 160 Neg Neg Neg Neg Neg Neg 121 Conf − − 160 14 Neg Neg Neg 54 3 Conf − 57 Neg Neg Neg Neg Neg Neg 128 Conf Eq − 640 Neg Neg Neg Neg Neg Neg 51 5 Conf Eq − 905 Neg Neg Neg Neg Neg Neg 33 Conf Eq − 452 Neg Neg Neg Neg Neg Neg 58 5 Conf Eq − 2560 Neg Neg Neg Neg Neg Neg 62 6 Conf Eq − 226 20 Neg Neg Neg Neg Neg 35 4 Conf + − 160 Neg Neg Neg Neg Neg Neg 36 Conf + − 40 Neg Neg Neg Neg Neg Neg 41 4 Conf + − 320 Neg Neg Neg Neg Neg Neg 65 13 Conf + − 80 14 Neg 14 Neg Neg Neg 67 4 Conf + − 1280 Neg Neg Neg Neg Neg 28 68 6 Conf + − 2560 Neg Neg Neg Neg Neg Neg 69 5 Conf + − 452 Neg Neg Neg Neg Neg Neg 70 6 Conf + − 640 Neg Neg Neg Neg Neg Neg 71 8 Conf + − 640 Neg Neg Neg Neg Neg 14 74 4 Conf + − 226 Neg Neg Neg Neg Neg Neg 1039 T. Bharucha et al. Table 4. Continued After IgG depletion (IgM-VNT) NAb titre Days of Before IgG Patient number illness Class depletion, JEV IgG JEV IgG JEV D1 D2 D3 D4 ZIK WN 75 14 Conf − 905 Neg Neg Neg Neg Neg Neg 79 5 Conf + − 640 Neg Neg Neg Neg Neg Neg 80 7 Conf + − 226 Neg Neg Neg Neg Neg Neg 81 6 Conf + − 640 Neg Neg Neg Neg Neg Neg 82 6 Conf + − 905 Neg Neg Neg Neg Neg Neg 86 Conf + − 2560 Neg Neg Neg Neg Neg 20 91 7 Conf + − 1280 Neg Neg Neg Neg Neg Neg 95 4 Conf + − 320 Neg Neg Neg Neg Neg 14 99 Conf + − 640 40 Neg Neg 20 Neg Neg 108 Conf + − 320 Neg Neg Neg 40 Neg Neg 109 Conf + − 113 Neg Neg Neg Neg Neg Neg 116 Conf + − 113 Neg Neg Neg Neg Neg Neg 122 Conf + − 1280 Neg Neg Neg Neg Neg Neg 125 Conf + − 160 Neg Neg 14 Neg Neg Neg 31 Comp − − 226 Neg Neg Neg 32 Comp − − 226 Neg Neg Neg 45 10 Comp − − 80 Neg Neg Neg 56 6 Unkn + − 80 Neg 40 20 Neg Neg Neg 77 6 Unkn + − 80 Neg 28 Neg Neg Neg Neg 85 Unkn + − 320 98 160 57 20 Neg Neg 78 4 Unkn + − 160 Neg Neg Neg Neg 80 Neg 39 3 Neg − − Neg Neg Neg Neg Neg Neg Neg 83 5 Neg − − Neg Neg Neg Neg Neg Neg Neg 48 3 Neg + − Neg 20 Neg Neg Neg Neg Neg 61 14 Neg + − Neg 14 14 Neg 14 Neg Neg 49 10 Neg + − Neg 14 Neg Neg Neg 120 Neg + − 20 40 Neg 20 14 Neg Neg NAb titre: NAb assessed by VNT, geometric mean calculated from duplicate results. neg: no NAb detected in duplicate samples (observation of cytopathic effect) for all serum dilutions tested (lowest = 20); NAb titre ≥40 considered as positive; D1–4: dengue virus 1–4; ZIK: Zika virus; WN: West Nile virus; class: classification for JE status according to the criteria set out in Figure 2 ; Conf: confirmed; Comp: compatible; Unkn: unknown; JEV IgG: = anti-JEG IgG detection by ELISA (Euroimmun); + : positive; Eq: equivocal; −: negative. Only one replicate tested or interpretable, the other samples were tested in duplicate. JEV IgG negative before depletion. Maximum dilution tested. Test not performed. provided the clinical samples. TB, NA and BP performed the experimental Supplementary data work. TB, NA, ADP, BP, NZ and XDL analysed and interpreted the data. Supplementary data are available at Transactions online. TB wrote the manuscript. All the authors edited successive drafts and approved the final version. Acknowledgements: We are very grateful to the patients and to Boun- Authors’ contributions: TB, ADP and XDL conceived the study. TB, NA, thaphany Bounxouei, the former Director of Mahosot Hospital, the late ADP, BP, XDL and NZ developed the methodology. SR, MV, MM, AC, OS, Rattanaphone Phetsouvanh, Director of the Microbiology Laboratory, and OP, ADP, JDP, CG and PNN designed and conducted the clinical study and the staff of the wards and Microbiology Laboratory of Mahosot Hospi- 1040 Transactions of the Royal Society of Tropical Medicine and Hygiene tal. We also thank Bounnak Saysanasongkham, the former Director of References the Department of Healthcare and Rehabilitation, Ministry of Health, and 1 World Health Organization. Japanese encephalitis vaccines: Bounkong Syhavong, Minister of Health, Lao PDR for their very kind help WHO position paper, February 2015–recommendations. Vaccine. and support. We thank the stakeholders of the SEAe project, members 2016;34(3):302–3. of the Unité des Virus Émergents (Christine Isnard and Camille Placidi) 2 Heffelfinger JD, Li X, Batmunkh N, et al. Japanese encephalitis surveil- and the CNR des Arbovirus (Patrick Gravier, Gilda Grard, Isabelle Leparc- lance and immunization –Asia and Western Pacific regions, 2016. Goffart and Mathilde Galla). We also thank Rodrigo Cachay, Eduardo Got- MMWR Morb Mortal Wkly Rep. 2017;66(22):579–83. tuzo and Humberto Guerra (Instituto de Medicina Tropical Alexander von Humboldt, Universidad Peruana Cayetano Heredia) for providing the Zika 3 Hills SL, Walter EB, Atmar RL, et al. Japanese encephalitis vaccine: virus patient samples. recommendations of the Advisory Committee on Immunization Prac- tices. MMWR Recomm Rep. 2019;68(2):1–33. Collaborators: We are grateful to all the SEAe study researchers, includ- 4 Pearce JC, Learoyd TP, Langendorf BJ, et al. Japanese encephali- ing Philippe Buchy, Em Bunnakea, Julien Cappelle, Mey Channa, Veronique tis: the vectors, ecology and potential for expansion. J Travel Med. 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New strains of Japanese encephali- manam, Viengmon Davong, Patrice Debré, Jean-François Delfraissy, Chris- tis virus circulating in Shanghai, China after a ten-year hiatus in local tian Devaux, Anousone Douangnouvong, Veasna Duong, Benoit Durand, mosquito surveillance. Parasites Vectors. 2019;12:14. Chanreaksmey Eng, Catherine Ferrant, Didier Fontenille, Lukas Hafner, Le 8 Ojha JK, Samantaray K, Mohanty S. Assess the coverage rate of Thanh Hai, Do Thu Huong, Marc Jouan, May July, Magali Lago, Jean- Japanese encephalitis vaccination and factors of non-compliance as Paul Moatti, Bernadette Murgue, Khin Yi Oo, MengHeng Oum, Khansouda- reported by parents of selected areas of Khurdha. Eur J Mol Clin Med. phone Phakhounthong, Anh Tuan Pham, Do Quyen, Malee Seephonelee, 2021;7(11):5049–60. Maud Seguy, Bountoy Sibounheunang, Kanarith Sim, Luong Minh Tan, Cho 9 Do LP, Bui TM, Hasebe F, et al. Molecular epidemiology of Japanese Thair, Win Thein, Phung Bich Thuy, Hervé Tissot-Dupont and Malavanh encephalitis in northern Vietnam, 1964–2011: genotype replacement. Vongsouvath. Virol J. 2015;12:51. 10 Bharucha T, Sengvilaipaseuth O, Vongsouvath M, et al. Develop- Funding: The work was supported by the University of Oxford and the ment of an improved RT-qPCR Assay for detection of Japanese Medical Research Council (grant MR/N013468/1). It was also supported encephalitis virus (JEV) RNA including a systematic review and by the Oxford Glycobiology endowment, the Institute of Research for comprehensive comparison with published methods. PLoS One. Development, Aix-Marseille University, the Wellcome Trust of Great Britain 2018;13(3):e0194412. and the European Union’s Horizon 2020 research, Fondation Total, Insti- tut Pasteur, International Network Institut Pasteur, Fondation Merieux, 11 Hills S, Dabbagh A, Jacobson J, et al. Evidence and rationale for the Aviesan Sud, Institut national de la santé et de la recherche médicale World Health Organization recommended standards for Japanese (Inserm), and innovation programme EVAg (grant agreement 653316). encephalitis surveillance. BMC Infect Dis. 2009;9:214. The Zika virus patient samples were provided by the EC-funded project 12 Dubot-Peres A, Sengvilaipaseuth O, Chanthongthip A, et al. How ZIKAlliance, Grant agreement no. 734548. many patients with anti-JEV IgM in cerebrospinal fluid really have Japanese encephalitis? Lancet Infect Dis. 2015;15(12): Competing interests: None declared. 1376–7. 13 Maeki T, Tajima S, Ikeda M, et al. Analysis of cross-reactivity between Ethical approval: Ethical clearance for the Laos CNS study was granted flaviviruses with sera of patients with Japanese encephalitis showed by the Ethical Review Committee of the former Faculty of Medical Sci- the importance of neutralization tests for the diagnosis of Japanese ences, National University of Laos (now University of Health Sciences) encephalitis. J Infect Chemother. 2019;25(10):786–90. and the Oxford University Tropical Ethics Research Committee, Oxford, 14 Hills S, Van Keulen A, Feser J, et al. Persistence of IgM antibodies after UK. For the blood donor samples, the protocol was presented to an eth- vaccination with live attenuated Japanese encephalitis vaccine. Am J ical committee (Comité de Protection des Personnes Sud Méditerranée Trop Med Hyg. 2020;104(2):576–9. I) and because no additional blood sampling was required, the com- 15 Fatima T, Rais A, Khan E, et al. Investigation of Japanese encephalitis mittee agreed that ethical approval was not required. The protocol is in virus as a cause of acute encephalitis in southern Pakistan, April 2015– agreement with the national regulations on personal data (Commission January 2018. PLoS One. 2020;15(6):e0234584. Nationale Informatique et Liberté), the collection of biological samples was declared to the French Ministry of Research and all data and sam- 16 Centers for Disease Control. Japanese encephalitis. Diagnostic ples were anonymized. For the Zika sera, ethical approval was granted by testing. Available from: https://www.cdc.gov/japaneseencephalitis/ the Institutional Ethics Committee of the Universidad Peruana Cayetano healthcareproviders/healthcareproviders-diagnostic.html [accessed Heredia (SIDISI 103488). 18 April 2022]. 17 Robinson JS, Featherstone D, Vasanthapuram R, et al. 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Journal

Transactions of The Royal Society of Tropical Medicine and Hygiene – Oxford University Press

Published: May 18, 2022

Keywords: diagnostics, flavivirus, Laos; neglected tropical disease; neurological infection, seroneutralization

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