Kremer JR, Langlet J, Skraber S, Weicherding P, Weber B, Cauchie HM, De Landtsheer S, Even J, Muller CP, Hoffmann L, Mossong J.

The genetic diversity of norovirus strains obtained from gastroenteritis outbreaks, sporadic case surveillance, and wastewater plants was compared in Luxembourg from October 2008 until June 2009. Except for GI.6 and GIV.1 strains detected exclusively in wastewater, all other genotypes were also found in human samples. Of the 9 NoV genotypes detected, only three (GII.4, GIIb/II.3, GIIc/II.12) were associated with institutional outbreaks. The majority of sequences from all sources belonged to genotype GII.4 including two potentially new sub-variants. Strains collected in the context of outbreaks may significantly underrepresent the overall genetic diversity of NoVs circulating in a country.
Institute of Immunology, National Health Laboratory and Centre de Recherche Public - Santé, 20A rue Auguste Lumière, L-1950 Luxembourg, Luxembourg Surveillance & Epidemiology of Infectious Diseases, National Health Laboratory, 42 rue du Laboratoire, L-191

Bernard Weber¹, Marisol Badiel², Yanet Alvarez-Otero², Philippe Thulliez³, José G. Montoya<sup>4,5


1</sup> Laboratoires Réunis, Junglinster, Luxembourg
² Fundacion Valle del Lili, Cali, Colombia
³ Laboratoire d'Immunoanalyses & Recherche sur la Toxoplasmose, Institut de Puériculture et de Périnatalogie (IPP), Paris, France
⁴ Palo Alto Medical Foundation-Toxoplasmosis Serology Laboratory, Palo Alto, CA, USA
⁵ Stanford University School of Medicine, Stanford, CA, USA

INTRODUCTION

Toxoplasmosis is caused by the unicellular protozoan parasite Toxoplasma gondii (T. gondii). In immunocompetent subjects, primary infection is usually asymptomatic or associated with self-limited symptoms such as fever, malaise, and cervical lymphadenopathy. Infection acquired during pregnancy is frequently associated with transmission of T. gondii to the fetus, resulting in congenital disease. In immunocompromised patients, reactivation of latent infection can cause life-threatening complications.

Infection with the parasite is widespread throughout the world, but the seroprevalence varies considerably between countries 10% to more than 90%) and population groups. Toxoplasmosis is more prevalent in some regions of Europe, in Latin America, and sub-Saharan Africa, than in Asia, North America, and Oceania. The impact of toxoplasmosis on public health is especially high in Latin America. Severe clinical forms with visceral localization, mainly pulmonary involvement, have been described after a primary infection in immunocompetent subjects in French Guiana. A high prevalence of ocular toxoplasmosis has also been reported in Brazil. T. gondii strains isolated from these subjects exhibited an atypical genotype, highly divergent from European and North American lineages. [...]


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Alain Menzel¹ und Eckart Meese<sup>2

1</sup> Laboratiores Réunis, Junglinster, Luxembourg

² Institut für Humangenetik, Universität des Saarland, Deutschland  

URO-NEWS 12·,2008,1-4  


Abstract:
Mithilfe der Untersuchung auf genetische Polymorphismen kann zum einen analysiert werden, ob bestimmte Gene im Zusammenhang mit einer Tumorform stehen, zum anderen könnte damit auch eine Typisierung möglich werden, die bei der Therapieplanung hilfreich ist.

Bittenbring J, Parisot F, Wabo A, Mueller M, Kerschenmeyer L, Kreuz M, Truemper L, Landt O, Menzel A, Pfreundschuh M, Roemer K.

BMC Cancer 2008, 8 :116  

ABSTRACT:
Background: SNP309 T/G (rs2279744) causes higher levels of MDM2, the most important negative regulator of the p53 tumor suppressor. SNP72 G/C (rs1042522) gives rise to a p53 protein with a greatly reduced capacity to induce apoptosis. Both polymorphisms have been implicated in cancer. The SNP309 G-allele has recently been reported to accelerate diffuse large B-cell lymphoma (DLBCL) formation in pre-menopausal women and suggested to constitute a genetic basis for estrogen affecting human tumorigenesis. Here we asked whether SNP309 and SNP72 are associated with DLBCL in women and are correlated with age of onset, diagnosis, or patient’s survival.

Methods: SNP309 and SNP72 were PCR-genotyped in a case-control study that included 512 controls and 311 patients diagnosed with aggressive NHL. Of these, 205 were diagnosed with DLBCL. 

Results: The age of onset was similar in men and women. The control and patients group showed similar SNP309 and SNP72 genotype frequencies. Importantly and in contrast to the previous findings, similar genotype frequencies were observed in female patients diagnosed by 51 years of age and those diagnosed later. Specifically, 3/20 female DLBCL patients diagnosed by 51 years of age were homozygous for SNP309 G and 2/20 DLBCL females in that age group were homozygous for SNP72 C. Neither SNP309 nor SNP72 had a significant influence on event-free and overall survival in multivariate analyses.

Conclusion: In contrast to the previous study on Ashkenazi Jewish Caucasians, DLBCL in pre-menopausal women of central European Caucasian ethnicity was not associated with SNP309 G. Neither SNP309 nor SNP72 seem to be correlated with age of onset, diagnosis, or survival of patients.

Olinger CM

The hepatitis B virus (HBV) is currently estimated to chronically infect almost 400 million people worldwide of which 65 millions live on the African continent. Based on the nucleotide distance between complete genomes, the circulating hepatitis B strains have been classified into different genotypes and subtypes most of which present a specific geographic distribution. Numerous phylogenetic studies have determined the genotypes and subtypes of HBV circulating in Africa, and have revealed a surprising picture. The most prevalent genotype found was genotype E, mostly in sub-Saharan Africa followed by genotype A distributed in sub-Saharan Africa but also in the South and East of Africa, and genotype D which was mostly found in North Africa. While the evolutionary history of genotype A has seen an obvious diversification with the appearance of several subtypes, genotype E did not undergo a similar evolutionary history. In fact, genotype E and A co-circulate in several countries but although genotype A strains show a high diversity from country to country, the diversity among genotype E strains found in the same countries is very low. While this low diversity suggests a recent appearance of genotype E in Africa it is paradoxical to the fact that the same genotype is found on more than a third of the African continent. This review summarizes the current knowledge about the HBV genotypes circulating in Africa and laboratory diagnosis.

Journal of Laboratory Medicine. July/August 2008

Olinger CM, Weber B, Otegbayo JA, Ammerlaan W, van der Taelem-Brulé N, Muller CP

The major neutralizing epitope, the “a” determinant of the hepatitis B virus (HBV) genotype E surface antigen (HBsAg) is most divergent from that of genotype A, which is used for preparing monoclonal antibodies used in commercially available HBV reagents. To evaluate the performance of the latest generation of HBsAg detection assays with respect to genotype E HBsAg. Three commercial assays were evaluated using sera from 200 Nigerian patients compared to the preS/S sequence of DNA positive samples. Out of 200 samples, 61 and 103 gave concordant positive and negative results between the three HBsAg assays. Of 36 samples with discordant results, 35 were con- Wrmed negative by neutralisation. One of the three assays showed signiWcantly high rate of false positives (29 of 35). DNA positive samples with no detectable HBsAg or reduced HBsAg detection signals (<75% of mean signal obtained with HBsAg positive samples) revealed several mutations (V14A, F46S, N48T, L49R, I49T, D51G, A53V, P54L, Q82P, F83C, L127P, A184V, T189I, S204N, V224A), mostly outside the a-determinant. Several of these mutations are found as wild type nucleotides normally in genotype A and only exceptionally in genotype E. All three assays showed comparable sensitivities for genotype E HBsAg detection (98.4–100%) but diVered considerably in speciWcity (84–99%). Failure to detect HBsAg antigen and diVerences in signal intensity were mainly associated with mutations in the preS/S gene outside the “a” determinant.

Medical Microbiology and Immunology. December 2007

Kessler HH, Santner B, Rabenau H, Berger A, Vince A, Lewinski C, Weber B, Pierer K, Stuenzner D, Mar

The AMPLICOR Enterovirus Test was evaluated with 103 cerebrospinal fluid (CSF) specimens. Twenty-seven CSF specimens were culture positive. With the AMPLICOR test, enterovirus RNA was detected in 34 specimens. Compared with culture, the AMPLICOR test gave a sensitivity of 96.3% and a specificity of 100%. The sensitivity of culture was 79.4% in comparison with the AMPLICOR test.

J Clin Microbiol 1997; 35: 976

Weber B, Behrens N , Doerr HW.


J Virol Meth 1997; 63: 137-143

Kutter D.

The Bayer-Technicon hematological devices differentiate leukocytes by their peroxidase activity and their volume, displaying them as separate clusters. Peroxidase deficiencies are manifested by the irregular location of these clusters. This makes it possible to identify persons totally or partially lacking myeloperoxidase. The deficiency is quantified by the myeloperoxidase index, which is expressed for every routine analysis and for which normal values were determined. Values of the myeloperoxidase index confirm varying degrees of deficiency and prevalence. Family studies using these degrees show that the hereditary pattern must be more complicated than the classical autosomal-recessive mode. A bigenic mode is suggested. While about half of the totally deficient individuals detected were free of typical symptoms, in the other half we found infectious complications that were sometimes life-threatening. The hematological devices allow the identification of persons suffering from eosinoperoxidase deficiency and from MPO deficiency of the monocytes. The latter symptom seems to indicate immaturity of these cells and may lead to unexpected diagnosis of malignancy.

J Mol Med 1998:76; 669-675.

Kutter D.


Bull Soc Lux Biol Clin 1998; 1

Kutter D.

The aim of the classical multiple test strip is to perform routine chemical analysis in one single operation, yielding maximum diagnostic and prognostic information. A maximum of 10 test areas should not be exceeded. The efficiency of the currently marketed test strips is discussed and improvements within acceptable price limits are suggested.

Clin Chim Acta. 2000; 297: 297-304

Hoy A, Leininger-Muller B, Kutter D, Siest G, Visvikis S.

Myeloperoxidase (MPO) is a glycoprotein released by activated polymorphonuclear neutrophils, which takes part in the defense of the organism through production of hypochlorous acid (HOCl), a potent oxidant. Since the discovery of MPO deficiency, initially regarded as rare and restricted to patients suffering from severe infections, MPO has attracted clinical attention. The development of new technologies allowing screening for this defect has permitted new advances in the comprehension of underlying mechanisms. Apart from its implications for host defense, the expression of MPO restricted to myeloid precursors makes MPO mRNA a good marker of acute myeloid leukemia. In addition, during the last few years, involvement of MPO has been described in numerous diseases such as atherosclerosis, lung cancer, Alzheimer's disease and multiple sclerosis. Both strong oxidative activity and MPO genetic polymorphism have been involved. This review summarizes the broad range of diseases involving MPO and points out the possible use of this protein as a new clinical marker and a future therapeutic target.

Clin Chem Lab Med 2002; 40: 2-8

Kutter D, Coulon N, Stirn F, Thoma M, Janecki J.

The double laser beam diffraction of spherized RBC used in the ADVIA 120 haematological analyser allows quantitation of cells aberrant not only by their volume but also by their haemoglobin concentration. The present investigation provides arguments for the identification of hyperchromic RBC as spherocytes, mainly the close relation between % hyperchromic cells and % lysed by the cryohaemolysis test. The percentage of hyperchromic erythrocytes may no longer be considered an instrumental artefact. Without allowing a definite diagnosis of hereditary spherocytosis, an increased percentage of hyperchromic cells indicates the degree of spherocytosis, making it an excellent automated and cost-free screening parameter for inherited and acquired corpuscular haemolysis.

Clin Lab 2002;48(3-4):163-70

Kutter D, Kremer A.

Economic assessment of iron deficiency. Replacement of serum iron and ferritin by other parameters. Serum iron and ferritin are still widely used parameters for the assessment of iron deficiency, in spite of their poor diagnostic performance. Compared to the combined use of ferritin, zinc protoporphyrin and soluble transferrin receptors an increase of the percentage of hypochromic erythrocytes (PEH) was found to be highly sensitive and specific for iron deficiency. We suggest to use it in a two phase strategy. Normal PEH excludes iron deficiency. Increased PEH may be confirmed by zinc protoporphyrin and/or soluble transferrin receptors.

Bull Soc Sci Med Grand Duche L

Thoma J, Stirn F, Kutter D.

A secondary peak (#C) included in the HbA1c-calculation by the HA-8140 HPLC (Menarini) shows a fairly good correlation with serum urea. The correlation with HbA1c and with serum glucose is at first glance significant but reveals at a closer look being biased by some incorrect assumptions. The difference between immunoturbidimetric determination (Tinaquant HbA1c II Roche) and HPLC shows a similar behaviour to urea as does the #C-peak of the chromatographic separation. This peak as well as the difference between both determinations of HbA1c could be attributed to carbamylated haemoglobin. The definitive identity has to be proven. This peak could be a monitoring tool for long-time urea. The integration of this peak into total HbA1c by the HA-8140 (Menarini) can lead to a false diagnosis of diabetes in non-diabetic patients with elevated urea.

Clin Lab 2000;46(5-6):261-8

Schmit JC, Weber B.

Potent new antiretroviral drugs combined with updated treatment strategies have now achieved efficient inhibition of HIV replication in most patients. The major targets for antiretroviral therapy are the reverse transcriptase and the protease of HIV. Up to date, 11 antiretroviral drugs have been licensed in the US. New nucleoside reverse transcriptase inhibitors (NRTI), i.e. abacavir, and adefovir dipivoxil proved to be effective in clinical trials. The antiretroviral activity and impact on clinical outcome of nonnucleoside reverse transcriptase inhibitors (NNRTI) are mostly short lived. Compared to NRTIs, protease inhibitors (PIs) have the highest antiretroviral activity. New PIs that conserve activity against mutant HIV strains or derivatives that have a simpler biosynthesis are being developed. A major drawback of highly active antiretroviral therapy is the selection of resistant mutants under suboptimal dosage, in advanced stages of disease or after pre-treatment with mono- or double-combination regimens. Monitoring of antiretroviral therapy is achieved by measurement of viral load using nucleic acid amplification techniques. The goal of antiretroviral therapy should be to reduce viral load below the detection limit of current assays in order to delay the emergence of resistant mutants and to maximally reduce disease progression. Recommendations for antiretroviral therapy and monitoring are evolving constantly due to the rapid progress in the development of active compounds and new insights into HIV pathogenesis. Resistance determination of HIV patient isolates seems to play a progressively increasing role in monitoring of antiretroviral treatment since treatment-naive patients may already harbour drug-resistant virus. Due to cross-resistance between different compounds, the choice of the treatment regimen should be guided by resistance patterns of HIV in order to warrant maximal and long-lasting efficiency.

Intervirology 1997;40(5-6):304-21

Berger A, Doerr HW, Scharrer I, Weber B

In 1994, hepatitis C virus (HCV) infection was transmitted to four HIV seropositive patients attending the Department of Angiology, University Clinics, Frankfurt am Main, by the administration of Gammagard. The patients were suffering from thrombocytopenia and received betweeen 20 and 30 g of the contaminated lot 93F21AB11. GBV-C/HGV RNA could be amplified from the Gammagard lot 93F21AB11 using 5'NCR and NS5 primer pairs. All the four patients were negative in the GBV-C/HGV RT-PCR prior to therapy and until the end of the follow-up period. GBV-C/HGV IgG antibodies to the putative envelope (E2) were detected using the E2 HGV-env kit (Boehringer-Mannheim, Germany) in Gammagard lot 93F21AB11 and in one patient before donation of immunoglobulin. Anti-E2 seroconversion was observed in one recipient, the other two patients remained anti-E2 seronegative until the end of the observation period. It is concluded that there is no direct evidence for transmission of GBV-C/HGV by contaminated intravenous immunoglobulin since GBV-C/HGV RNA was not detected in the recipients up to 1 year after administration.

J Med Virol 1997 Sep;53(1):25-30

Berger A, Doerr HW, Weber B

Hepatitis B virus (HBV) and human immunodeficiency virus (HIV) account for millions of cases of vertical infections worldwide. Laboratory diagnosis is essential for the detection of potentially infectious carriers. HBeAg represents the best serological marker for HBV replication. Since 10% of HBeAg-negative carriers transmit the virus to their children, determination of viral DNA is more reliable for the assessment of the risk to vertical infection. Risk assessment of vertical HIV transmission and monitoring AZT therapy during pregnancy are achieved by determination of HIV-1 viral load and CD4+ cell count. HIV-1 RNA or cDNA detection permits a nearly 100% sensitive diagnosis of congenital HIV infection already 2 weeks after birth. While qualitative HBV DNA determination should be limited only to anti-HBe carriers in order to assess infectiosity, HIV-1 RNA measurement represents in combination with the CD4+ cell count the best prognostic marker for vertical HIV infection and for the follow-up of infected children.

Intervirology 1998;41(4-5):201-7

Weber B, Michl U, Muhlbacher A, Paggi G, Bossi V

The new automated Enzymun-Test anti-HBc Plus for the detection of antibodies to hepatitis B virus (HBV) core antigen (anti-HBc) after pretreatment with reducing agents dithiothreitol (25 degrees C; + DTT) or potassium bisulfite (37 degrees C; + MBS) was evaluated by testing 571 serum and plasma samples. The panel included dilution series of reference standards and samples from chronic carriers, one seroconversion panel, samples from patients in acute or chronic stage of the disease or resolved hepatitis B, preselected sera from blood donors that were initially reactive in an anti-HBc assay without pretreatment, potentially cross-reactive serum samples obtained from patients suffering from other diseases or with passed infections other than HBV, pregnant women and individuals with HBV vaccination. The IMx CORE(TM) (Abbott Diagnostics) served as reference assay. Discrepant samples were further investigated with two different commercial anti-HBc enzyme immunoassays, other HBV-specific serological markers and HBV DNA hybridization. The sensitivity of the Enzymun-Test Anti-HBc Plus (25 degrees + DTT, and 37 degrees C; + MBS) in comparison to the reference assay was 100%. The IMx CORE showed a twofold higher sensitivity than Enzymun-Test Anti-HBc Plus for anti-HBc detection in dilution series of serum samples from HBV carriers. The agreement in terms of specificity of the Enzymun-Test Anti-HBc Plus (25 degrees C; + DTT, and 37 degrees C; + MBS) and in comparison to IMx CORE was 97.4 and 96.4%, respectively. After resolution of discrepant results (3 samples were tested false negative with IMx CORE), the agreement in terms of specificity of the Enzymun-Test Anti-HBc Plus (25 degrees C; + DTT, and 37 degrees C; + MBS) in comparison to the combination of the comparative assays was 98.3 and 97.4%, respectively. In conclusion, the new automated Enzymun-Test Anti-HBc Plus with sample pretreatment with DTT (25 degrees C; + DTT) or MBS (37 degrees C; + MBS) permits a highly sensitive and specific detection of anti-HBc in diagnostic virology and blood donation testing.
Intervirology 1998;41(1):17-23

Rabenau H, Knoll B, Allwinn R, Doerr HW, Weber B.

A variable rate of false-positive results may be observed with commercial enzyme immunoassays (EIAs) for the detection of rotavirus and adenovirus antigen in stool specimens, depending on the quality of the reagents and the presence of potentially interfering substances in stool samples. The present study was performed in an attempt to improve the specificity of current commercial rotavirus and adenovirus EIAs without significant loss of sensitivity by optimizing the cut-off value. A collective of 174 stool samples obtained from children suffering from acute gastroenteritis was tested. Electron microscopy (EM) and PAGE were used as reference methods for rotavirus detection. For the evaluation of the adenovirus kits, virus isolation in cell culture and the polymerase chain reaction served as reference standards. The highest sensitivity for rotavirus and adenovirus detection was achieved by the Ridascreen(R) Rotavirus and Ridascreen Adenovirus. However, the Ridascreen(R) Rotavirus and Ridascreen Adenovirus produced the highest number of false-positive results (n = 9) for each rotavirus and adenovirus detection. Cross-reactivities to coronaviruses and reoviruses were observed with the rotavirus antigen EIAs. For Rotazyme II, Ridascreen Rotavirus and Ridascreen Adenovirus, the specificity could be markedly increased without loss of sensitivity by doubling the cut-off value. For the alternative immunoassays, which were overall more specific, it was not possible to significantly decrease the rate of false-positive results without impairment of sensitivity by raising the cut-off value. In conclusion, at least for some rotavirus and adenovirus antigen EIAs, the cut-off value set by the manufacturer may not permit an optimal differentiation between true-positive and -negative samples. By raising the cut-off value from 50 to 100%, the specificity of two rotavirus antigen and one adenovirus antigen EIA can be improved markedly without significant loss of sensitivity.
Intervirology 1998;41(2-3):55-62

Weber B, Gurtler L, Thorstensson R, Michl U, Muhlbacher A, Burgisser P, Villaescusa R, Eiras A, Gabr

Fourth-generation assays for the simultaneous detection of human immunodeficiency virus (HIV) antigen and antibody that were available on the international market until now have antigen detection modules with relatively poor sensitivity and produce a higher rate of false-positive results than third-generation enzyme immunoassays (EIAs). The new Cobas Core HIV Combi EIA with an improved sensitivity for HIV p24 antigen was compared to alternative fourth- and third-generation assays, the p24 antigen test, and HIV type 1 (HIV-1) RNA reverse transcriptase PCR (RT-PCR). A total of 94 seroconversion panels (n = 709 sera), samples from the acute phase of infection after seroconversion (n = 32), anti-HIV-1-positive specimens (n = 730) from patients in different stages of the disease, 462 subtyped samples from different geographical locations, anti-HIV-2-positive sera (n = 302), dilutions of cell culture supernatants (n = 62) from cells infected with different HIV-1 subtypes, selected performance panels from Boston Biomedica Inc., 7,579 unselected samples from blood donors, 303 unselected daily routine samples, 997 specimens from hospitalized patients, and potentially interfering samples (n = 1,222) were tested with Cobas Core HIV Combi EIA. The new assay showed a sensitivity comparable to that of the Abbott HIV-1 AG Monoclonal A for early detection of HIV infection in seroconversion panels. The mean time delay of Cobas Core HIV Combi EIA (last negative sample plus 1 day) in comparison to that for HIV-1 RT-PCR for 87 panels tested with both methods was 2.75 days. The diagnostic window was reduced with Cobas Core HIV Combi EIA by between 3.6 and 5.7 days from that for third-generation assays. The specificities of Cobas Core HIV Combi EIA in blood donors were 99.84 and 99.85% (after repeated testing). Overall, 30 repeatedly reactive false-positive results out of 10,031 HIV-negative samples were obtained with Cobas Core HIV Combi EIA. Our results show that a fourth-generation assay with improved specificity such as Cobas Core HIV Combi EIA is suitable for blood donor screening because of its low number of false positives and because it detects HIV p24 antigen with a sensitivity comparable to that of single-antigen assays.
J Clin Microbiol 2002 Jun;40(6):1938-46

Weber B, Berger A, Rabenau H, Doerr HW.

Combined antigen and antibody screening (fourth-generation) assays reduce the diagnostic window period between the time of human immunodeficiency virus (HIV) infection and laboratory diagnosis by 4 days, on average, in comparison to antibody-only (third generation) enzyme immunoassays (EIAs). The aim of the present study was to assess whether the new VIDAS HIV DUO Ultra (Biomerieux, Marcy-l'Etoile, France) showed an improved sensitivity and specificity in comparison to licensed fourth-generation assays. A total of 16 seroconversion panels, 15 cell culture supernatants infected with different HIV type 1 (HIV-1) subtypes, and 257 potentially cross-reactive serum samples were tested with VIDAS DUO HIV Ultra, Genscreen Plus HIV Ag-Ab, Enzygnost HIV Integral, Enzymun-Test HIV Combi, Genscreen HIV 1/2, version 2 (third-generation EIA), and Genetic Systems HIV-1 Ag EIA (p24 antigen assay). VIDAS HIV DUO Ultra showed a comparable sensitivity to the single p24 antigen assay in seroconversion panels and a dilution series of virus lysates. The diagnostic window was reduced with VIDAS HIV DUO Ultra by 3.82 days, on average, in comparison with the fourth-generation assay with the lowest sensitivity of the antigen detection module. HIV-1 infection was detected 5.88 days earlier than with third-generation EIA. The mean time delay between reverse transcription-PCR and VIDAS HIV DUO Ultra was only 2.31 days. The specificity of fourth-generation assays after retesting ranged between 98.1 and 100%. In conclusion, VIDAS HIV DUO Ultra can replace single-antigen screening for laboratory diagnosis and screening of HIV infection in blood donors. There was no evidence for a second diagnostic window due to impaired sensitivity of the antibody detection module of all the fourth-generation EIAs evaluated in the present study. The specificity after initial and/or repeated testing of VIDAS HIV DUO Ultra was equivalent to that of a third-generation assay.
J Clin Microbiol 2002 Apr;40(4):1420-6

Rabenau HF, Buxbaum S, Preiser W, Weber B, Doerr HW.

Herpes simplex virus (HSV) types 1 and 2 are widespread human infectious agents that are responsible for persistent and latent infections. HSV type 2 (HSV-2) infection is usually transmitted sexually, while HSV type I (HSV-1) is commonly acquired by saliva contact during childhood. In a retrospective study, sera from more than 4,800 patients were analyzed for HSV type-specific IgG antibodies. In people older than 15 years, the seroprevalence of HSV-1 showed no statistically significant discrepancy between the control group (76.3% in females and 75.2% in males), HIV-infected patients (82.8% in females and 84.3% in males), and organ transplant (OTX) recipients (90.3% in females and 86.3% in males) (P>0.05). Age-related analysis of the control group showed that there is an age-dependent increase of HSV-1 seroprevalence in both sexes, reaching its peak in those aged 40 years and older (women 85.4%, men 82.8%). The only age group in which there is a significantly higher seropositivity rate in women than in men is in those aged between 15 and 39 years, with 70.8% versus 63.7% (P<0.05). As with HSV-1, there is an age-related increase of the HSV-2 seroprevalence; however, this increase starts later in life, with the onset of sexual activity. The HSV-2 prevalence across all age groups was highest in female prostitutes (78.0%) and among HIV-infected patients (women 64.1%, men 54.3%); this contrasts with the control group (overall women 17%, men 12.5%; those above 15 years of age, women 18%, men 13.8%) and the OTX patients (women 22.6%, men 9.8%). In the control group the rate of positivity increases with age and peaks in the group older than 40 years (24.2% in women and 16.2% in men). In females the seroprevalence is always elevated compared with males. The data presented show that female sex and older age are independent predictors of HSV-2 seropositivity, while immunosuppression is not. Our additional data show no evidence of a statistically significant humoral HSV-1/HSV-2 cross-immunity. People with HSV-1 serum antibodies have no lower risk of HSV-2 seropositivity than those lacking antibodies to HSV-1. The same is true when investigating HSV-1 seroprevalence rates in HSV-2-seropositive or -negative individuals, retrospectively.
Med Microbiol Immunol (Berl) 2002 Mar;190(4):153-60

Weber B, Berger A, Rabenau H.

Recombinant antigen-based enzyme immunoassays (EIAs) for the detection of human cytomegalovirus (HCMV) specific antibody are believed to yield a higher sensitivity and specificity than virus lysate EIAs. The aim of the present study was to evaluate the accuracy of newly established HCMV assays (Copalis CMV Multiplex, Sorin; Cobas Core CMV IgG and IgM EIAs, Roche Diagnostics; Anti-HCMV recombinant IgG, gB-IgG, IgM and IgA, Biotest; and ETI-CYTOK-G PLUS and M reverse PLUS, Sorin) based on recombinant antigens and/or virus lysate for laboratory diagnosis of HCMV infection. For the assessment of sensitivity, follow-up samples from patients suffering from active HCMV infection were tested. Testing a large number of potentially interfering samples challenged the specificity of the assays. There was no statistically significant difference in the performance of HCMV IgG assays. The results were more heterogeneous for the detection of serological markers of active infection (HCMV IgM, HCMV IgA and anti-CM2). The sensitivities of the different assays ranged between 40.5 and 71.4%. A variable number (17.8-1.7%) of false-positive results were obtained among potentially interfering serum samples. Two of the recombinant antigen based assays showed a high degree of interference with EBV VCA-IgM-positive sera. The best performance was achieved with ETI-CYTOK-M reverse PLUS since it combined the highest sensitivity with specificity. Commercially available assays based on recombinant antigens showed, overall, a poorer performance than the virus lysate EIA.
J Virol Methods 2001 Aug;96(2):157-70

Weber B, Melchior W, Gehrke R, Doerr HW, Berger A, Rabenau H.

Isolated reactivity to hepatitis B virus (HBV) core antigen (anti-HBc) is observed relatively frequently in immunocompromised individuals, intravenous drug abusers (IVDA), and in the presence of HCV infection. The reason for the lack of HBsAg is not clear. The aim of the present study was to investigate which factors (genetic variability of S gene, low-level HBsAg, and immune complexes may be responsible for the failure of HBsAg detection with commercial HBsAg screening assays. Dilution series of two recombinant HBsAg escape mutants and dilutions of serum samples from chronic HBV carriers with multiple insertions in the a determinant and different HBsAg subtypes were tested with a highly sensitive assay that detects wild-type HBsAg (Elecsys HBsAg, Roche Diagnostics, Penzberg, Germany) and two assays that detect HBV wild-type and escape mutants (Murex HBsAg Version 3, Murex and Enzygnost HBsAg 5.0, Dade Behring, Marburg, Germany). Elecsys HBsAg showed in comparison to Murex HBsAg Version 3 and Enzygnost HBsAg 5.0 a reduced sensitivity for escape mutant detection. On the other hand, the best performance for HBsAg subtype detection was obtained with Elecsys HBsAg. In the second part of the study, a selected panel of isolated anti-HBc reactive (n = 104) serum samples (AxSYM Core) was submitted to testing by Elecsys HBsAg, Murex HBsAg Version 3, Enzygnost HBsAg 5.0, and HBsAg detection after immune complex dissociation (ICD) and anti-HBs determination with two different assays (AxSYM Ausab and Elecsys Anti-HBs). To assess the specificity of anti-HBc test results, all the samples were tested by a second anti-HBc assay (Elecsys Anti-HBc). Quantitative HBV DNA detection was undertaken with a commercially available HBV PCR assay (Amplicor HBV Monitor). HCV infection was present in 65.4% of anti-HBc only reactive individuals. Five AxSYM Core positive samples were negative by Elecsys Anti-HBc. Overall, 15 (14.4%) AxSYM Ausab negative samples gave positive results with Elecsys Anti-HBs (median value: 21 IU/ml). No low-level HBsAg carrier was detected among the isolated anti-HBc reactive individuals with Elecsys HBsAg. There was no evidence for the presence of immune complexes. Only one sample was repeatedly reactive by the Murex HBsAg, suggesting that the a mutant form of HBsAg was responsible for the isolated anti-HBc reactivity, however neutralisation assay was not interpretable and HBV DNA PCR was negative. Fifteen (14.4%) anti-HBc only positive individuals were HBV DNA carriers with concentrations ranging from 800 to more than >4,000,000 copies of viral DNA/ml. In conclusion, the most probable explanations for isolated anti-HBc reactivity in our study group are a possible interference of HBsAg synthesis by HCV infection (65.4%) and divergence of results of anti-HBs assays (14.4%). There is no evidence for the presence of low-level HBsAg carriers and immune complexes. HBsAg mutants cannot be excluded definitively by the test strategy used in the present evaluation. Copyright 2001 Wiley-Liss, Inc.
J Med Virol 2001 Jul;64(3):312

Rabenau HF, Kohler E, Peters M, Doerr HW, Weber B.

The aim of the present study was to evaluate the diagnostic accuracy of serology by using new assays for the detection of genus and species-specific IgG, IgM, IgA and secretory IgA antibody in female sex workers. Cervical swabs and first void urine (FVU) from 314 female sex workers were submitted to nucleic acid amplification by ligase chain reaction (LCx, Abbott). Concomittantly, blood samples were tested for the presence of IgG, IgM and IgA antibodies using a genus-specific assay (rELISA, Medac) and species-specific test (SeroCT, Orgenics). Chlamydia trachomatis infection was detected in a total of 30 (9.6%) female sex workers by LCR. With rELISA, seroprevalences for IgG, IgM, and IgA antibody to Chlamydia were 88.9%, 19.1% and 62.7%, respectively. IgG and IgA antibody prevalences against C. trachomatis (SeroCT) were 65.0% and 23.9%, respectively. In comparison to the positive LCR results obtained from cervical swab and/or FVU, the sensitivity of rELISA for Chlamydia IgG, IgA and IgM detection was 93.3%, 83.3% and 16.7%, respectively. With SeroCT, the sensitivity for C. trachomatis-specific IgG and IgA detection was 86.7% and 33.3%, respectively. The specificities of both serologic tests in comparison to LCR were very low. In conclusion, the correlation of serology with active C. trachomatis infection of the lower genital tract is very low. According to our results, serologic testing for Chlamydia can exclude active infection of the lower genital tract with a high reliability (> or = 95%). However, detection of C. trachomatis can only be reliably achieved by nucleic acid amplification assays.
Infection 2000 Mar-Apr;28(2):97-102

Berger A, Doerr HW, Rabenau HF, Weber B.

Although isolated antibody to hepatitis B core antigen (anti-HBc) is frequently nonspecific or may be the only serological marker of past self-limiting hepatitis B, where antibodies against the surface antigen have disappeared, isolated anti-HBc seropositivity is frequently associated with chronic hepatitis B in HIV- and HCV-infected individuals. Of 5,520 samples that tested positive for anti-HBc (IMx and AxSYM CORE, Abbott, Delkenheim, Germany) at the Institute of Virology, University Clinic Frankfurt during the time interval from January 1994 to February 1996, 643 (11.6%) were isolated anti-HBc-reactive in the IMx and AxSYM CORE assays (inhibition values >90%). There was a statistically significant association between isolated anti-HBc seropositivity and HCV and HIV/HCV coinfection (p < 0.05). A total of 190 samples were available for further testing. Six (3.2%) of 190 isolated anti-HBc-positive samples were considered false-positive since they were only positive in the AxSYM or IMx CORE assay and a linear decrease of the measured signal could not be observed in dilution series. Of 184 serum samples tested with nested PCR using primers of the S genome region, only 6 (3.3%) were HBV DNA-positive. Anti-HBc-IgM antibody could be detected in 3 (1.6 %) of the tested samples using the IMx CORE-M. With the more sensitive VIDAS HBc IgM specific IgM antibody was detected in 15 (8.5%) of 177 samples at concentrations ranging from 10 to >200 Paul Ehrlich Institute U/ml. HIV or HCV coinfection was present in 28.1% and 37.5% of isolated anti-HBc-positive individuals, respectively. We conclude from our observations that only a limited proportion of anti-HBc-isolated individuals are potentially infectious, however anti-HBc-IgM which is detectable in any form of liver disease associated with HBV infection was present in more than 8% of the individuals. Of isolated anti-HBc-positive sera 37% were positive for anti-HCV, suggesting that anti-HCV antibody testing should be performed in isolated anti-HBc-positive individuals.
Intervirology 2000;43(2):71-6

Weber B, Fall EM, Berger A, Doerr HW.

BACKGROUND: Screening of blood donors for human cytomegalovirus (HCMV) infection is usually performed by the combined detection of specific IgG and IgM antibody. However, in most of the cases of primary infection HCMV IgG seroconversion is observed concomitantly to IgM production and HCMV IgM antibody detection for blood donor screening is subject to a relatively high frequency of false positive results. OBJECTIVE: In the present study a newly established HCMV IgG ELISA based on recombinant antigens (anti-HCMV recombinant IgG ELISA, Biotest) was evaluated in terms of sensitivity and specificity for blood donor screening. STUDY DESIGN: A total of 442 serum samples including follow-up sera of five patients suffering from primary HCMV infection, selected seropositive and seronegative blood donors and routine specimens were comparatively investigated with three HCMV antibody ELISAs (anti-HCMV recombinant IgG ELISA, Biotest; Enzygnost anti-CMV/IgG + IgM, Dade Behring; and Captia CMV-TA, Centocor). RESULTS: IgG seroconversion was detected with anti-HCMV recombinant IgG ELISA as early as IgM in all five patients suffering from primary infection. The alternative ELISAs were less sensitive, detecting seroconversion one to three bleeds later in 2 (Enzygnost anti-CMV/IgG + IgM) and 4 patients (Captia CMV-TA), respectively. Anti-HCMV recombinant IgG ELISA showed a 99.1% agreement with Enzygnost anti-CMV/IgG + IgM and/or Western blot in the preselected blood donors and routine specimens. Relatively high numbers of false negative (n=20) and positive results (n=7) were obtained with Captia CMV-TA. CONCLUSIONS: Our preliminary data suggest that HCMV antibody screening of blood donors can be performed reliably by detection of specific IgG provided that a highly sensitive assay system is used.
J Clin Virol 1999 Dec;14(3):173-81

Gurtler L, Muhlbacher A, Michl U, Hofmann H, Paggi GG, Bossi V, Thorstensson R, G-Villaescusa R, Eir

In order to reduce the window phase between time of human immunodeficiency virus (HIV) infection and laboratory diagnosis, new fourth generation screening assays which permit a simultaneous detection of HIV antigen and antibody have been developed. In a multicenter study, a new automated fourth generation assay, Enzymun-Test HIV Combi (Boehringer Mannheim GmbH) was compared to third generation assay, p24 antigen tests and Western blot. A total of 37 seroconversion panels, samples of the early infection (n = 42), HIV-1 antibody positive sera, including subtypes A E, and O (n = 1118), HIV-2 positive samples (n = 252) and cell culture supernatants infected with different HIV-1 subtypes and HIV-2 (n = 50), blood donors (n = 6649), hospitalized patients (n = 475), HIV neg. sera with indeterminate Western blot (n = 32), potentially cross reactive serum samples (n = 435) and HIV negative specimens from Cameroon (n = 68) were tested. A total of 16 of 29 seroconversions were detected on average 8.5 days earlier with Enzymun-Test HIV Combi than HIV-1/HIV-2 3rd generation EIA (Abbott Laboratories). Overall, in the 29 panels investigated comparatively with the two assays, the mean time delay between Enzymun-Test HIV Combi and HIV-1/HIV-2 3rd generation EIA was 4.7 days. HIV antigen was detected in three out of 35 seroconversions one bleed earlier with HIV-1 Ag Monoclonal than with Enzymun-Test HIV Combi. Enzymun-Test HIV Combi showed a sensitivity of 100% for HIV antibody detection for HIV-1 group M and O and HIV-2 positive specimens. While p24 antigen of different HIV-1 subtypes was detected with Enzymun-Test HIV Combi in all the 49 cell culture supernatants, HIV Ag was not detected in an HIV-2 virus lysate. A total of 66 false positive results out of 7659 HIV negative samples were obtained with the Enzymun-Test HIV Combi. The specificity for unselected blood donors was 99.6%. The Enzymun-Test HIV Combi permits an earlier diagnosis of HIV infection than third generation assays through the detection of p24 antigen, which may be present in serum samples from individuals with recent HIV infection prior to seroconversion and it shows an excellent sensitivity for antibodies to all known HIV-1 subtypes and HIV-2. The specificity in blood donors and hospitalized patients is comparable to that of other assays.
J Virol Methods 1998 Nov;75(1):27-38

Weber B, Muhlbacher A, Michl U, Paggi G, Bossi V, Sargento C, Camacho R, Fall EH, Berger A, Schmitt

Although human immunodeficiency virus (HIV) antigen assays are of limited value for monitoring antiretroviral therapy, they play an important role for confirmatory testing of fourth generation HIV screening enzyme immunoassay (EIA) reactive samples. In a multicenter study, a new automated rapid p24 antigen assay, Elecsys HIV Ag (Roche Diagnostics Boehringer Mannheim GmbH, Penzberg, Germany), was compared to FDA licensed tests (Abbott HIV-1 Ag monoclonal and Coulter HIV-1 p24 antigen assay). In the evaluation 27 seroconversion panels were included, sera from the acute phase of infection, single and follow-up samples from HIV antibody positive patients, dilution series of HIV antigen positive standards, sera and cell culture supernatants infected with different HIV-1 subtypes (A-H, and O) HIV-2 and recombinant HIV-1 (gag/env) isolates. To challenge the specificity of the new assay, 2565 unselected blood donors, sera from pregnant women, dialysis and hospitalized patients and 407 potentially cross-reactive samples were investigated. Acute HIV infection was detected in three to eight seroconversion panels earlier with Elecsys HIV Ag than with the alternative assays. Higher numbers of serum samples from HIV infected patients tested positive by Elecsys HIV Ag than with the comparative assays. All HIV-1 subtypes and HIV-2 isolates were recognized with Elecsys HIV Ag. Abbott HIV-1 Ag monoclonal and Coulter HIV-1 p24 antigen assay showed a variable sensitivity for the different HIV-1 subtypes. The specificity of Elecsys HIV Ag and Coulter HIV-1 p24 antigen assay were 99.8 and 99.93%, respectively. All the eight sera that were false reactive by Elecsys HIV Ag were tested negative with the Elecsys HIV Ag Neutralization Test. In conclusion, Elecsys HIV Ag was more sensitive than the alternative assays and showed a high specificity in combination with the neutralization assay. The very short incubation time of 18 min and the fully automated procedure of Elecsys HIV Ag which permits direct testing from the primary patient blood collection tube, represent a major improvement for routine laboratory diagnosis in comparison to the alternative assays.
J Virol Methods 1999 Mar;78(1-2):61-70

Weber B, Bayer A, Kirch P, Schluter V, Schlieper D, Melchior W.

The performance of hepatitis B virus (HBV) surface antigen (HBsAg) screening assays is continuously improved in order to reduce the residual risk of transfusion-associated hepatitis B. In a multicenter study, a new automated rapid screening assay, Elecsys HBsAg (Roche Diagnostics), was compared to well-established tests (Auszyme Monoclonal [overnight incubation] version B and IMx HBsAg [Abbott]). Included in the evaluation were 23 seroconversion panels; sera from the acute and chronic phases of infection; dilution series of various HBsAg standards, HBV subtypes, and S gene mutants; and isolated anti-HBV core antigen-positive samples. To challenge the specificity of the new assay, sera from HBsAg-negative blood donors, pregnant women, and dialysis and hospitalized patients and potentially cross-reactive samples were investigated. Elecsys HBsAg showed a higher sensitivity for HBsAg subtypes ad, ay, adw2, adw4, ayw1, ayw2, ayw4, and adr detection in dilution series of different standards or sera than Auszyme Monoclonal version B and/or IMx HBsAg. Acute hepatitis B was detected in 11 to 16 of 23 seroconversion panels between 2 and 16 days earlier with Elecsys HBsAg than with the alternative assays. Elecsys HBsAg and Auszyme Monoclonal version B detected HBsAg surface mutants with equal sensitivity. The sensitivity and specificity of Elecsys HBsAg were 100%. Auszyme Monoclonal version B had a 99.9% specificity, and its sensitivity was 96.6%. IMx HBsAg showed a poorer sensitivity and specificity than the other assays. In conclusion, Elecsys HBsAg permits earlier detection of acute hepatitis B and different HBV subtypes than the alternative assays. By using highly sensitive HBsAg screening assays, low-level HBsAg carriers among isolated anti-HBV core antigen-positive individuals can be detected.
J Clin Microbiol 1999 Aug;37(8):2639-47

Weber B, Fall EH, Berger A, Doerr HW.

In order to reduce the diagnostic window between the time of human immunodeficiency virus (HIV) infection and laboratory diagnosis, new screening enzyme-linked immunosorbent assays (ELISAs) which permit the simultaneous detection of HIV antigen and antibody have been developed. Two fourth-generation assays, HIV DUO (Biomerieux) and HIV Combi (Boehringer Mannheim), for the combined detection of HIV antigen and antibody, were compared with a third-generation assay (HIV-1/HIV-2 3rd Generation Plus enzyme immunoassay [EIA]; Abbott) and a p24 antigen test (HIV-1 Ag monoclonal; Abbott). A total of 17 seroconversion panels, 15 cell culture supernatants infected with different HIV type 1 (HIV-1) subtypes, and 255 potentially cross-reactive serum samples were tested. Ten seroconversions were detected an average of 8.1 days earlier with HIV DUO and 7.5 days earlier with HIV Combi than with the third-generation ELISA. Overall, in the 17 seroconversion panels tested, HIV DUO detected HIV-1 infection an average of 4.8 days and HIV Combi detected infection an average of 4.4 days earlier than HIV-1/HIV-2 3rd Generation Plus EIA. HIV antigen was detected with HIV DUO and HIV Combi in all of the 15 cell culture supernatants infected with different HIV-1 subtypes, including subtype O. With fourth-generation assays, considerably fewer false-positive results (n = 4 to 6) were obtained, in comparison with the third-generation EIA (n = 18). Fourth-generation assays permit an earlier diagnosis of HIV infection than third-generation antibody screening assays through the detection of p24 antigen, which may be present in serum samples from individuals with recent HIV infection prior to seroconversion.In order to reduce the diagnostic window between the time of human immunodeficiency virus (HIV) infection and laboratory diagnosis, new screening enzyme-linked immunosorbent assays (ELISAs) which permit the simultaneous detection of HIV antigen and antibody have been developed. Two fourth-generation assays, HIV DUO (Biomerieux) and HIV Combi (Boehringer Mannheim), for the combined detection of HIV antigen and antibody, were compared with a third-generation assay (HIV-1/HIV-2 3rd Generation Plus enzyme immunoassay [EIA]; Abbott) and a p24 antigen test (HIV-1 Ag monoclonal; Abbott). A total of 17 seroconversion panels, 15 cell culture supernatants infected with different HIV type 1 (HIV-1) subtypes, and 255 potentially cross-reactive serum samples were tested. Ten seroconversions were detected an average of 8.1 days earlier with HIV DUO and 7.5 days earlier with HIV Combi than with the third-generation ELISA. Overall, in the 17 seroconversion panels tested, HIV DUO detected HIV-1 infection an average of 4.8 days and HIV Combi detected infection an average of 4.4 days earlier than HIV-1/HIV-2 3rd Generation Plus EIA. HIV antigen was detected with HIV DUO and HIV Combi in all of the 15 cell culture supernatants infected with different HIV-1 subtypes, including subtype O. With fourth-generation assays, considerably fewer false-positive results (n = 4 to 6) were obtained, in comparison with the third-generation EIA (n = 18). Fourth-generation assays permit an earlier diagnosis of HIV infection than third-generation antibody screening assays through the detection of p24 antigen, which may be present in serum samples from individuals with recent HIV infection prior to seroconversion.In order to reduce the diagnostic window between the time of human immunodeficiency virus (HIV) infection and laboratory diagnosis, new screening enzyme-linked immunosorbent assays (ELISAs) which permit the simultaneous detection of HIV antigen and antibody have been developed. Two fourth-generation assays, HIV DUO (Biomerieux) and HIV Combi (Boehringer Mannheim), for the combined detection of HIV antigen and antibody, were compared with a third-generation assay (HIV-1/HIV-2 3rd Generation Plus enzyme immunoassay [EIA]; Abbott) and a p24 antigen test (HIV-1 Ag monoclonal; Abbott)
J Clin Microbiol 1998 Aug;36(8):2235-9

Kutter D, Devaquet P, Vanderstocken G, Paulus JM, Marchal V, Gothot A.

A group of 100 totally or subtotally myeloperoxidase (MPO)-deficient individuals was compared to a reference population of 118 probands selected at random. Data for a protective effect of the deficiency against cardiovascular damage are presented. On the other hand, a significantly higher occurrence of severe infections and chronic inflammatory processes was noted among the deficient patients. An increased incidence of cancer among the MPO-deficient individuals was not demonstrated.
Acta Haematol 2000;104(1):10-5

Kutter D, Janecki J, Verstraeten L.

The ADVIA (Bayer) haematological system differentiates leukocytes by their volume and their peroxidase (MPO and EPO) activity. It thus allows specific counting of the eosinophils and detection of totally EPO and MPO deficient individuals. EPO activity is determined by the coordinates of the eosinophil cluster on the outprint. A reliable method of quantitation by simple angle measure is suggested. It allows recognition of partial deficiencies. Among approximately 100,000 persons, 29 cases of total EPO deficiency were detected. No typical or specific pathology of the defect was noted. Family studies showed heterozygous parents and offspring to be partially deficient. The frequency of these partial deficiencies in the general population is studied. The results exclude a simple autosomal recessive monogenic transmission of the defect. A model for bigenic transmission is suggested.The ADVIA (Bayer) haematological system differentiates leukocytes by their volume and their peroxidase (MPO and EPO) activity. It thus allows specific counting of the eosinophils and detection of totally EPO and MPO deficient individuals. EPO activity is determined by the coordinates of the eosinophil cluster on the outprint. A reliable method of quantitation by simple angle measure is suggested. It allows recognition of partial deficiencies. Among approximately 100,000 persons, 29 cases of total EPO deficiency were detected. No typical or specific pathology of the defect was noted. Family studies showed heterozygous parents and offspring to be partially deficient. The frequency of these partial deficiencies in the general population is studied. The results exclude a simple autosomal recessive monogenic transmission of the defect. A model for bigenic transmission is suggested.The ADVIA (Bayer) haematological system differentiates leukocytes by their volume and their peroxidase (MPO and EPO) activity. It thus allows specific counting of the eosinophils and detection of totally EPO and MPO deficient individuals. EPO activity is determined by the coordinates of the eosinophil cluster on the outprint. A reliable method of quantitation by simple angle measure is suggested. It allows recognition of partial deficiencies. Among approximately 100,000 persons, 29 cases of total EPO deficiency were detected. No typical or specific pathology of the defect was noted. Family studies showed heterozygous parents and offspring to be partially deficient. The frequency of these partial deficiencies in the general population is studied. The results exclude a simple autosomal recessive monogenic transmission of the defect. A model for bigenic transmission is suggested.
Redox Rep 2000;5(4):225-8

Kouri T, Laippala P, Kutter D, Gant V, Hallander H, Guder WG.

Analytical quality specifications for ordinal scale measurements have not been presented so far. Criteria are suggested for multiproperty (multiple) urine test strips based on upper limits of healthy reference intervals, analytical performance and statistical tests applicable to ordinal scales. Trueness (accuracy) can be evaluated against an acceptable comparison method by applying sensitivity and specificity concepts, and defining a grey zone with a lower detection limit and an upper confirmation limit. Concordance (agreement) of two or more ordinal scale categories should be evaluated by subtracting random agreement, using Kappa statistics. Repeatability (precision) can be calculated for categorized results using binomial statistics.
Scand J Clin Lab Invest 1999 N

Dolphe Kutter, Jerzy Janecki, Luc Verstraeten

The ADVIA (Bayer) haematological system differentiates leukocytes by their volume and their peroxidase (MPO and EPO) activity. It thus allows specific counting of the eosinophils and detection of totally EPO and MPO deficient individuals. EPO activity is determined by the coordinates of the eosinophil cluster on the outprint. A reliable method of quantitation by simple angle measure is suggested. It allows recognition of partial deficiencies. Among approximately 100,000 persons, 29 cases of total EPO deficiency were detected. No typical or specific pathology of the defect was noted. Family studies showed heterozygous parents and offspring to be partially deficient. The frequency of these partial deficiencies in the general population is studied. The results exclude a simple autosomal recessive monogenic transmission of the defect. A model for bigenic transmission is suggested.
Redox Report 2000; 5: 225-228

Weber B.

Recent developments in the laboratory diagnosis of hepatitis B virus infection include the optimization of key serologic markers, including hepatitis B virus surface antigen and antihepatitis B virus core antibody, as well as the development of automated nucleic acid amplification assays. There is still a lack of standardization for nucleic acid amplification assays that are used for the monitoring of antiviral therapy and follow-up of chronic infection and the clinical significance of hepatitis B virus DNA levels need to be clarified. Although highly sensitive automated nucleic acid amplification assays for blood donor screening are available, their implementation is still subject to discussion and certain countries rejected hepatitis B virus DNA testing for blood donation due to poor cost effectiveness. Genetic variability of hepatitis B virus constitutes a major challenge for diagnosis of hepatitis B virus infection, particularly with regard to hepatitis B virus surface antigen detection, antihepatitis B virus surface antigen quantification and nucleic acid amplification assays. The performances of hepatitis B virus surface antigen enzyme immunoassays in regard to genotype and surface antigen variability need to be further improved. Polyclonal antibody-based hepatitis B virus surface antigen enzyme immunoassays, although they cannot guarantee 100% sensitivity, demonstrate superior S gene mutant recognition to assays using monoclonal capture and tracer antibodies. Isolated antihepatitis B virus core reactivity is an unusual but frequent result, which requires a test algorithm for resolution and hepatitis B virus DNA detection with sensitive nucleic acid amplification assays in order to exclude occult hepatitis B virus infection.
Expert Rev Mol Diagn. 2005 Jan;5(1):75-91

Weber B

The genetic variability of hepatitis B virus (HBV) represents a challenge for the sensitivity of immunologic and molecular based assays. Based on sequence divergence in the entire genome of >8%, HBV genomes have been classified into eight groups designated A to H. The genotypes of HBV have distinct geographical distributions. Although preliminary clinical studies seem to indicate that there is an association between HBV genotype and natural history of infection and response to antiviral therapy, further evaluations on larger collectives of patients are necessary to give a clearer picture of the subject. The analytical sensitivity of HBsAg and anti-HBs assays may be dependent on HBV genotype or subtype. The influence of genotypic variability on the sensitivity of nucleic acid amplification tests (NAT) has so far been poorly investigated. Preliminary results show that new real-time NAT detect genotypes A to G with an equal sensitivity. Different mechanisms intervening at the translational or post-translational level, including conformational changes, hydrophobic changes, insertion of basic residues and reduced synthesis or secretion of HBsAg may account solely or in conjunction for escape mutations to the immune response and to detection in HBsAg immunassays. The clinical significance of S-gene mutants, needs in analogy to that of HBV genotypes, to be further investigated. HBV mutants are stable over time and can be transmitted horizontally or vertically. The sensitivity of HBsAg assays for mutant detection is continuously improved. Immunoassays based on polyclonal capture antibody show the highest sensitivity for the recognition of recombinant mutants or serum samples harboring mutant forms of HBsAg. However, they do not guarantee full sensitivity. Detection of HBsAg needs to be improved by the introduction of new HBsAg assays able to recognize so far described S-gene mutants and with a lower detection threshold than current immunoassays in order to detect smallest amounts of HBsAg in low level carriers. There is also a need for more complete epidemiological data on the prevalence of HBsAg mutants and strategies for the (differential) screening of mutants need to be developed and evaluated.
J Clin Virol. 2005 Feb;32(2):102-12.

Weber B, Muhlbacher A, Melchior W.

The issue of HBV DNA screening on blood donations is controversially discussed since the economic impact of post-transfusion hepatitis B is expected to be relatively low. We report on a case of HBsAg negative unapparent acute HBV infection, which was detected by HBV NAT testing on 96-member maxi-pools with a commercially available NAT assay, which has a detection threshold of 3 IU/mL of plasma. The presence of an HBsAg escape mutant could be excluded by sequencing the amplified DNA. Follow-up testing showed the presence of an acute HBV infection (anti-HBc-IgM positive) and finally anti-HBs seroconversion. Although the reduction of the diagnostic window with NAT screening on maxi-pools may be relatively low, it may help to improve the residual risk of blood donation, especially in asymptomatic HBV infection, where the HBsAg positive period may be very short and low levels of circulating surface antigen are present. It would also permit to detect occult HBV infection in chronic carriers who are HBsAg negative. Since the viral load in chronic isolated anti-HBc positive carriers is low, there is a potential risk for failure of HBV DNA detection with pool-PCR in blood donors. Anti-HBc screening would reduce the residual risk.
J Clin Virol. 2005 Jan;32(1):67-70.

Weber B

New screening enzyme immunoassays, which permit the simultaneous detection of HIV antigens reduce the diagnostic window period between the time of immunodeficiency virus (HIV) infection and seroconversion. The VIDAS HIV DUO Ultra is an enzyme-linked fluorescent assay (ELFA) for the screening of HIV infection. It is performed with the fully automated VIDAS or mini-VIDAS instruments, which are so-called walk away systems. The detection limit is 3 pg of HIV-1 p24 Ag/mL serum. HIV antibody is detected with the same sensitivity as stand-alone third-generation antibody tests. The total incubation time is about 2 h. Results are calculated, interpreted, and printed by the VIDAS instrument. Usually, fourth-generation assays demand a special algorithm for the analysis of reactive samples. For the anti-HIV part of the assay, confirmation of reactivity should be done with an assay that lacks the p24-antigen detection module and when reactivity persists subsequently by immunoblot. For the p24-antigen part, confirmation of reactivity should be analyzed in an assay that lacks the anti-HIV detection part.
Methods Mol Biol. 2005;304:245-55.

Weber B and Berger A

Highly sensitive qualitative and quantitative automated commercially available nucleic acid amplification tests (NAT) for the detection of hepatitis B virus (HBV) infection have only been developed in the last years. The potential indications for HBV NAT are: follow-up of chronic hepatitis B, therapy and antiviral resistance monitoring, determination of infectivity and transmission risk, detection of occult (HBsAg negative and HBV DNA positive) infection and mutant virus which may escape serologic diagnosis, blood donor screening and resolution of unusual or discordant serologic constellations. Although NAT are now widely implemented in the routine diagnosis of clinical laboratories, there are several important issues, which need to be further investigated. There is still a lack of standardization for NAT that are used for the monitoring antiviral therapy and follow-up of chronic infection and the clinical significance of HBV DNA levels needs to be clarified. The influence of genetic variability in terms of genotype variation has so far been poorly investigated. Although highly sensitive automated NAT for blood donor screening are available, their implementation is still subject to discussion and certain countries rejected HBV DNA testing for blood donation for reasons of poor cost-effectiveness.
J Lab Med 2005; 29:33-43

Weber B, Thorstensson R, Tanprasert S, Schmitt U, Melchior W.

BACKGROUND AND OBJECTIVES: The influence of genetic variability on the sensitivity of serological diagnosis of human immunodeficiency virus (HIV) infection has, to date, been poorly investigated. The aim of the present study was to assess whether fourth-generation assays for the combined detection of HIV antigen and antibodies to HIV (anti-HIV) permit a reduction of the diagnostic window in comparison to third-generation antibody enzyme immunoassays (EIAs), which so far have shown a poor sensitivity for detection of HIV-1 non-subtype B primary infections. MATERIALS AND METHODS: Three patients with primary HIV-1 subtype E (CRF01-AE) infection were tested with different third- and fourth-generation assays, stand-alone HIV antigen (Ag) EIAs and reverse transcription-polymerase chain reaction (RT-PCR). Additionally, virus lysates from HIV-1 Group M and O and HIV-2, at concentrations of p24 Ag close to the detection limit of licensed HIV Ag EIAs, were investigated with fourth-generation EIAs and HIV Ag EIAs. RESULTS: In the first blood donor, the most sensitive fourth-generation assay detected HIV-1 infection 11 days earlier than five of the eight third-generation antibody assays. Fourth-generation EIAs, with a high sensitivity for HIV antigen, detected HIV-1 subtype E infection simultaneously or 4 days later than HIV-1 RT-PCR on pooled samples. Low concentrations of virus lysates of different HIV-1 subtypes A-H and group O, tested positive with fourth-generation EIAs, with a high sensitivity of the antigen-detection module. CONCLUSIONS: Fourth-generation EIAs, especially those with a high sensitivity for HIV-1 p24 antigen, reduce the diagnostic window for primary HIV-1 subtype E infection in comparison with third-generation antibody-screening assays. These preliminary data from seroconversions and virus lysates indicate that the genetic diversity of HIV-1 does not represent a major challenge for the most sensitive EIAs of this new assay generation.
Vox Sang. 2003 Aug;85(2):73-9.

Weber B

The genetic variability of hepatitis B virus (HBV) represents a challenge for the sensitivity of immunodiagnosis, especially for the detection of surface antigen (HBsAg). There are two types of variants of HBV. Naturally occurring variants are the results of random changes selected over years of population pressure. These variants include HBV genotypes and unusual sequences, which may be poorly detected by immunoassays. The selected variants are mutants that arise in individuals under medically (vaccine, hepatitis B immune globulin and antiviral therapy) or naturally (chronic hepatitis B) induced immune pressure. HBV S-gene mutants have been identified in successfully immunised people worldwide. Based on the assumption that current vaccines containing S protein do not cross-protect against S-gene mutants, a mathematical model predicts the disappearance of wild type HBV in areas with HBsAg endemicity and the emergence of S gene mutant in approximately 100 years as a consequence of universal HBV vaccination. Mutant viruses may escape detection by commercial HBsAg kits. There are several reports on HBsAg negative carriers (HBV-DNA positive) of S-gene mutants with immunosilent infection or "unusual" serologic constellations. Although S gene mutants have been found to be associated with a more severe clinical course of HBV infection and hepatocellular carcinoma (HCC), the clinical significance of the genetic variability of HBV genotypes and HBsAg mutants needs to be further investigated. Detection of HBsAg needs to be improved by the introduction of new HBsAg assays able to recognize so far described S gene mutants and with a lower detection threshold than current immunoassays in order to detect smallest amounts of HBsAg in low level carriers. There is also a need for more complete epidemiological data on the prevalence of HBsAg mutants in Western Europe and assays for the (differential) screening of mutants need to be developed and evaluated.
J Lab Med 2003; 28: 56-69

Weber B.


AIDS. 2003 Apr 11;17(6):931-3.

Weber B, Dengler T, Berger A, Doerr HW, Rabenau H.

In recent years the diagnostic industry has developed new automated immunoassays for the qualitative detection of hepatitis B virus (HBV) surface antigen (HBsAg) in serum and plasma samples that are performed on analyzers that permit a high-speed throughput, random access, and primary tube sampling. The aim of the present study was the evaluation of two new automated HBsAg screening assays, IMMULITE HBsAg and IMMULITE 2000 HBsAg, from Diagnostic Products Corporation. The new HBsAg assays were compared to well-established tests (Auszyme Monoclonal [overnight incubation, version B], IMx HBsAg, AxSYM HBsAg, and Prism HBsAg [all from Abbott] and Elecsys HBsAg [Roche Diagnostics]). In the evaluation were included seroconversion panels, sera from the acute and chronic phases of infection, dilution series of various HBsAg standards, HBV subtypes and S gene mutants. To challenge the specificity of the new assays, sera from HBsAg-negative blood donors, pregnant women, and dialysis and hospitalized patients and potentially cross-reactive samples were investigated. IMMULITE HBsAg and IMMULITE 2000 HBsAg, although not as sensitive as the Elecsys HBsAg assay, were equivalent to the AxSYM HBsAg assay and showed a higher sensitivity than the Auszyme Monoclonal B and IMx HBsAg systems for detection of acute infection in seroconversion panels. The specificities (100%) of both IMMULITE assays on unselected blood donors and potentially interfering samples were comparable to those of the alternative assays after repeated testing. In conclusion, the new IMMULITE HBsAg and IMMULITE 2000 HBsAg assays show a good sensitivity for HBsAg detection compared to other well-established tests. The specificity on repeatedly tested samples was equivalent to that of the alternative assays. The rapid turnaround time, primary tube sampling, and on-board dilution make it an interesting assay system for clinical laboratory diagnosis.
J Clin Microbiol. 2003 Jan;41(1):135-43.

Weber B, Meier T, Enders G.


J Clin Virol. 2002 Dec;25(3):357-9.

Weber B


J Clin Microbiol. 2002 Nov;40(11):4402-3

Weber B

The presence of isolated antibody to hepatitis B virus (HBV) core antigen (anti-HBc) is the most frequent so-called unusual seroconstellation observed in HBV serology. Its clinical significance is still relatively unclear. In the last decade, its diagnostic significance has been intensively investigated. Actually, false positive results can be excluded through adequate confirmatory testing. Isolated anti-HBc reactivity is generally observed after resolved HBV infection (loss of anti-HBs or low-level anti-HBs). This serological pattern is also very frequently associated with HBsAg negative chronic low-level HBV DNA carriage. The reasons for the absence of HBsAg detection are inhibition of expression by HCV co-infection, and/or low-level HBsAg synthesis under the limit of detection of screening assays, presence of immune complexes and probably also in a minority of cases HBsAg mutants. While anti-HBc screening of blood donations might further reduce the HBV related residual risk, it is not used worldwide because of its relatively poor cost-.effectiveness and the burden of exclusion of donors through non-specific test results. Combined anti-HBc/HBsAg testing with a highly sensitive and specific assay is probably technically achievable and would permit to detect the rare cases of HBV DNA negative potentially infectious donations at a reasonable financial burden.
J Lab Med 2002; 26: 451-458

Kutter D, Gulbis B.

Permanent significant hyperchromia, equivalent to hyperspherocytosis, the leading symptom of hereditary spherocytosis (HS), has become accessible to routine screening by hematologic automats using double angle laser technology. This has resulted in the discovery of a much higher incidence of this anomaly. In previous investigations we suggested the permanence of significant hyperchromia as obligate criterion for the diagnosis HS. Intercurrent normal percentages of hyperchromic RBC rather pointed to secondary, non-hereditary spherocytosis. Describing 6 typical cases, we demonstrate that occasional normal percentages of hyperchromic red blood cells occurring during phases of anemia and/or iron deficiency are compatible with the diagnosis of HS.
Clin Lab. 2005;51(7-8):411-8.

Kutter D.

Modern double beam laser technique allows screening for hereditary spherocytosis in the course of routine hematology. An incidence of 1:150 men and 1:800 women has been determined. The anomaly is symptomless in the majority of the cases. This explains the discrepancy between our values and the incidence of 1:5,000 reported in the literature. The diagnosis of hereditary spherocytosis should be reported to the physician and the patient, as it may be wayleading in case of unexpected, unspecific complications such as anemia, jaundice, cholelithiasis, liver cell damage and iron overload. Regular monitoring of plasma ferritin and glucose is recommended.
Bull Soc Sci Med Grand Duche Luxemb. 2005;(1):7-22