Graham M. Cleator

Detection of BK virus and JC virus DNA in urine samples from immunocompromised (HIV-infected) and immunocompetent (HIV-non-infected) patients using polymerase chain reaction and microplate hybridisation

BACKGROUNDnThe majority of the human population is infected with two human polyomaviruses BK virus (BKV) and JC virus (JCV) during childhood. After initial infection both viruses persist within renal system. Reactivation of both viruses may be linked with immunodeficiency or immunosuppressive therapy.nnnOBJECTIVEnTo evaluate the relationship between immunodeficiency and viruria, prevalence of BK and JC viruria over time was investigated in a cohort of HIV seropositive individuals at different stages of disease. The excretion in this group was compared with virus excretion in their HIV seronegative partners and in an unselected cohort of patients attending a Genito-Urinary Medicine (GUM) clinic.nnnSTUDY DESIGNnThe excretion of BKV and JCV DNA in multiple urine samples from HIV-infected patients at different stages of disease and their HIV-negative partners, and in single samples from a cohort of patients at a GUM clinic was investigated. A microplate hybridisation method was developed to increase both the sensitivity and specificity of detection of the PCR product. The method was also applied to estimate the DNA copy numbers of BKV and JCV in urine samples.nnnRESULTSnWithin the HIV group, the level of immunosuppression (CD4+ category) was not associated with JCV viruria. By contrast, there was a modest correlation between immunodeficiency as indicated by a decline in CD4+ count and BKV viruria. Shedding of both BKV and JCV DNA together in urine samples of HIV-infected patients was much higher than in control groups (P = 0.02), indicating that HIV infection may associate with polyomavirus reactivation. The incidence of flu-like syndrome was much higher in HIV-infected asymptomatic individuals than acquired immunodeficiency syndrome (AIDS)-related complex (ARC)/AIDS patients. In general, the concentration of BKV DNA viruria (DNA copy number) was dependent to CD4+ counts (P = 0.008) while concentration of JCV DNA was independent to CD4+ cell count (P = 0.54). The prevalence of BKV and JCV DNA in patients who were infected with C. trachomatis was 9/50 (18%) and 11/50 (22%), respectively. BKV and JCV DNA was detected in 3/19 (15%) and 2/19 (10%) of patients who were infected with N. gonorrhoea. Results suggested that persons infected with C. trachomatis were more likely to show BKV and JCV viruria.nnnCONCLUSIONnThese results confirm that shedding of BK and JC viruses in urine is not exclusively found in immunosupression, it may also occur in healthy individuals. The frequency of virus excretion is however, apparently increased in HIV-infected patients, although no firm statistical difference could be established. One of the interesting aspects of these findings was the relatively high incidence of BKV and JCV viruria in both control groups, i.e. HIV-negative partners of HIV-infected patients and patients attending a GUM clinic.

Specific diagnostic methods for herpesvirus infections of the central nervous system: A consensus review by the European Union Concerted Action on Virus Meningitis and Encephalitis

BACKGROUNDnHerpesvirus infections of the central nervous system are often severe but are fortunately rare. The incidence of these infections has however, increased in recent years as a consequence of an increase in the number of immune-compromised individuals. New diagnostic procedures have improved our ability to diagnose these infections and herpesviruses may yet be implicated as the cause of further neurological diseases with no known aetiology. Methodological standards for selection and evaluation of patient materials are essential to the provision of reliable diagnosis, yet few studies have addressed this important issue.nnnOBJECTIVESnTo describe and define methodological standards and reference methodology for diagnosis of herpesvirus infections of the CNS.nnnSTUDY DESIGNnInformation gathered by literature review.nnnRESULTSnOnly for herpes simplex encephalitis is there sufficient data to allow the definition of reference methodology. Good methodological standards exist but few studies have adhered to these standards. As methods for the detection of specific intrathecal antibody synthesis are well established yet under-used in diagnostic virology, the principle of these measurements is reviewed in some detail.nnnCONCLUSIONSnHerpesvirus infections of the CNS are of increasing importance. High quality, multi-centre studies are needed to establish the value of the new diagnostic test procedures if further improvement in the diagnostic sensitivity and specificity of these procedures is to be achieved.

An international external quality assessment of nucleic acid amplification of herpes simplex virus

BACKGROUNDnThere is an increasing awareness of the need for external quality control of diagnostic virology.nnnOBJECTIVESnTo assess the quality of nucleic acid amplification tests (NAT) of herpes simplex within Europe.nnnSTUDY DESIGNnHerpes simplex virus (HSV) proficiency panels were produced at the Swedish Institute for Infectious Disease Control on behalf of the European Union Concerted Action for Quality Control of Nucleic Acid Amplification in 1999 and 2000. Nine reference laboratories evaluated the production process. Each panel consisted of 12 coded samples with various concentrations of inactivated, freeze-dried HSV type 1 (HSV-1), and HSV type 2 (HSV-2), or negative controls. Positive samples included HSV-1 and HSV-2 in a range of concentrations (2 x 10(2) to 2 x 10(7) genome copies per ml) similar to those found in cerebrospinal fluids from patients with HSV encephalitis.nnnRESULTSnSixty-six participants reported a total of 76 data sets for panel 1, and 71 reported 78 data sets for panel 2. The majority of the participants employed qualitative in-house polymerase chain reaction (PCR) methods, either in a single, nested or semi-nested format. For panel 2, 9 laboratories reported use of real-time PCR in contrast to 3 for panel 1. Three laboratories submitted quantitative results on both panels. Thirty percent of the data sets had correct results for the entire panel 1. In 6 data sets (8%) a total of 11 false positive results were reported. For panel 2, 28% of the data sets had correct result. Nineteen false positive results were reported in 14 data sets (18%), but most of the incorrect results reflected a lack of test sensitivity.nnnCONCLUSIONSnThe relatively high frequency of false positive results and the large number of false-negative results, albeit at low copy number, stress the need for improvement in the quality of HSV NAT and for external quality control programmes.

European Proficiency Testing Program for Molecular Detection and Quantitation of Hepatitis B Virus DNA

ABSTRACT External quality control of hepatitis B virus (HBV) DNA detection remains an important issue. This study reports and compares the results obtained from two different proficiency panels for both the qualitative and quantitative assessment of HBV DNA. The panels were designed by the European Union Quality Control Concerted Action, prepared by Boston Biomedica, Inc., and distributed in May 1999 (panel 1) and February 2000 (panel 2). Each contained two negative samples and six positive samples with 103 to 107 copies/ml (panel 1) or 103 to 2 × 106 copies of HBV DNA per ml (panel 2). For panel 1, 42 laboratories submitted 20 qualitative (all in-house PCRs) and 37 quantitative (87% commercial assays) data sets. For panel 2, 51 laboratories submitted 25 qualitative (all in-house PCRs) and 47 quantitative (94% commercial assays) data sets. Five data sets (8.8%) in panel 1 and two data sets (2.8%) in panel 2 contained totals of six and two false-positives, respectively, corresponding to false-positive result rates of 5.3% for panel 1 and 1.4% for panel 2. The false-negative result rates of 10.5% for panel 1 and 17.4% for panel 2 were dependent on the detection levels of the assays employed as well as panel composition. In the qualitative analysis of all data sets, 47.4% (panel 1) and 51.4% (panel 2) had all samples correct. An adequate or better score (all correct or only the weak-positive sample missed) was obtained with 77.2% of the panel 1 samples and 68.1% of the panel 2 samples. In the quantitative analysis, 57.1% (panel 1) and 42.6% (panel 2) of the data sets achieved an adequate or better score (positive results within the acceptable range of the geometric mean ± 0.5 log10 of all positive results). These results demonstrate that while the qualitative performance of HBV detection has considerably improved compared to that of a previously published HBV proficiency study, the detection levels of many commercial quantitative assays are still too high to allow adequate quantitation of all relevant clinical samples.

Detection of BK virus in urine by polymerase chain reaction: a comparison of DNA extraction methods.

Using specimens spiked with BK virus, several DNA extraction methods were evaluated for their ability to remove polymerase chain reaction (PCR) inhibitors from urine samples. It was found that PCR inhibition could be completely overcome by extracting samples with 30% polyethylene glycol (PEG) in 3 M sodium chloride, and partially overcome by extracting samples with guanidine thiocyanate in the presence of high salt concentrations. The nature of the sample inhibition was investigated, leading to the conclusion that both urea and unidentified non-proteinaceous DNA associated substances inhibit BKV DNA amplification from urine.

Reliability of Nucleic Acid Amplification Methods for Detection of Chlamydia trachomatis in Urine: Results of the First International Collaborative Quality Control Study among 96 Laboratories

ABSTRACT The first European Quality Control Concerted Action study was organized to assess the ability of laboratories to detect Chlamydia trachomatis in a panel of urine samples by nucleic acid amplification tests (NATs). The panel consisted of lyophilized urine samples, including three negative, two strongly positive, and five weakly positive samples. Ninety-six laboratories in 22 countries participated with a total of 102 data sets. Of 204 strongly positive samples 199 (97.5%) were correctly reported, and of 506 weakly positive samples 466 (92.1%) were correctly reported. In 74 (72.5%) data sets correct results were reported on all samples, and 17 data sets (16.7%) showed either one false-negative or one false-positive result. In another 11 data sets, two or more incorrect results were reported, and two data sets reported a false-positive result on one negative sample. The Roche COBAS Amplicor test was performed in 44 (43%) data sets, the Abbott LCx assay was performed in 31 (30%) data sets, the Roche Amplicor manual assay was performed in 9 (9%) data sets, an in-house PCR was performed in 9 (9%) data sets, the Becton Dickinson ProbeTec ET assay was performed in 5 (4.9%) data sets, and the GenProbe TMA assay was performed in 4 (3.9%) data sets. The results of the Roche Amplicor manual (95.6% correct), COBAS Amplicor (97.0%), and Abbott LCx (94.8%) tests were comparable (P = 0.48). The results with the in-house PCR, BD ProbeTec ET, and GenProbe TMA tests were reported correctly in 88.6, 98, and 92.5% of the tests, respectively. Freeze-drying of clinical urine specimens proved to be a successful method for generating standardized, stable, and easy-to-transport samples for the detection of C. trachomatis by using NATs. Although the results, especially the specificity, for this proficiency panel were better than most quality control studies, sensitivity problems occurred frequently, underlining the need for good laboratory practice and reference reagents to monitor the performance of these assays.

Association of acute parvovirus B19 infection with new onset of acute lymphoblastic and myeloblastic leukaemia

Aims: To investigate the association of acute parvovirus B19 infection with new onset of acute lymphoblastic and myeloblastic leukaemia. Methods: Cerebrospinal fluid (CSF) samples from patients with acute myelogenous leukaemia (AML) at diagnosis (n = 2) and acute lymphoblastic leukaemia (ALL) at diagnosis (n = 14) were analysed for parvovirus B19 DNA by means of nested polymerase chain reaction. In addition, samples from patients with benign intracranial hypertension (BIH) (n = 10) and hydrocephalus (n = 13) were tested as controls. Results: Four leukaemia cases were positive—common ALL (n = 2), null cell ALL (n =1), and M7 AML (n = 1)—whereas all controls were negative (Yates corrected χ2 value, 3.97; p = 0.046; odds ratio, 16.92; confidence interval, 1.03 to 77.18). All four patients were significantly anaemic, but none was encephalitic or had evidence of central nervous system leukaemia. In three of these patients, serum tumour necrosis α, interferon γ, interleukin 6, granulocyte–macrophage colony stimulating factor (range, 34.93–3800.06pg/ml), and macrophage chemoattractant protein 1 were detectable. All of these four patients carried at least one of the HLA-DRB1 alleles, which have been associated with symptomatic parvovirus B19 infection. Conclusion: Erythroid suppression and immune cell proliferation are both associated with B19 infection and may also be important in the pathogenesis of acute leukaemia.

External Quality Assessment Program for Qualitative and Quantitative Detection of Hepatitis C Virus RNA in Diagnostic Virology

ABSTRACT To assess the performance of laboratories in detecting and quantifying hepatitis C virus (HCV) RNA levels in HCV-infected patients, we distributed two proficiency panels for qualitative and quantitative HCV RNA testing. The panels were designed by the European Union Quality Control Concerted Action, prepared by Boston Biomedica Inc., and distributed in May 1999 (panel 1) and February 2000 (panel 2). Each panel consisted of two negative samples and six positive samples, with HCV RNA target levels from 200 to 500,000 copies/ml. Panel 1 had four samples with at least 50,000 copies/ml, and panel 2 had two samples with at least 50,000 copies/ml. Fifty-seven laboratories submitted 45 qualitative and 35 quantitative data sets on panel 1, and 81 laboratories submitted 75 qualitative and 48 quantitative data sets on panel 2. In both panels, about two-thirds of the qualitative data sets and >90% of the quantitative data sets were obtained with commercial assays. With each panel, two data sets gave one false-positive result, corresponding to false-positivity rates of 1.3% and 0.8% for panel 1 and panel 2, respectively. Samples containing at least 50,000 copies/ml were found positive in 97% and 99% of the cases with panel 1 and panel 2, respectively. In contrast, the positive samples containing ≤5,000 copies/ml were reported positive in only 71% and 77% of the cases with panel 1 and panel 2, respectively. Adequate or better scores on qualitative results (all results correct or only the low-positive samples missed) were obtained in 84% (panel 1) and 80% (panel 2) of the data sets. In the analysis of quantitative results, 60% (panel 1) and 73% (panel 2) of the data sets obtained an adequate or better score (≥80% of the positive results within the range of the geometric mean ± 0.5 log10). Our results indicate that considerable improvements in molecular detection and quantitation of HCV have been achieved, particularly through the use of commercial assays. However, the lowest detection levels of many assays are still too high, and further standardization is still needed. Finally, this study underlines the importance of proficiency panels for monitoring the quality of diagnostic laboratories.

The detection of intrathecal synthesis of anti-herpes simplex IgG antibodies: Comparison between an antigen-mediated immunoblotting technique and antibody index calculations

The detection of intrathecal antibody synthesis was compared by the calculation of antibody indices (AI) derived from ELISA techniques with the detection of virus‐specific oligoclonal IgGs by an antigen‐mediated capillary blot technique. Twenty‐seven paired serum and cerebrospinal fluid (CSF) samples were examined from 15 immunocompetent patients with herpes simplex virus encephalitis (HSE) diagnosed by PCR on early CSF samples. These techniques were also applied to paired samples from 20 multiple sclerosis (MS) patients, 10 patients with other inflammatory neurological diseases and 10 patients with non inflammatory neurological disorders. There was a good correlation between the results obtained by AI and those obtained by immunoblotting, especially in HSE (2 discordant results out of 27). Discrepancies were more frequent (25%) in MS patients where a “polyspecific” reaction characterized by low affinity antibodies is known to occur. Some of the discrepancies could, in part, be due to serological cross‐reaction with varicella zoster virus. J. Med. Virol. 53:324–331, 1997.

Multiplex polymerase chain reaction for adenovirus and herpes simplex virus in eye swabs

Adenoviruses and herpes simplex virus (HSV) can cause clinically indistinguishable episodes of acute eye disease. Adenovirus infection is associated with nosocomial outbreaks and HSV may result in episodes of recurrent ocular inflammation. In a comparison of multiplex PCR for the two viral DNAs and virus isolation in cell culture, identical results were obtained for 18 of 20 specimens (positive for adenovirus in 5, HSV in 5, and negative in 8). One specimen was falsely negative for each viral DNA. Inclusion of human beta-globin primers in the adenovirus-HSV reaction was precluded by a consequential 10--100-fold reduction in sensitivity for the two viral targets and by the failure of beta-globin DNA amplification at the annealing temperature (45 degrees C) required to ensure detection of adenoviruses of serotypes 7 and 11 with the selected adenovirus primers. A single-target beta-globin PCR gave positive results with 19 of the 20 specimens prepared by treatment with proteinase K lysis buffer, indicating the effectiveness of this simple DNA extraction procedure. Nonetheless, the availability of effective antiviral therapy for HSV made monitoring for extraction failure using human primers crucial to avoid false-negative results for HSV DNA. Adenovirus-HSV PCR has considerable potential for the rapid diagnosis of viral eye disease particularly if beta-globin primers can be included in the reaction.