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Does proficiency testing improve the quality of hantavirus serodiagnostics? Experiences with INSTAND EQA schemes
International Journal of Medical Microbiology 305 (2015) 607–611
Contents lists available at ScienceDirect
International Journal of Medical Microbiology journal homepage: www.elsevier.com/locate/ijmm
Does proficiency testing improve the quality of hantavirus serodiagnostics? Experiences with INSTAND EQA schemes Jörg Hofmann a,∗ , Hans-Peter Grunert b , Oliver Donoso-Mantke c , Heinz Zeichhardt c,d , Detlev H. Kruger a a National Consultant Laboratory for Hantaviruses, Institute of Medical Virology, Helmut-Ruska-Haus, Charité—University Medicine, Charitéplatz 1, 10117 Berlin, Germany b GBD Gesellschaft für Biotechnologische Diagnostik mbH, Potsdamer Chaussee 80, 14129 Berlin, Germany c INSTAND e.V., Society for Promoting Quality Assurance in Medical Laboratories, Ubierstrasse 20, 40223 Düsseldorf, Germany d Institute of Virology, Campus Benjamin Franklin, Charité—University Medicine, Hindenburgdamm 27, 12203 Berlin, Germany
a r t i c l e
i n f o
Keywords: Hantavirus Puumala virus Dobrava-Belgrade virus Proficiency testing
a b s t r a c t Hantavirus infections in Germany appear periodically with peak numbers every 2–3 years. The reported cases in the years 2007, 2010 and 2012 exceeded many times over those in the years in-between. In order to reveal faults of certain in vitro diagnostic assays (IVDs), to harmonize the performances of the individual assays and to improve the users’ competence in interpreting the results, the National Consiliary Laboratory for Hantaviruses and INSTAND e.V. (Society for Promoting Quality Assurance in Medical Laboratories e.V.) established an external quality assessment (EQA) scheme for proficiency testing of hantavirus serodiagnostics. The first EQA scheme (pilot study) started in March 2009 with 58 participating laboratories from Germany and neighboring countries. Twice a year four serum samples were sent out to the participants to investigate whether the sample reflects an acute or past infection and to distinguish between infections with the hantavirus types Puumala virus (PUUV) and Dobrava-Belgrade virus (DOBV), both endemic in Central Europe. In addition, samples negative for anti-hantavirus antibodies were tested in order to examine the specificity of the IVDs applied in the participating laboratories. An increasing number of laboratories participated, with a maximum of 92 in March 2014. When summarizing in total 2592 test results, the laboratories reached an overall specificity of 96.7% and a sensitivity of 95% in their detection of a hantavirus infection. A correct distinction between acute and past infections was forwarded in 90–96% of replies of laboratories. Exact serotyping (PUUV vs. DOBV) of the infection was reported in 81–96% of replies with the lowest accuracy for past DOBV infections; cross-reactivities between diagnostic antigens of the two viruses as well as persistent IgM titers in humans may interfere with exact testing. The EQAs revealed acceptable results for the serodiagnostic of hantavirus infection including serotyping but further improvement is still needed. © 2015 Elsevier GmbH. All rights reserved.
1. Introduction Hantaviruses, members of Bunyaviridae family, are emerging viruses which cause Hemorrhagic Fever with Renal Syndrome (HFRS) in Asia and Europe and Hantavirus Cardiopulmonary Syndrome in the Americas (Vaheri et al., 2013). In Central Europe, Puumala virus (PUUV) and Dobrava-Belgrade virus (DOBV) are the two main pathogenic hantaviruses circulating in rodent hosts and transmitted to humans (Klempa et al., 2013). In Germany, a total of 7252 symptomatic HFRS cases were reported from 2007 to 2012
∗ Corresponding author. E-mail address: [email protected] (J. Hofmann). http://dx.doi.org/10.1016/j.ijmm.2015.08.009 1438-4221/© 2015 Elsevier GmbH. All rights reserved.
with increasing peak activities in the outbreak years 2007 (1687 cases), 2010 (2016 cases), and 2012 (2824 cases). During outbreak years, the hantavirus disease belongs to the group of the 5 most frequent notifiable virus diseases in Germany (www3.rki.de/SurvStat/ QueryForm.aspx). Large hantavirus outbreaks in Germany are caused by PUUV infections and a detailed molecular epidemiological characterization of PUUV strains from the different outbreak regions of the country has been established (Ettinger et al., 2012; Hofmann et al., 2008). Moreover, infections by the DOBV Kurkino genotype lead to additional cases of hantavirus disease (Hofmann et al., 2014). Since hantaviruses are strongly host-associated, infections of humans are linked to the particular virus type circulating in the geographic area where infected rodents live. Thus, PUUV infections appear mostly
608
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in South and Southwest Germany, but DOBV infections in the North and Northeast, corresponding to the natural habitats of the rodent hosts, the bank vole (Myodes glareolus) and striped field mouse (Apodemus agrarius), respectively. Hantavirus diagnostic is based on seroassays, as enzyme immune assay (EIA), immunoblot, immunofluorescence assay (IFA), and rapid immunochromatographic strip assay. (Meisel et al., 2006; Vaheri et al., 2008). The antigen source in PUUV assays usually consists in a homologous (PUUV) nucleocapsid protein, while for DOBV assays – particularly in older IVDs – a heterologous (Hantaan virus) nucleocapsid antigen is used. DOBV and Hantaan virus nucleocapsid proteins share some homology in their amino acid sequences (Klempa et al., 2005), however, such cross-reactions complicate a reliable antibody particularly in low-titer sera (Meisel et al., 2006). Confirmatory assays – like the focus reduction neutralization test (FRNT) requiring BSL-3 conditions or RT-PCR assays – can be performed by highly specialized groups only but not by the majority of diagnostic laboratories. Moreover, whereas serotyping by FRNT is reported to be most specific when using sera of convalescent patients, the occurrence of detectable virus genomes in the blood is restricted to the very early phase after disease onset (Kruger et al., 2011). Therefore, the routine diagnostics is based on use of the above mentioned seroassays. Since hantavirus disease is notifiable in Germany according to the Infection Protection Act, a country-wide network of qualified laboratories is needed which uses assays of high reliability. We have established an external quality assessment (EQA) system and determined the diagnostic performance of more than 50 laboratories in Germany and neighboring countries twice a year. 2. Material and methods Serum samples were obtained from patients who were diagnosed in the National Consiliary Laboratory to be either IgG+/IgM− or IgG+/IgM+ seropositive for either PUUV or DOBV. After approval of the physician in charge, the patients were asked for giving a blood donation. The patients completed a disease-specific questionnaire, including potential infection source, clinical course, travel activities, which was later used as additional information for the participating laboratories. Certain acutely infected patients
were sampled again one year later providing valuable followup sera. In any case all clinical samples were taken from the patients after informed consent including clarification that the samples are exclusively used for EQA schemes and completely anonymized in order to exclude any traceability to personal data of the patients. Donated blood materials were processed at the GBD Gesellschaft für Biotechnologische Diagnostik mbH, Berlin. The resulting sera were characterized at the National Consiliary Laboratory and the GBD and were tested by PCR to be negative for hantavirus RNA in order to exclude infectivity especially in samples representing acute hantavirus infection (DOBV or PUUV). These well-investigated samples were sent to up to eight experienced target value laboratories for serological confirming the sample properties. If concordant results were obtained, the individual sample was included into the proficiency testing panel. In addition, samples negative for anti-hantavirus antibodies were tested in order to examine the specificity of the IVDs applied by the participating laboratories. According to the amount of the test material certain samples were used in more than one panel. The results reported by the participating laboratories and the correct interpretations were made available (in anonymous form) to every individual laboratory and also to non-participants on the homepage of INSTAND (http://www.instandev.de/en/eqas.html). Thus, information on the performance of single assays was distributed publicly. 3. Results Each EQA scheme comprised four samples from PUUV-infected, DOBV-infected, or non-infected individuals. Table 1 summarizes the number of participating laboratories (increasing from 58 in the pilot study of March 2009 to 92 in the EQA scheme of March 2014), the composition of each 4-sera panel, and the assays used by the participants during the 11 EQA schemes. The number of participating laboratories increased by about one-third after 2012, the year with multiple outbreaks and the highest number of reported cases. Fig. 1 gives an overview about the assays used by the participants. More than 50% of all analyses were performed exclusively by immunoblot assays, numerically followed by a combination of EIA and immunoblot (16% of analyses). The proportion of the single
Table 1 Number of participants, characteristics of the panel composition, and IVDs used in the EQAs from March 2009 to March 2014.
Participants (n) Sera provided in the panel Acute PUUV Past PUUV Acute DOBV Past DOBV Negative Assays used by participants EIA only IFA only Blot only EIA + IFA EIA + RT EIA + Blot IFA + Blot EIA + IFA + Blot EIA + RT + Blot EIA + IFA + RT RT + Blot RT + EIA + IFA + Blot IFA + RT IFA + RT + Blot
Pilot study
Sep 09
Mrz 10
Sep 10
Mrz 11
Sep 11
Mrz 12
Sep 12
Mrz 13
Sep 13
Mrz 14
Mrz 09 58
55
64
64
71
69
71
71
83
81
92
2 0 1 0 1
1 0 1 1 1
1 1 0 2 0
0 1 2 1 0
0 2 1 1 0
0 2 1 1 0
1 0 1 1 1
0 1 2 1 0
0 1 1 1 1
0 1 2 1 0
1 1 1 0 1
9 5 28 3 1 7 3 2 0 0 0 0 0 0
8 5 26 2 1 6 3 4 0 0 0 0 0 0
9 3 32 2 1 8 2 4 1 0 0 0 1 1
11 3 32 3 0 7 3 3 0 0 0 0 1 1
9 4 37 2 0 10 3 3 1 1 0 0 1 0
9 3 35 2 1 8 4 3 2 1 0 0 1 0
8 3 36 2 0 11 3 3 2 1 1 1 0 0
10 3 34 2 1 13 3 4 1 0 0 0 0 0
10 2 44 1 0 15 4 4 1 0 0 0 2 0
9 3 40 1 0 16 3 5 1 0 2 0 0 1
10 3 50 1 1 17 4 3 1 0 2 0 0 0
PUUV: Puumala virus, DOBV: Dobrava-Belgrade virus, EIA: enzyme immunoassay, IFA: immunofluorescence assay, RT: rapid test.
J. Hofmann et al. / International Journal of Medical Microbiology 305 (2015) 607–611
361/401
643/714
837/873
706/735
272/282 222/240
90
85 271/334
Correct results (in %)
95
2176/2291
291/301
assays or assay combinations used remained stable with only slight deviation between 2009 and 2014. For reasons of clarity and comprehensibility assay combinations used by only a few participants were summarized as “others” (for details see legend to Fig. 1) During 11 EQA schemes, four hantavirus-antibody negative specimens were included into the panels as negative controls. A total of 301 results were reported regarding these specimens; 291/301 answers were correct, however, 10/301 (3.3%) results were false-positive. On the basis of the hantavirus antibodypositive specimens distributed to the participants, 2291 results were reported. Out of them, 2176 replies were correct (“hantavirus antibody-positive”, no matter whether acute vs. past infection or what virus type) but 115/2291 (5.0%) were false negative (Fig. 2, left two columns). Furthermore, Fig. 2 shows the outcome of testing of the defined sera (representing acute PUUV infection, past PUUV infection, acute DOBV infection, or past DOBV infection) according to these criteria: (i) differentiation acute vs. past infection (black columns) and (ii) typing of PUUV vs. DOBV infection (grey columns). It should be mentioned that not all laboratories attempted to differentiate the
569/593
virus type. The identification of the virus type seems to be more difficult than the distinction between acute and past infection. The value of correct distinction of acute vs. past hantavirus infection, i.e. without typing for PUUV vs. DOBV infection, varies between about 96% for sera from acute PUUV, past PUUV, and acute DOBV infections on the one hand and 90% for sera from past DOBV infections on the other. Typing of the involved virus, however, was most successful for sera from past PUUV infections (96%) and showed the lowest success ratio for sera from past DOBV infections (81%)—see Fig. 2. As shown in Table 1, at least 14 different assays or assay combinations were used by the laboratories. However, routine diagnostics might not be performed with more than one assay, unless a laboratory finds a reactive sample which needs confirmatory testing. The results obtained with the different test formats were subdivided in those for the detection of acute vs. past PUUV and DOBV infections. One limitation was the different number of tests that were performed with each test format. Therefore, only the performances of the four most frequently used test combinations were compared (Fig. 3). The majority of laboratories used immunoblots to analyze the samples; however, this test format has some difficulties with the detection of past DOBV infections. The low performance of “EIA only” is remarkable. Behind the format EIA several enzyme immunoassays are summarized. Fifteen different IgG-EIA assays were used in the 09/2013 EQA scheme, including four home-brewed assays. The most frequently used assays are shown in supplementary Table S1. Especially those which use heterologous (Hantaan virus) antigens for the detection of DOBV antibody might be responsible for the high error rate. The differentiation of serotype specific antibodies is difficult because of the antigenic relatedness of hantavirus nucleocapsid proteins which frequently results in cross-reactions and, thus, potential misinterpretation of diagnostic data. Here we analyzed the cross-reaction in immunoblot assays because more than 95% participants used the same commercially available blot assay. Fig. 4 shows the reported data for sera representing acute and past PUUV and DOBV infections in the IgG immunoblot (excluding data from the pilot study and of samples with inconsistent results). PUUV IgG positive samples, independent on whether representing acute or
Fig. 1. Overview about single assays or assay combinations used by the participants. A total of 14 different assays or assay combinations were used by the participants. “12% Others” summarizes the following assay combinations: EIA and IFA, EIA and rapid test, EIA and rapid test and blot, rapid test and blot, and IFA and RT.
100
609
80
75
70 hantavirus negave
hantavirus posive
acute PUUV
past PUUV
acute DOBV
past DOBV
status of infecon
Fig. 2. Number of correct results of all proficiency tests. The grey bars indicate correct results regarding the overall hantavirus serostatus. Black columns represent results for acute vs. past hantavirus infection, and light grey bars depict those for differentiation of PUUV vs. DOBV infection. The numbers above the bars indicate correct results/participants. Note: Some participating laboratories did not serotype all samples.
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Fig. 3. Correctness of results depending on diagnostic assays used. The bars on the left side indicate correct results for hantavirus-antibody positivity (without typing), all other bars on the right side indicate correct results for hantavirus typing depending on the assay or assay combination used.
χ2 = 62.65, p < 0.001
Number of crossreactivity reporting participants / all participants (in %)
80
χ2 = 60.77, p < 0.001
167/220
70 358/577 60
50
314/693
40
106/307
30
20
10
0
acute PUUV
acute DOBV
past PUUV
past DOBV
status of infection
Fig. 4. Cross-reactivity of antibodies in the immunoblot assay. Above the bars the numbers of participants who reported cross-reactivity with the heterologous antigen as compared to all participants are given. Data collected were entered into Microsoft Excel (Microsoft Corp., Billingham, WA, USA). Cross-tables were calculated using EpiInfo 7 (http://wwwn.cdc.gov/epiinfo/). Results with respect to categorized variables were analyzed by Mantel–Haenszel test. A p-value <0.001 was considered to indicate statistical significance.
past infection, showed a higher proportion of cross-reactions with DOBV antigen than DOBV-positive sera with PUUV antigen. This difference turned out to be statistically significant. Comparable results were also obtained for EIA or IFA, however, due to the heterogeneity of applied EIA systems and the limited number of IFA-derived results, the data did not allow reliable statistical analyses. In spite of the high cross-reactions of DOBV or PUUV antigens with heterologous serotype specific antibodies observed with all applied test formats, immunoblots used alone or in combination with other test formats reliably led to correct differentiation between DOBV and PUUV infections, both acute and past. In this regard taking into account the reaction intensities of the individual antigen specific bands is helpful.
4. Discussion Hantavirus infections get increasing attention by patients and physicians but also by the diagnostic laboratories and IVD manufacturers. With increasing public awareness and increasing screening requests, reliable assays and test performances for the detection of hantavirus infections are needed. To ensure a high standard of the laboratory diagnostics of those emerging viruses like hantaviruses is an important demand for IVD manufacturers. EQA scheme can reveal the competence of the laboratories and the value of proficiency test formats applied. Another very valuable effect of external quality assessment is the training of the users. Especially before 2012, many inquiries
J. Hofmann et al. / International Journal of Medical Microbiology 305 (2015) 607–611
from public health institutions and clinics about putative double infections by different hantaviruses reached the National Consiliary Laboratory in Germany. These queries were caused by misinterpretations of immunoblot results due to cross-reaction of antibodies with more than one specific hantavirus antigen. Also previous travel activities of hantavirus-infected patients are of importance to interpret test results. Therefore, the participants of our EQA schemes obtained short anamnestic information with each sample that should help to appraise the test results. The decreasing number of inquiries to the National Consiliary Laboratory regarding interpretation of test results may serve as a suitable indicator for the increased competence in hantavirus diagnostics. Here we presented an overview of the results of 11 EQA schemes over a time period of 5 years involving a maximum of 92 participating diagnostic laboratories. In total, 3.3% of results were false positive for sera representing negative immune status and 5.0% were false negative for sera representing acute and past hantavirus infections (see Fig. 2). This shows an over-all specificity of 96.7% and sensitivity of 95% of the hantavirus serodiagnostics (without differentiation between serotype specific antibodies) by the participating laboratories. A correct distinction between acute and past sera was made in 90–96% of replies of laboratories. A recently published European external quality assessment study including 28 “expert laboratories” showed 88% correctness of hantavirus specific IgG detection but only 62% of IgM detection (Escadafal et al., 2012). These data and the results presented here show that there is some potential for further improvement of the test formats. Along with the proficiency tests, five new or improved IgG and IgM assays appeared on the market. On the other hand, three IgG and one IgM test were no longer used in the most recent proficiency tests. Due to the evident cross-reactivity of hantavirus antigens, not in all cases definitive antibody typing can be performed by serological standard approaches. Studying a large PUUV outbreak in Southern Germany, some sera could not be typed by EIA or immunoblot approaches but only by IFA and FRNT (Schilling et al., 2007). Among the sera of DOBV-infected patients from Northeastern Germany, 65/86 samples showed crossreactivity with PUUV antigen in EIA, but with much lower titers than with DOBV antigen (Hofmann et al., 2014). In our actual study, it seems that past DOBV infections are less efficiently recognized in terms of acute vs. past infections (90%) and PUUV vs. DOBV infections (81%) when compared with the other serum groups (acute PUUV, past PUUV, acute DOBV). One reason could be the long persistence of IgM in sera of some DOBV-infected patients which can be detected as late as 1 or 2 years after the clinical phase (Hofmann et al., 2014; Meisel et al., 2006). It should be mentioned that the only
611
way for an exact virus typing is by molecular approaches. Thus, the establishment of a proficiency test for hantaviral RNA is one of the future tasks. Acknowledgements Dipl.-Ing. Vanessa Lindig is gratefully acknowledged for the analyses of all proficiency testings and PD Dr. Werner Hopfenmueller for the consulting in statistical analyses. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ijmm.2015.08. 009. References Escadafal, C., Avsic-Zupanc, T., Vapalahti, O., Niklasson, B., Teichmann, A., Niedrig, M., Donoso-Mantke, O., 2012. Second external quality assurance study for the serological diagnosis of hantaviruses in Europe. PLoS Negl. Trop. Dis. 6 (4), e1607. Ettinger, J., Hofmann, J., Enders, M., Tewald, F., Oehme, R.M., Rosenfeld, U.M., Ali, H.S., Schlegel, M., Essbauer, S., Osterberg, A., Jacob, J., Reil, D., Klempa, B., Ulrich, R.G., Kruger, D.H., 2012. Multiple synchronous outbreaks of Puumala virus Germany, 2010. Emerg. Infect. Dis. 18 (9), 1461–1464. Hofmann, J., Meisel, H., Klempa, B., Vesenbeckh, S.M., Beck, R., Michel, D., Schmidt-Chanasit, J., Ulrich, R.G., Grund, S., Enders, G., Kruger, D.H., 2008. Hantavirus outbreak Germany, 2007. Emerg. Infect. Dis. 14 (5), 850–852. Hofmann, J., Meier, M., Enders, M., Fuhrer, A., Ettinger, J., Klempa, B., Schmidt, S., Ulrich, R.G., Kruger, D.H., 2014. Hantavirus disease in Germany due to infection with Dobrava-Belgrade virus genotype Kurkino. Clin. Microbiol. Infect. 20 (10), O648–O655. Klempa, B., Stanko, M., Labuda, M., Ulrich, R., Meisel, H., Kruger, D.H., 2005. Central European Dobrava Hantavirus isolate from a striped field mouse (Apodemus agrarius). J. Clin. Microbiol. 43 (6), 2756–2763. Klempa, B., Radosa, L., Kruger, D.H., 2013. The broad spectrum of hantaviruses and their hosts in Central Europe. Acta Virol. 57 (2), 130–137. Kruger, D.H., Schonrich, G., Klempa, B., 2011. Human pathogenic hantaviruses and prevention of infection. Hum. Vaccine 7 (6), 685–693. Meisel, H., Wolbert, A., Razanskiene, A., Marg, A., Kazaks, A., Sasnauskas, K., Pauli, G., Ulrich, R., Kruger, D.H., 2006. Development of novel immunoglobulin G (IgG) IgA, and IgM enzyme immunoassays based on recombinant Puumala and Dobrava hantavirus nucleocapsid proteins. Clin. Vaccine Immunol. 13 (12), 1349–1357. Schilling, S., Emmerich, P., Klempa, B., Auste, B., Schnaith, E., Schmitz, H., Kruger, D.H., Gunther, S., Meisel, H., 2007. Hantavirus disease outbreak in Germany: limitations of routine serological diagnostics and clustering of virus sequences of human and rodent origin. J. Clin. Microbiol. 45 (9), 3008–3014. Vaheri, A., Vapalahti, O., Plyusnin, A., 2008. How to diagnose hantavirus infections and detect them in rodents and insectivores. Rev. Med. Virol. 18 (4), 277–288. Vaheri, A., Henttonen, H., Voutilainen, L., Mustonen, J., Sironen, T., Vapalahti, O., 2013. Hantavirus infections in Europe and their impact on public health. Rev. Med. Virol. 23 (1), 35–49.
Contents lists available at ScienceDirect
International Journal of Medical Microbiology journal homepage: www.elsevier.com/locate/ijmm
Does proficiency testing improve the quality of hantavirus serodiagnostics? Experiences with INSTAND EQA schemes Jörg Hofmann a,∗ , Hans-Peter Grunert b , Oliver Donoso-Mantke c , Heinz Zeichhardt c,d , Detlev H. Kruger a a National Consultant Laboratory for Hantaviruses, Institute of Medical Virology, Helmut-Ruska-Haus, Charité—University Medicine, Charitéplatz 1, 10117 Berlin, Germany b GBD Gesellschaft für Biotechnologische Diagnostik mbH, Potsdamer Chaussee 80, 14129 Berlin, Germany c INSTAND e.V., Society for Promoting Quality Assurance in Medical Laboratories, Ubierstrasse 20, 40223 Düsseldorf, Germany d Institute of Virology, Campus Benjamin Franklin, Charité—University Medicine, Hindenburgdamm 27, 12203 Berlin, Germany
a r t i c l e
i n f o
Keywords: Hantavirus Puumala virus Dobrava-Belgrade virus Proficiency testing
a b s t r a c t Hantavirus infections in Germany appear periodically with peak numbers every 2–3 years. The reported cases in the years 2007, 2010 and 2012 exceeded many times over those in the years in-between. In order to reveal faults of certain in vitro diagnostic assays (IVDs), to harmonize the performances of the individual assays and to improve the users’ competence in interpreting the results, the National Consiliary Laboratory for Hantaviruses and INSTAND e.V. (Society for Promoting Quality Assurance in Medical Laboratories e.V.) established an external quality assessment (EQA) scheme for proficiency testing of hantavirus serodiagnostics. The first EQA scheme (pilot study) started in March 2009 with 58 participating laboratories from Germany and neighboring countries. Twice a year four serum samples were sent out to the participants to investigate whether the sample reflects an acute or past infection and to distinguish between infections with the hantavirus types Puumala virus (PUUV) and Dobrava-Belgrade virus (DOBV), both endemic in Central Europe. In addition, samples negative for anti-hantavirus antibodies were tested in order to examine the specificity of the IVDs applied in the participating laboratories. An increasing number of laboratories participated, with a maximum of 92 in March 2014. When summarizing in total 2592 test results, the laboratories reached an overall specificity of 96.7% and a sensitivity of 95% in their detection of a hantavirus infection. A correct distinction between acute and past infections was forwarded in 90–96% of replies of laboratories. Exact serotyping (PUUV vs. DOBV) of the infection was reported in 81–96% of replies with the lowest accuracy for past DOBV infections; cross-reactivities between diagnostic antigens of the two viruses as well as persistent IgM titers in humans may interfere with exact testing. The EQAs revealed acceptable results for the serodiagnostic of hantavirus infection including serotyping but further improvement is still needed. © 2015 Elsevier GmbH. All rights reserved.
1. Introduction Hantaviruses, members of Bunyaviridae family, are emerging viruses which cause Hemorrhagic Fever with Renal Syndrome (HFRS) in Asia and Europe and Hantavirus Cardiopulmonary Syndrome in the Americas (Vaheri et al., 2013). In Central Europe, Puumala virus (PUUV) and Dobrava-Belgrade virus (DOBV) are the two main pathogenic hantaviruses circulating in rodent hosts and transmitted to humans (Klempa et al., 2013). In Germany, a total of 7252 symptomatic HFRS cases were reported from 2007 to 2012
∗ Corresponding author. E-mail address: [email protected] (J. Hofmann). http://dx.doi.org/10.1016/j.ijmm.2015.08.009 1438-4221/© 2015 Elsevier GmbH. All rights reserved.
with increasing peak activities in the outbreak years 2007 (1687 cases), 2010 (2016 cases), and 2012 (2824 cases). During outbreak years, the hantavirus disease belongs to the group of the 5 most frequent notifiable virus diseases in Germany (www3.rki.de/SurvStat/ QueryForm.aspx). Large hantavirus outbreaks in Germany are caused by PUUV infections and a detailed molecular epidemiological characterization of PUUV strains from the different outbreak regions of the country has been established (Ettinger et al., 2012; Hofmann et al., 2008). Moreover, infections by the DOBV Kurkino genotype lead to additional cases of hantavirus disease (Hofmann et al., 2014). Since hantaviruses are strongly host-associated, infections of humans are linked to the particular virus type circulating in the geographic area where infected rodents live. Thus, PUUV infections appear mostly
608
J. Hofmann et al. / International Journal of Medical Microbiology 305 (2015) 607–611
in South and Southwest Germany, but DOBV infections in the North and Northeast, corresponding to the natural habitats of the rodent hosts, the bank vole (Myodes glareolus) and striped field mouse (Apodemus agrarius), respectively. Hantavirus diagnostic is based on seroassays, as enzyme immune assay (EIA), immunoblot, immunofluorescence assay (IFA), and rapid immunochromatographic strip assay. (Meisel et al., 2006; Vaheri et al., 2008). The antigen source in PUUV assays usually consists in a homologous (PUUV) nucleocapsid protein, while for DOBV assays – particularly in older IVDs – a heterologous (Hantaan virus) nucleocapsid antigen is used. DOBV and Hantaan virus nucleocapsid proteins share some homology in their amino acid sequences (Klempa et al., 2005), however, such cross-reactions complicate a reliable antibody particularly in low-titer sera (Meisel et al., 2006). Confirmatory assays – like the focus reduction neutralization test (FRNT) requiring BSL-3 conditions or RT-PCR assays – can be performed by highly specialized groups only but not by the majority of diagnostic laboratories. Moreover, whereas serotyping by FRNT is reported to be most specific when using sera of convalescent patients, the occurrence of detectable virus genomes in the blood is restricted to the very early phase after disease onset (Kruger et al., 2011). Therefore, the routine diagnostics is based on use of the above mentioned seroassays. Since hantavirus disease is notifiable in Germany according to the Infection Protection Act, a country-wide network of qualified laboratories is needed which uses assays of high reliability. We have established an external quality assessment (EQA) system and determined the diagnostic performance of more than 50 laboratories in Germany and neighboring countries twice a year. 2. Material and methods Serum samples were obtained from patients who were diagnosed in the National Consiliary Laboratory to be either IgG+/IgM− or IgG+/IgM+ seropositive for either PUUV or DOBV. After approval of the physician in charge, the patients were asked for giving a blood donation. The patients completed a disease-specific questionnaire, including potential infection source, clinical course, travel activities, which was later used as additional information for the participating laboratories. Certain acutely infected patients
were sampled again one year later providing valuable followup sera. In any case all clinical samples were taken from the patients after informed consent including clarification that the samples are exclusively used for EQA schemes and completely anonymized in order to exclude any traceability to personal data of the patients. Donated blood materials were processed at the GBD Gesellschaft für Biotechnologische Diagnostik mbH, Berlin. The resulting sera were characterized at the National Consiliary Laboratory and the GBD and were tested by PCR to be negative for hantavirus RNA in order to exclude infectivity especially in samples representing acute hantavirus infection (DOBV or PUUV). These well-investigated samples were sent to up to eight experienced target value laboratories for serological confirming the sample properties. If concordant results were obtained, the individual sample was included into the proficiency testing panel. In addition, samples negative for anti-hantavirus antibodies were tested in order to examine the specificity of the IVDs applied by the participating laboratories. According to the amount of the test material certain samples were used in more than one panel. The results reported by the participating laboratories and the correct interpretations were made available (in anonymous form) to every individual laboratory and also to non-participants on the homepage of INSTAND (http://www.instandev.de/en/eqas.html). Thus, information on the performance of single assays was distributed publicly. 3. Results Each EQA scheme comprised four samples from PUUV-infected, DOBV-infected, or non-infected individuals. Table 1 summarizes the number of participating laboratories (increasing from 58 in the pilot study of March 2009 to 92 in the EQA scheme of March 2014), the composition of each 4-sera panel, and the assays used by the participants during the 11 EQA schemes. The number of participating laboratories increased by about one-third after 2012, the year with multiple outbreaks and the highest number of reported cases. Fig. 1 gives an overview about the assays used by the participants. More than 50% of all analyses were performed exclusively by immunoblot assays, numerically followed by a combination of EIA and immunoblot (16% of analyses). The proportion of the single
Table 1 Number of participants, characteristics of the panel composition, and IVDs used in the EQAs from March 2009 to March 2014.
Participants (n) Sera provided in the panel Acute PUUV Past PUUV Acute DOBV Past DOBV Negative Assays used by participants EIA only IFA only Blot only EIA + IFA EIA + RT EIA + Blot IFA + Blot EIA + IFA + Blot EIA + RT + Blot EIA + IFA + RT RT + Blot RT + EIA + IFA + Blot IFA + RT IFA + RT + Blot
Pilot study
Sep 09
Mrz 10
Sep 10
Mrz 11
Sep 11
Mrz 12
Sep 12
Mrz 13
Sep 13
Mrz 14
Mrz 09 58
55
64
64
71
69
71
71
83
81
92
2 0 1 0 1
1 0 1 1 1
1 1 0 2 0
0 1 2 1 0
0 2 1 1 0
0 2 1 1 0
1 0 1 1 1
0 1 2 1 0
0 1 1 1 1
0 1 2 1 0
1 1 1 0 1
9 5 28 3 1 7 3 2 0 0 0 0 0 0
8 5 26 2 1 6 3 4 0 0 0 0 0 0
9 3 32 2 1 8 2 4 1 0 0 0 1 1
11 3 32 3 0 7 3 3 0 0 0 0 1 1
9 4 37 2 0 10 3 3 1 1 0 0 1 0
9 3 35 2 1 8 4 3 2 1 0 0 1 0
8 3 36 2 0 11 3 3 2 1 1 1 0 0
10 3 34 2 1 13 3 4 1 0 0 0 0 0
10 2 44 1 0 15 4 4 1 0 0 0 2 0
9 3 40 1 0 16 3 5 1 0 2 0 0 1
10 3 50 1 1 17 4 3 1 0 2 0 0 0
PUUV: Puumala virus, DOBV: Dobrava-Belgrade virus, EIA: enzyme immunoassay, IFA: immunofluorescence assay, RT: rapid test.
J. Hofmann et al. / International Journal of Medical Microbiology 305 (2015) 607–611
361/401
643/714
837/873
706/735
272/282 222/240
90
85 271/334
Correct results (in %)
95
2176/2291
291/301
assays or assay combinations used remained stable with only slight deviation between 2009 and 2014. For reasons of clarity and comprehensibility assay combinations used by only a few participants were summarized as “others” (for details see legend to Fig. 1) During 11 EQA schemes, four hantavirus-antibody negative specimens were included into the panels as negative controls. A total of 301 results were reported regarding these specimens; 291/301 answers were correct, however, 10/301 (3.3%) results were false-positive. On the basis of the hantavirus antibodypositive specimens distributed to the participants, 2291 results were reported. Out of them, 2176 replies were correct (“hantavirus antibody-positive”, no matter whether acute vs. past infection or what virus type) but 115/2291 (5.0%) were false negative (Fig. 2, left two columns). Furthermore, Fig. 2 shows the outcome of testing of the defined sera (representing acute PUUV infection, past PUUV infection, acute DOBV infection, or past DOBV infection) according to these criteria: (i) differentiation acute vs. past infection (black columns) and (ii) typing of PUUV vs. DOBV infection (grey columns). It should be mentioned that not all laboratories attempted to differentiate the
569/593
virus type. The identification of the virus type seems to be more difficult than the distinction between acute and past infection. The value of correct distinction of acute vs. past hantavirus infection, i.e. without typing for PUUV vs. DOBV infection, varies between about 96% for sera from acute PUUV, past PUUV, and acute DOBV infections on the one hand and 90% for sera from past DOBV infections on the other. Typing of the involved virus, however, was most successful for sera from past PUUV infections (96%) and showed the lowest success ratio for sera from past DOBV infections (81%)—see Fig. 2. As shown in Table 1, at least 14 different assays or assay combinations were used by the laboratories. However, routine diagnostics might not be performed with more than one assay, unless a laboratory finds a reactive sample which needs confirmatory testing. The results obtained with the different test formats were subdivided in those for the detection of acute vs. past PUUV and DOBV infections. One limitation was the different number of tests that were performed with each test format. Therefore, only the performances of the four most frequently used test combinations were compared (Fig. 3). The majority of laboratories used immunoblots to analyze the samples; however, this test format has some difficulties with the detection of past DOBV infections. The low performance of “EIA only” is remarkable. Behind the format EIA several enzyme immunoassays are summarized. Fifteen different IgG-EIA assays were used in the 09/2013 EQA scheme, including four home-brewed assays. The most frequently used assays are shown in supplementary Table S1. Especially those which use heterologous (Hantaan virus) antigens for the detection of DOBV antibody might be responsible for the high error rate. The differentiation of serotype specific antibodies is difficult because of the antigenic relatedness of hantavirus nucleocapsid proteins which frequently results in cross-reactions and, thus, potential misinterpretation of diagnostic data. Here we analyzed the cross-reaction in immunoblot assays because more than 95% participants used the same commercially available blot assay. Fig. 4 shows the reported data for sera representing acute and past PUUV and DOBV infections in the IgG immunoblot (excluding data from the pilot study and of samples with inconsistent results). PUUV IgG positive samples, independent on whether representing acute or
Fig. 1. Overview about single assays or assay combinations used by the participants. A total of 14 different assays or assay combinations were used by the participants. “12% Others” summarizes the following assay combinations: EIA and IFA, EIA and rapid test, EIA and rapid test and blot, rapid test and blot, and IFA and RT.
100
609
80
75
70 hantavirus negave
hantavirus posive
acute PUUV
past PUUV
acute DOBV
past DOBV
status of infecon
Fig. 2. Number of correct results of all proficiency tests. The grey bars indicate correct results regarding the overall hantavirus serostatus. Black columns represent results for acute vs. past hantavirus infection, and light grey bars depict those for differentiation of PUUV vs. DOBV infection. The numbers above the bars indicate correct results/participants. Note: Some participating laboratories did not serotype all samples.
610
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Fig. 3. Correctness of results depending on diagnostic assays used. The bars on the left side indicate correct results for hantavirus-antibody positivity (without typing), all other bars on the right side indicate correct results for hantavirus typing depending on the assay or assay combination used.
χ2 = 62.65, p < 0.001
Number of crossreactivity reporting participants / all participants (in %)
80
χ2 = 60.77, p < 0.001
167/220
70 358/577 60
50
314/693
40
106/307
30
20
10
0
acute PUUV
acute DOBV
past PUUV
past DOBV
status of infection
Fig. 4. Cross-reactivity of antibodies in the immunoblot assay. Above the bars the numbers of participants who reported cross-reactivity with the heterologous antigen as compared to all participants are given. Data collected were entered into Microsoft Excel (Microsoft Corp., Billingham, WA, USA). Cross-tables were calculated using EpiInfo 7 (http://wwwn.cdc.gov/epiinfo/). Results with respect to categorized variables were analyzed by Mantel–Haenszel test. A p-value <0.001 was considered to indicate statistical significance.
past infection, showed a higher proportion of cross-reactions with DOBV antigen than DOBV-positive sera with PUUV antigen. This difference turned out to be statistically significant. Comparable results were also obtained for EIA or IFA, however, due to the heterogeneity of applied EIA systems and the limited number of IFA-derived results, the data did not allow reliable statistical analyses. In spite of the high cross-reactions of DOBV or PUUV antigens with heterologous serotype specific antibodies observed with all applied test formats, immunoblots used alone or in combination with other test formats reliably led to correct differentiation between DOBV and PUUV infections, both acute and past. In this regard taking into account the reaction intensities of the individual antigen specific bands is helpful.
4. Discussion Hantavirus infections get increasing attention by patients and physicians but also by the diagnostic laboratories and IVD manufacturers. With increasing public awareness and increasing screening requests, reliable assays and test performances for the detection of hantavirus infections are needed. To ensure a high standard of the laboratory diagnostics of those emerging viruses like hantaviruses is an important demand for IVD manufacturers. EQA scheme can reveal the competence of the laboratories and the value of proficiency test formats applied. Another very valuable effect of external quality assessment is the training of the users. Especially before 2012, many inquiries
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from public health institutions and clinics about putative double infections by different hantaviruses reached the National Consiliary Laboratory in Germany. These queries were caused by misinterpretations of immunoblot results due to cross-reaction of antibodies with more than one specific hantavirus antigen. Also previous travel activities of hantavirus-infected patients are of importance to interpret test results. Therefore, the participants of our EQA schemes obtained short anamnestic information with each sample that should help to appraise the test results. The decreasing number of inquiries to the National Consiliary Laboratory regarding interpretation of test results may serve as a suitable indicator for the increased competence in hantavirus diagnostics. Here we presented an overview of the results of 11 EQA schemes over a time period of 5 years involving a maximum of 92 participating diagnostic laboratories. In total, 3.3% of results were false positive for sera representing negative immune status and 5.0% were false negative for sera representing acute and past hantavirus infections (see Fig. 2). This shows an over-all specificity of 96.7% and sensitivity of 95% of the hantavirus serodiagnostics (without differentiation between serotype specific antibodies) by the participating laboratories. A correct distinction between acute and past sera was made in 90–96% of replies of laboratories. A recently published European external quality assessment study including 28 “expert laboratories” showed 88% correctness of hantavirus specific IgG detection but only 62% of IgM detection (Escadafal et al., 2012). These data and the results presented here show that there is some potential for further improvement of the test formats. Along with the proficiency tests, five new or improved IgG and IgM assays appeared on the market. On the other hand, three IgG and one IgM test were no longer used in the most recent proficiency tests. Due to the evident cross-reactivity of hantavirus antigens, not in all cases definitive antibody typing can be performed by serological standard approaches. Studying a large PUUV outbreak in Southern Germany, some sera could not be typed by EIA or immunoblot approaches but only by IFA and FRNT (Schilling et al., 2007). Among the sera of DOBV-infected patients from Northeastern Germany, 65/86 samples showed crossreactivity with PUUV antigen in EIA, but with much lower titers than with DOBV antigen (Hofmann et al., 2014). In our actual study, it seems that past DOBV infections are less efficiently recognized in terms of acute vs. past infections (90%) and PUUV vs. DOBV infections (81%) when compared with the other serum groups (acute PUUV, past PUUV, acute DOBV). One reason could be the long persistence of IgM in sera of some DOBV-infected patients which can be detected as late as 1 or 2 years after the clinical phase (Hofmann et al., 2014; Meisel et al., 2006). It should be mentioned that the only
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way for an exact virus typing is by molecular approaches. Thus, the establishment of a proficiency test for hantaviral RNA is one of the future tasks. Acknowledgements Dipl.-Ing. Vanessa Lindig is gratefully acknowledged for the analyses of all proficiency testings and PD Dr. Werner Hopfenmueller for the consulting in statistical analyses. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ijmm.2015.08. 009. References Escadafal, C., Avsic-Zupanc, T., Vapalahti, O., Niklasson, B., Teichmann, A., Niedrig, M., Donoso-Mantke, O., 2012. Second external quality assurance study for the serological diagnosis of hantaviruses in Europe. PLoS Negl. Trop. Dis. 6 (4), e1607. Ettinger, J., Hofmann, J., Enders, M., Tewald, F., Oehme, R.M., Rosenfeld, U.M., Ali, H.S., Schlegel, M., Essbauer, S., Osterberg, A., Jacob, J., Reil, D., Klempa, B., Ulrich, R.G., Kruger, D.H., 2012. Multiple synchronous outbreaks of Puumala virus Germany, 2010. Emerg. Infect. Dis. 18 (9), 1461–1464. Hofmann, J., Meisel, H., Klempa, B., Vesenbeckh, S.M., Beck, R., Michel, D., Schmidt-Chanasit, J., Ulrich, R.G., Grund, S., Enders, G., Kruger, D.H., 2008. Hantavirus outbreak Germany, 2007. Emerg. Infect. Dis. 14 (5), 850–852. Hofmann, J., Meier, M., Enders, M., Fuhrer, A., Ettinger, J., Klempa, B., Schmidt, S., Ulrich, R.G., Kruger, D.H., 2014. Hantavirus disease in Germany due to infection with Dobrava-Belgrade virus genotype Kurkino. Clin. Microbiol. Infect. 20 (10), O648–O655. Klempa, B., Stanko, M., Labuda, M., Ulrich, R., Meisel, H., Kruger, D.H., 2005. Central European Dobrava Hantavirus isolate from a striped field mouse (Apodemus agrarius). J. Clin. Microbiol. 43 (6), 2756–2763. Klempa, B., Radosa, L., Kruger, D.H., 2013. The broad spectrum of hantaviruses and their hosts in Central Europe. Acta Virol. 57 (2), 130–137. Kruger, D.H., Schonrich, G., Klempa, B., 2011. Human pathogenic hantaviruses and prevention of infection. Hum. Vaccine 7 (6), 685–693. Meisel, H., Wolbert, A., Razanskiene, A., Marg, A., Kazaks, A., Sasnauskas, K., Pauli, G., Ulrich, R., Kruger, D.H., 2006. Development of novel immunoglobulin G (IgG) IgA, and IgM enzyme immunoassays based on recombinant Puumala and Dobrava hantavirus nucleocapsid proteins. Clin. Vaccine Immunol. 13 (12), 1349–1357. Schilling, S., Emmerich, P., Klempa, B., Auste, B., Schnaith, E., Schmitz, H., Kruger, D.H., Gunther, S., Meisel, H., 2007. Hantavirus disease outbreak in Germany: limitations of routine serological diagnostics and clustering of virus sequences of human and rodent origin. J. Clin. Microbiol. 45 (9), 3008–3014. Vaheri, A., Vapalahti, O., Plyusnin, A., 2008. How to diagnose hantavirus infections and detect them in rodents and insectivores. Rev. Med. Virol. 18 (4), 277–288. Vaheri, A., Henttonen, H., Voutilainen, L., Mustonen, J., Sironen, T., Vapalahti, O., 2013. Hantavirus infections in Europe and their impact on public health. Rev. Med. Virol. 23 (1), 35–49.