First results of a national external quality assessment scheme for the detection of SARS-CoV-2 genome sequences.

BACKGROUND: Broad and decentralised testing of SARS-CoV-2 RNA genomes is a WHO-recommended strategy to contain the SARS-CoV-2 pandemic by identifying infected cases in order to minimize onward transmission. With the need to increase the test capacities in Austria, nation-wide numerous laboratories rapidly implemented assays for molecular detection of SARS-CoV-2 based on real-time RT-PCR assays. The objective of this study was to monitor reliability of the laboratory results for SARS-CoV-2 RNA detection through an external quality assessment (EQA) scheme. METHODS: For this, the Center for Virology, Medical University of Vienna was tasked by the Federal Ministry of Social Affairs, Health, Care and Consumer Protection to perform the first Austrian EQA on SARS-CoV-2 which was organised in cooperation with the Austrian Association for Quality Assurance and Standardization of Medical and Diagnostic Tests (ÖQUASTA). Data were analysed on the basis of qualitative outcome of testing in relation to the nucleic acid (NA) extraction and detection methods used. RESULTS AND CONCLUSION: A total of 52 laboratories participated, contributing results from 67 test panels comprising 42 distinct combinations of NA extraction and PCR reagents. By testing 3 positive (CT values: S1, 28.4; S2, 33.6; S3, 38.5) and 1 negative sample, no false-positive results were obtained by any of the laboratories. Otherwise, 40/67 tests (60 %) detected all positive samples correctly as positive, but 25/67 tests (37 %) did not detect the weakest positive sample (S3), and 3 % reported S2 and S3 as false-negative. Improvement in test sensitivity by focusing on NA extraction and/or PCR-based detection is recommended.

Transmission of HIV Drug Resistance and the Predicted Effect on Current First-line Regimens in Europe.

BACKGROUND: Numerous studies have shown that baseline drug resistance patterns may influence the outcome of antiretroviral therapy. Therefore, guidelines recommend drug resistance testing to guide the choice of initial regimen. In addition to optimizing individual patient management, these baseline resistance data enable transmitted drug resistance (TDR) to be surveyed for public health purposes. The SPREAD program systematically collects data to gain insight into TDR occurring in Europe since 2001. METHODS: Demographic, clinical, and virological data from 4140 antiretroviral-naive human immunodeficiency virus (HIV)-infected individuals from 26 countries who were newly diagnosed between 2008 and 2010 were analyzed. Evidence of TDR was defined using the WHO list for surveillance of drug resistance mutations. Prevalence of TDR was assessed over time by comparing the results to SPREAD data from 2002 to 2007. Baseline susceptibility to antiretroviral drugs was predicted using the Stanford HIVdb program version 7.0. RESULTS: The overall prevalence of TDR did not change significantly over time and was 8.3% (95% confidence interval, 7.2%-9.5%) in 2008-2010. The most frequent indicators of TDR were nucleoside reverse transcriptase inhibitor (NRTI) mutations (4.5%), followed by nonnucleoside reverse transcriptase inhibitor (NNRTI) mutations (2.9%) and protease inhibitor mutations (2.0%). Baseline mutations were most predictive of reduced susceptibility to initial NNRTI-based regimens: 4.5% and 6.5% of patient isolates were predicted to have resistance to regimens containing efavirenz or rilpivirine, respectively, independent of current NRTI backbones. CONCLUSIONS: Although TDR was highest for NRTIs, the impact of baseline drug resistance patterns on susceptibility was largest for NNRTIs. The prevalence of TDR assessed by epidemiological surveys does not clearly indicate to what degree susceptibility to different drug classes is affected.

Primary resistance to integrase strand-transfer inhibitors in Europe.

OBJECTIVES: The objective of this study was to define the natural genotypic variation of the HIV-1 integrase gene across Europe for epidemiological surveillance of integrase strand-transfer inhibitor (InSTI) resistance. METHODS: This was a multicentre, cross-sectional study within the European SPREAD HIV resistance surveillance programme. A representative set of 300 samples was selected from 1950 naive HIV-positive subjects newly diagnosed in 2006-07. The prevalence of InSTI resistance was evaluated using quality-controlled baseline population sequencing of integrase. Signature raltegravir, elvitegravir and dolutegravir resistance mutations were defined according to the IAS-USA 2014 list. In addition, all integrase substitutions relative to HXB2 were identified, including those with a Stanford HIVdb score ≥ 10 to at least one InSTI. To rule out circulation of minority InSTI-resistant HIV, 65 samples were selected for 454 integrase sequencing. RESULTS: For the population sequencing analysis, 278 samples were retrieved and successfully analysed. No signature resistance mutations to any of the InSTIs were detected. Eleven (4%) subjects had mutations at resistance-associated positions with an HIVdb score ≥ 10. Of the 56 samples successfully analysed with 454 sequencing, no InSTI signature mutations were detected, whereas integrase substitutions with an HIVdb score ≥ 10 were found in 8 (14.3%) individuals. CONCLUSIONS: No signature InSTI-resistant variants were circulating in Europe before the introduction of InSTIs. However, polymorphisms contributing to InSTI resistance were not rare. As InSTI use becomes more widespread, continuous surveillance of primary InSTI resistance is warranted. These data will be key to modelling the kinetics of InSTI resistance transmission in Europe in the coming years.

High CXCL-16 levels correlate with symptomatic disease in lung transplant recipients with human cytomegalovirus replication in the allograft.

Human cytomegalovirus (HCMV) is an important pathogen in lung transplant recipients (LTRs). In LTRs, HCMV may replicate in the transplanted lung, and this is indicated by HCMV DNA detection in the bronchoalveolar lavage fluid (BALF). Local replication may occur without causing clinical symptoms or, in some patients, it may lead to symptomatic HCMV disease. In the present study, we analyzed whether HCMV replication in the allograft induces CXCL-16, a chemokine that may play a key role in the regulation of mucosal immunity, and investigated whether CXCL-16 levels in BALF can be used to differentiate LTRs with asymptomatic HCMV replication from patients who simultaneously develop disease. In total, BALF samples from 57 LTRs, of whom 8 developed HCMV disease, were assessed for CXCL-16 levels using a quantitative enzyme-linked immunosorbent assay. We found that HCMV replication in the lung triggered a significant rise in CXCL-16 levels in the BALF (p < 0.001, Wilcoxon signed-rank test). Furthermore, the CXCL-16 increase, induced by HCMV, was significantly lower in LTRs who did not develop HCMV disease (p < 0.001, Mann-Whitney U-test). Thus, CXCL-16 is a potential marker that may contribute to identify those LTRs in whom local HCMV replication in the lung remains asymptomatic.

Analysis of plasma surfactant protein D levels in lung transplant recipients.

In lung transplant recipients (LTRs), severe clinical complications, such as microbial infections of the lung or transplant rejection, may occur. Surfactant protein D (SP-D) is a C-type lectin that is mainly produced in alveolar type II cells. Plasma SP-D levels are usually low, but may increase when the lung-blood barrier is impaired. In this study, we analyzed whether plasma SP-D concentrations reflect rejection or infection of the lung allograft. An enzyme-linked immunosorbent assay was used to measure SP-D levels in plasma samples from 58 LTRs during intervals without pathologic respiratory findings and during episodes of acute cellular rejection (ACR), microbial colonization, and microbial pneumonia. Median plasma SP-D levels were significantly increased during episodes of microbial pneumonia, but not in the absence of pathologic respiratory findings, during microbial colonization, or during ACR up to grade A2-A3 (P < 0.05). During pneumonia, an increased plasma SP-D level was detected in 60% of LTRs and this was further associated with a significantly higher risk for the patients to develop stage III bronchiolitis obliterans syndrome (BOS III) or to die within the subsequent 6 months after pneumonia (P = 0.0093). All patients with a plasma SP-D level of >300 ng/mL during pneumonia developed BOS III and/or died within 6 months of follow-up (P = 0.001). The determination of SP-D levels in plasma during pneumonia in LTRs may be of prognostic value and warrants further evaluation.

Association of human cytomegalovirus DNAaemia and specific granzyme B responses in lung transplant recipients.

In lung transplant recipients (LTRs), human cytomegalovirus (HCMV) DNAaemia could be associated with HCMV disease and reduced allograft survival. In the present study we analysed whether or not HCMV-specific granzyme B (Grz-B) responses indicating CD8(+) T cell cytotoxicity exert an impact on HCMV DNAaemia and relate to specific interferon (IFN)-γ secretion. HCMV-specific Grz-B responses were quantitated by enzyme-linked immunosorbent assay (ELISA) in 70 samples from 39 HCMV seropositive LTRs who were prospectively investigated for HCMV DNA plasma levels and IFN-γ kinetics using a standardized CD8(+) T cell assay (QuantiFERON®-CMV assay). In all LTRs who were protected from HCMV DNAaemia by early and persistent IFN-γ responses, Grz-B responses were also detected. In LTRs who developed episodes of HCMV DNAaemia, the Grz-B responses which were detected prior to viral DNA detection differed significantly in patients who experienced episodes with high (exceeding 1000 copies/ml) and low plasma DNA levels (P = 0·0290, Fisher's exact test). Furthermore, the extent of Grz-B release prior to viral DNAaemia correlated statistically with the detected levels of IFN-γ (P < 0·0001, Spearman's rank test). Of note, simultaneous detection of Grz-B and IFN-γ secretion was associated significantly with protection from high HCMV DNA plasma levels during the subsequent follow-up (P = 0·0057, Fisher's exact test), and this association was stronger than for IFN-γ detection alone. We conclude that, in addition to IFN-γ responses, Grz-B secretion by CD8(+) T cells is essential to control HCMV replication and a simultaneous measurement of IFN-γ and Grz-B could contribute to the immune monitoring of LTRs.

Prospective analysis of human cytomegalovirus DNAemia and specific CD8+ T cell responses in lung transplant recipients.

In lung transplant recipients (LuTRs), human cytomegalovirus (HCMV) DNAemia may be associated with HCMV disease and reduced survival of the allograft. Because T cells are essential for controlling HCMV replication, we investigated in this prospective study whether the kinetics of plasma HCMV DNA loads in LuTRs are associated with HCMV-specific CD8+ T cell responses, which were longitudinally assessed using a standardized assay. Sixty-seven LuTRs were monitored during the first year posttransplantation, with a mean of 17 HCMV DNA PCR quantifications and 11.5 CD8+ T cell tests performed per patient. HCMV-specific CD8+ T cell responses displayed variable kinetics in different patients, differed significantly before the onset of HCMV DNAemia in LuTRs who subsequently experienced episodes of DNAemia with high (>1000 copies/mL) and low plasma DNA levels (p = 0.0046, Fisher's exact test), and were absent before HCMV disease. In HCMV-seropositive LuTRs, high-level DNAemia requiring preemptive therapy occurred more frequently when HCMV-specific CD8+ T cell responses fluctuated, were detected only after HCMV DNA detection, or remained undetectable (p = 0.0392, Fisher's exact test). Thus, our data indicate that HCMV-specific CD8+ T cells influence the magnitude of HCMV DNAemia episodes, and we propose that a standardized measurement of CD8+ T cell immunity might contribute to monitoring the immune status of LuTRs posttransplantation.

Human cytomegalovirus infection in lung transplant recipients triggers a CXCL-10 response.

Human cytomegalovirus (HCMV) causes significant morbidity in lung transplant recipients (LTRs). The clinical effects of HCMV replication are determined partly by a type 1 T-helper cell (Th1) response. Because the chemokine interferon-inducible protein of 10 kilodaltons (IP-10, CXCL-10) induces a Th1 response, we investigated whether HCMV triggers IP-10 in LTRs. The IP-10 concentration and HCMV DNA load were determined in 107 plasma and 46 bronchoalveolar lavage fluid (BALF) samples from 36 LTRs. Initial HCMV detection posttransplantation was significantly associated with increased plasma IP-10, regardless of whether the patients showed HCMV DNAemia (p = 0.001) or HCMV replication only in the allograft (p < 0.0001). In subsequent episodes of HCMV detection, plasma IP-10 increased regardless of whether HCMV was detected in blood (p = 0.0078) or only in BALF (p < 0.0001) and decreased after successful antiviral therapy (p = 0.0005). Furthermore, levels of HCMV DNA and IP-10 correlated statistically (p = 0.0033). Increased IP-10 levels in HCMV-positive BALF samples were significantly associated with severe airflow obstruction, as indicated by a decrease in forced expiratory volume in one second (FEV1). Our data indicate that HCMV replication in LTRs evokes a plasma IP-10 response and that, when an IP-10 response is observed in BALF, it is associated with inflammatory airway obstruction in the allograft.

HIV-1 drug-resistance patterns among patients on failing treatment in a large number of European countries.

BACKGROUND: Information about patterns of HIV-1 drug resistance among treatment-exposed patients is crucial for the development of novel effective drugs. Currently no system exists that monitors patterns of resistance in patients failing therapy. METHODS: The study included 1,988 HIV-1 sequences from patients experiencing therapy failure collected between 2000 and 2004 in 15 European countries. Genotypic resistance was interpreted using the ANRS algorithm. Phenotypic resistance was predicted using the Virco geno- to phenotype system. RESULTS: 80.7% of the sequences included at least one drug-resistance mutation. Mutations were found for NRTIs (73.5%), NNRTIs (48.5%), and protease inhibitors (35.8%). Ninety percent of sequences with genotypic resistance harbored M184V, M41L, K103N, D67N, and/or T215Y. Among NRTIs, resistance was most frequently predicted for lamivudine. About half of all sequences had reduced susceptibility for NNRTIs. Resistance to most boosted protease inhibitors was found in < 25%. No sequence had resistance to all currently available drugs. CONCLUSION: Levels of resistance among patients with therapy failure were high. The patterns of resistance reflect resistance to drugs available for a longer time. Fully suppressive regimens can be designed even for the most mutated HIV because boosted protease inhibitors have remained active against most circulating viruses and new drug classes have become available.

Comparison of virtual phenotype and HIV-SEQ program (Stanford) interpretation for predicting drug resistance of HIV strains.

OBJECTIVE: Drug resistant HIV strains can be identified by genotypic analysis. The prediction of resistance from the HIV pol sequence, however, requires expert interpretation which is currently provided by various interpretation programs. In the present study, the comparability of two of these programs was investigated. METHODS: One hundred and six HIV sequences obtained from patient samples were subjected to interpretation by the Stanford HIV-SEQ program (http://hivdb.stanford.edu) and, in parallel, by virtual phenotype analysis (VircoNET). RESULTS: The overall concordance between the two assays was high with respect to nonnucleoside reverse transcriptase inhibitors (NNRTIs) and protease inhibitors. Highly discrepant results were obtained from 22 samples (1.5% of all comparisons), and these discrepancies were significantly associated with the interpretation of nucleoside reverse transcriptase inhibitors (NRTIs) (P < 0.01), especially regarding resistance to zalcitabine (ddC), didanosine (ddI) and abacavir (ABC). Nearly all of these discrepant samples showed a sensitive virtual phenotype. Mutations at codons 69 and 74 were associated with a discrepant interpretation for ddC. CONCLUSIONS: The prediction of resistance to certain NRTIs from a given HIV sequence may be contradictory and requires further investigation.

Screening for possible failure of herpes simplex virus PCR in cerebrospinal fluid for the diagnosis of herpes simplex encephalitis.

The objectives of this study were to evaluate the reliability of herpes simplex virus (HSV) PCR testing in cerebrospinal fluid (CSF) for the detection of herpes simplex encephalitis. This was done by examining retrospectively the clinical follow-up of a large group of patients tested routinely by HSV-PCR. In addition, an attempt was made to assess the incidence of herpes simplex encephalitis in a central European population. CSF samples from 1,427 patients from all Vienna hospitals were submitted for HSV-PCR testing during a period of 4 years and 8 months. Herpes simplex encephalitis was detected by PCR in 12 cases and by serological methods in one additional patient. Retrospective analysis of the course of disease, which was possible in 799 PCR-negative patients, led to the identification of three additional cases in which herpes simplex encephalitis appears to have occurred despite negative PCR results. Failure of the PCR in these patients is most likely due to the time of obtaining CSF during the course of disease. A high specificity of the assay was demonstrated by the lack of false positive results in any of the 708 cases in which other causes for the neurological symptoms had been identified in the follow-up. The incidence of herpes simplex encephalitis in the population of Vienna was between 1 case/469,000-577,000 individuals/year. The highest annual incidence was detected in the age group between 3 months and 3 years, which, however, could not be confirmed statistically.

Comparison of sequence analysis and the INNO-LiPA HBV DR line probe assay for detection of lamivudine-resistant hepatitis B virus strains in patients under various clinical conditions.

A line probe assay (INNO-LiPA HBV DR) detecting drug-resistant hepatitis B virus (HBV) strains was evaluated. Results concordant with sequence analysis were obtained with 48 of 56 serum samples from HBV-infected patients undergoing lamivudine therapy. In eight cases, additional minor subpopulations could be identified by the line probe assay.

Twelve months of lamivudine treatment for chronic hepatitis B virus infection in renal transplant recipients.

BACKGROUND: Chronic hepatitis B virus (HBV) infection increases morbidity and mortality in renal transplant recipients (RTR). Lamivudine has shown promising results in patients with chronic hepatitis B, but experience with its use in RTR is limited. METHODS: In a prospective, open labeled, uncontrolled trial, 19 HBsAg(+) RTR were treated with lamivudine for 12 months. HBV-serologic analysis, HBV-DNA quantitation, and HBV genome sequence analysis were performed every 3 months. RESULTS: At baseline 16 patients were HBV DNA(+), 12 patients were HBeAg(+)/Ab (-). After 3 months HBV DNA was negative in 80% of patients. In the 3 patients with elevated liver enzymes, normal values were achieved within 12 weeks. At 12 months 4 of 8 HBeAg(+)/Ab(-) patients on treatment showed HBeAb, two of them with loss of HBeAg. Three patients developed mutations of the HBV polymerase gene associated with lamivudine resistance. CONCLUSIONS: Lamivudine is safe and effective in HB-sAg(+) RTR, the rate of HBe-seroconversion and of lamivudine-resistance is comparable to that of nonimmunosuppressed patients.

Monitoring the virus load can predict the emergence of drug-resistant hepatitis B virus strains in renal transplantation patients during lamivudine therapy.

The development of resistant hepatitis B virus (HBV) strains during lamivudine treatment has been described repeatedly. To investigate whether the development of such resistant HBV strains can be predicted in an early phase of therapy, the HBV loads of 11 renal transplantation patients were screened at 3-month intervals by a quantitative HBV polymerase chain reaction (PCR) assay. Lamivudine resistance was detected by sequence analysis. Five patients developed resistance to lamivudine in the 12-15-month follow-up period. In all of them, a virus load of 1x103 HBV DNA copies still was detectable after 3 months of therapy. This was statistically significantly different from those patients who did not develop lamivudine resistance within the observation period, all of whom had no HBV DNA detectable after 3 months of treatment (P=.0022). Thus, virus load testing by use of a sensitive PCR assay allows the early prediction of the emergence of lamivudine-resistant HBV strains.

Comparison of line probe assay (LIPA) and sequence analysis for detection of HIV-1 drug resistance.

The identification of HIV strains that are resistant to antiretroviral drugs, which emerge during a patient's therapy or are already present in infected individuals prior to treatment, is of increasing importance for the clinical management of HIV-infected persons. Two different methods were compared for the genotypic detection of resistance development in the HIV-1 reverse transcriptase (RT) gene, the commonly used sequence analysis, and the commercially available RT-line immunoprobe assay (LIPA), which can detect mutations at six separate codons of the RT gene, which are known to confer resistance to certain nucleoside inhibitors. Eighty serum samples from HIV-1-infected persons, some of whom were receiving antiretroviral therapy, were investigated in parallel by sequencing as well as by LIPA. LIPA results agreed with sequence data in the vast majority of the cases. However, in 40% of the samples, LIPA failed to yield evaluable results for one or more of the codon positions. In particular, LIPA detection rate was low at codon 41 (75%), whereas at codons 69/70, 74, 184, and 215 results were obtained from 90%-95% of the samples. A number of mutations in the vicinity of the respective codons were detected by sequencing, and these may have been responsible for the LIPA hybridization failure. There remained a number of samples, however, where no explanation for the lack of hybridization could be derived from sequence data. Our results indicate that the use of the LIPA does not eliminate the need for sequence analysis for detection of drug-resistant HIV strains.

Introduction of HIV-1 subtypes C, E and A into Austria.

BACKGROUND: Different subtypes of HIV-1 are prevalent in various geographical regions. Knowledge of their distribution is of importance with respect to possible differences in biological properties (such as reported for subtype E) as well as to diagnostic problems that may arise when specific subtypes are not recognized by standard serological assays. OBJECTIVES: The objectives of this study were to investigate the presence of the five major subtypes of HIV-1 (A-E) in the Austrian population and to estimate the prevalence of the individual subtypes in different risk groups. STUDY DESIGN: Serum samples from 88 HIV-1 positive patients were tested for the presence of subtype-specific antibodies using a peptide ELISA. RESULTS: The majority of the patients were shown to be infected with HIV-1 subtype B, but infections with subtypes A, C, and E were also detected in the Austrian population, primarily in the heterosexual transmission group. While subtypes A and C were probably imported from different African countries, subtype E appears to have been introduced by sex tourists returning from Thailand. CONCLUSION: Introduction of HIV subtypes other than B from Africa and Asia into Austria has already occurred and will certainly increase within the next few years.

Possible influence of the mutant CCR5 Allele on vertical transmission of HIV-1.

A possible correlation between the rate of vertical transmission of HIV-1 and the presence of the defective HIV co-receptor gene delta 32ccr5 in the chromosomes of infants born to HIV-positive mothers was assessed. The prevalence and genotypic distribution of the delta 32ccr5 gene were studied in 451 uninfected and 225 HIV-1-infected adults and 79 children born to HIV-1-positive mothers in Austria (45 uninfected and 34 infected by vertical transmission). As expected in a Caucasian population, the delta 32ccr5 allele was found in uninfected Austrians at a frequency of 10% (17.3% heterozygotes and 1.3% delta 32ccr5/ delta 32ccr5 homozygotes, consistent with the expected Hardy-Weinberg distribution). The mutant allele frequency was 11.1% in uninfected children (17.8% heterozygotes, 2.2% homozygotes) and 9.6% in HIV-positive adults (19.1% heterozygotes but no delta 32ccr5/delta 32ccr5 homozygotes). Among the group of 34 vertically infected children, however, there were only two heterozygotes and no delta 32ccr5/delta 32ccr5 homozygotes, corresponding to a significantly reduced mutant allele frequency of 2.9% (P = 0.05 compared to HIV-negative children). These results suggest that CCR5/delta 32ccr5 heterozygous children are less susceptible to vertical transmission of HIV-1. The data also support the hypothesis that delta 32ccr5 homozygous individuals are resistant to HIV-1 infection.

Resistance--clinical significance and diagnostics.

One of the most important factors, inhibiting the success of antiretroviral treatment of HIV infections is the development of drug resistant HIV strains in the patients during therapy. Detection and identification of such resistant virus strains is therefore becoming increasingly important for the clinical management of the patients, as well as for the detection of the transmission of already resistant virus strains. In the following paper different methods for the genotypic and phenotypic screening for resistant HIV-strains are compared and the problems of resistance testing are discussed.

Identification of tick-borne encephalitis virus ribonucleic acid in tick suspensions and in clinical specimens by a reverse transcription-nested polymerase chain reaction assay.

BACKGROUND: Tick-borne encephalitis virus (TBEV) is a major human pathogenic flavivirus. Sensitive assays for the detection of viral RNA may be valuable both for the identification of virus in ticks as well as for diagnostic purposes. OBJECTIVES: (1) The development of a sensitive polymerase chain reaction (PCR) test system for the detection of TBEV-RNA and its application to the identification of infected ticks; and (2) evaluation of the PCR assay for diagnostic purposes, i.e., detection of TBE virus RNA in blood and in cerebrospinal fluid (CSF) of TBE patients. STUDY DESIGN: (1) Establishment of a TBEV-specific reverse transcription (RT)-nested PCR assay and evaluation of its sensitivity; (2) comparison of the PCR assay with that of virus isolation from tick suspensions; and (3) investigation of 105; serum and CSF samples from patients with serologically confirmed TBE by RT-nested PCR. RESULTS: An RT-nested PCR assay was established with a detection limit of 100-1000 copies of TBEV RNA. All tick suspensions from which the virus could be isolated by inoculation of suckling mice also screened positive in the PCR assay. Of the 105 clinical samples investigated, only one serum and one CSF sample were positive by PCR assay, and these were both obtained very early in the course of the disease. CONCLUSIONS: The PCR assay described is valuable for the detection of TBEV in tick suspensions and can substitute for the usual virus isolation procedure in which suckling mice are inoculated. Its application for diagnostic purposes, however, does not seem to provide a significant improvement over serological diagnosis. Only in very rare cases, when a sample is drawn extremely early in the course of disease, may TBEV RNA be detected in serum or CSF before the appearance of specific IgM antibodies and thus allow an earlier diagnosis.

Antibodies to the nonstructural protein of parvovirus B19 in persistently infected patients: implications for pathogenesis.

Three patients with persistent parvovirus B19 infection, as documented by the prolonged presence of IgM directed to the viral capsid proteins and detection of viral DNA in serum by dot-blot hybridization or polymerase chain reaction (PCR), were investigated for the presence of antibodies to the nonstructural protein NS-1 of parvovirus B19. This was done by using an ELISA based on recombinant NS-1 protein. Whereas control sera displayed no reactivity, sera from persistently infected patients showed a strong specific antibody response to NS-1. Patients were followed for 3-18 months, during which IgM titers declined but IgG directed to the nonstructural protein remained detectable. The appearance of NS-1-specific antibodies might indicate an altered course of viral infection leading to the establishment of persistently active infection and subsequent destruction of cells of nonerythroid lineage.