Detection of bacterial contamination in platelet concentrates from Brazilian donors by molecular amplification of the ribosomal 16S gene.

OBJECTIVE: The aim of our work was to establish a semi-automated high-throughput DNA amplification method for the universal screening of bacteria in platelet concentrates (PCs). BACKGROUND: Among cases of transfusion transmission of infectious agents, bacterial contamination ranks first in the number of events, morbidity and mortality. Transmission occurs mainly by transfused PCs. Automated culture is adopted by some blood banks for screening of bacterial contamination, but this procedure is expensive and has a relatively long turnaround time. METHODS: PCs were spiked with suspensions of five different bacterial species in a final concentration of 1 and 10 colony-forming units (CFU) per millilitre. After incubation, the presence of bacteria was investigated by real-time polymerase chain reaction (PCR) and by the Enhanced Bacterial Detection System (eBDS, Pall) assay as a reference method. Real-time PCR amplification was performed with a set of universal primers and probes targeting the 16S rRNA gene. Co-amplification of human mitochondrial DNA served as an internal control. RESULTS: Using the real-time PCR method, it was possible to detect the presence of all bacterial species tested with an initial concentration of 10 CFU mL-1 24 h after contamination, except for Staphylococcus hominis. The PCR assay also detected, at 24 h, the presence of Serratia marcescens and Enterobacter cloacae with an initial concentration of 1 CFU mL-1 . CONCLUSIONS: The real-time PCR assay may be a reliable alternative to conventional culture methods in the screening of bacterial contamination of PCs, enabling bacterial detection even with a low initial concentration of microorganisms.

Quantitation of hepatitis delta virus using a single-step internally controlled real-time RT-qPCR and a full-length genomic RNA calibration standard.

Quantitation of circulating hepatitis delta virus (HDV) RNA is important for assessing the response to antiviral therapy and for understanding the complex dynamic interactions between hepatitis B virus (HBV) and HDV replication. Although several PCR assays for HDV RNA have been described none of them incorporate an internal control or use a full-length RNA calibration standard for absolute quantitation. This study describes the development and evaluation of a novel single-step real-time RT-qPCR assay for HDV RNA quantitation which incorporates a Brome Mosaic virus internal control to prevent false negatives and under-reporting due to inhibitors or due to inefficient RNA purification, reverse transcription or PCR amplification. The assay has a dynamic range of ≥7log(10) and is designed to detect all HDV genotypes. The 95% detection limit is ∼3800 HDV RNA copies/ml, 700 copies/ml being detectable in 20% of repeats. Both intra-assay and inter-assay variability are low (CV 8% and 17%, respectively). Plasma HDV RNA was detected in 75% of 59 HDV antibody-positive samples with titres ranging from 8.4×10(4) to 4.4×10(8) copies/ml. The assay described provides a reliable and sensitive quantitative system for therapeutic monitoring and for studying the dynamic interplay between hepatitis B virus replication and HDV viral load.

Quantification of reverse transcriptase in ALS and elimination of a novel retroviral candidate.

BACKGROUND: Retroviral involvement in amyotrophic lateral sclerosis (ALS) has been suspected for several years since the recognition that both murine and human retroviruses can cause ALS-like syndromes. Nonquantitative studies have demonstrated the retroviral enzyme reverse transcriptase (RT) in ALS patients' sera, but the amount and source of RT activity are unknown. We therefore developed a quantitative assay to study RT levels in ALS and examined the possibility that the recently discovered human gammaretrovirus XMRV (xenotropic MuLV-related virus) might be the source of the RT activity. METHODS: A quantitative product-enhanced RT assay was used to measure RT activity levels in serum and CSF. XMRV sequences were sought by PCR analysis of DNA and RNA extracted from blood. RESULTS: Fifty percent of ALS patients' sera contained >6 x 10(-8) RT units/mL as opposed to 7% of control sera (p = 0.008). The levels of RT activity in ALS patients were comparable to the levels observed in patients infected with HIV. RT activity was detected in only 1 of 25 CSF samples tested. XMRV sequences were not found in any of 25 nucleic acid extracts obtained from ALS patients' blood. CONCLUSIONS: These findings further support the concept of retroviral involvement in amyotrophic lateral sclerosis (ALS) and demonstrate that serum is more suitable than CSF for assay of reverse transcriptase (RT) activity in this disease. The levels of serum RT activity detected are comparable to those found in HIV infection. XMRV is not detectable in the blood of ALS patients, and the agent responsible for ALS-associated RT activity therefore remains unidentified.

Development and evaluation of a real-time RT-PCR assay for quantification of cell-free human immunodeficiency virus type 2 using a Brome Mosaic Virus internal control.

Quantification of cell-free virus in plasma is important for monitoring disease progression and for assessing the response to antiretroviral therapy in both human immunodeficiency type 1 and type 2 (HIV-1, HIV-2) infections. Although commercial assays suitable for HIV-1 quantification have been used for more than a decade, no commercial assays are yet available for the measurement of cell-free HIV-2. We have therefore developed a novel real-time RT-PCR assay which, unlike previously described 'in house' assays, incorporates a Brome Mosaic Virus (BMV) internal control to minimise the risk of generating false-negative or falsely low results due to unrecognised problems with viral RNA purification, cDNA synthesis or PCR amplification. The assay has a dynamic range of >5 log10, detects the clinically important HIV-2 subtypes A and B with high sensitivity and shows no cross reactivity with HIV-1. The 95% detection limit is approximately 100 HIV-2 RNA copies/ml and both the inter-assay and intra-assay variability are low (CV% at 1.8 x 10(5) copies/ml, 13.3% and 5.7%, respectively). Overall, plasma HIV-2 RNA was detected in 38% of 167 unselected HIV-2 antibody-positive samples analysed over a 2 year period. The assay described provides an ideal system for studying viral replication in HIV-2 infected patients and for monitoring antiretroviral therapy.

Real-time PCR quantitation of hepatitis B virus DNA using automated sample preparation and murine cytomegalovirus internal control.

Quantitation of circulating hepatitis B virus (HBV) DNA is important for monitoring disease progression and for assessing the response to antiviral therapy. Several commercial and 'in house' assays for HBV DNA quantitation have been described but many of these have limitations of relatively low sensitivity and limited dynamic range. This study describes the development and evaluation of a FRET-based real-time PCR assay designed to overcome these limitations and to provide accurate quantitation of DNA from all eight genotypes of HBV (A-H). The assay employs a fully automated nucleic acid extraction system permitting high-sample throughput with minimal 'hands-on' time and incorporates a murine cytomegalovirus (mCMV) internal control to prevent false negative results and under-reporting due to unrecognised problems with viral lysis, DNA purification or PCR amplification. Sensitivity, assessed by Probit analysis at the 95% detection level, was 24.4 IU/ml, associated with an extremely wide dynamic range (approximately 9 log10). Coefficients of variation were low for both intra-assay and inter-assay variability (CV%, 7-11%) and quantitative data correlated well (R2 = 0.97) with the Digene hybrid capture assay. This assay provides an ideal system for therapeutic monitoring and for studying the relationship between HBV viral load and stage of disease.

Detection of serum reverse transcriptase activity in patients with ALS and unaffected blood relatives.

BACKGROUND: Retroviral involvement in the etiology of sporadic ALS has been suspected for several years since the recognition that both murine and human retroviruses can cause motor neuron disease-like syndromes. In a pilot study, an increased prevalence of a retroviral marker (reverse transcriptase [RT] activity) was demonstrated in the serum of British patients with ALS. The current investigation was designed to confirm and extend these findings in a geographically distinct patient cohort under blinded testing conditions. METHODS: A highly sensitive product-enhanced RT assay was employed to test coded sera obtained from 30 American patients with sporadic ALS and from 14 of their blood relatives, 16 of their spouses, and 28 nonrelated, nonspousal control subjects. RESULTS: Serum RT activity was detected in a higher proportion of ALS patients (47%) than in non-blood-related controls (18%; p = 0.008). The prevalence of RT activity in the serum of spousal controls (13%) was similar to that in other non-blood-related controls. Unexpectedly, the prevalence of serum RT activity in blood relatives of ALS patients (43%) approached that in the ALS patients themselves. CONCLUSIONS: These results confirm that patients with ALS have a significantly higher prevalence of serum reverse transcriptase (RT) activity than that seen in unrelated control subjects. The finding of a similarly increased prevalence in blood relatives of ALS patients raises the possibility that the observed RT activity might be due to an inherited endogenous retrovirus.

Mimicry between the hepatitis C virus polyprotein and antigenic targets of nuclear and smooth muscle antibodies in chronic hepatitis C virus infection.

Autoantibodies to smooth muscle (SMA) and nuclear components (ANA) arise in the natural course of chronic infection with hepatitis C virus. In view of the growing evidence for 'molecular mimicry' as a mechanism of autoimmunity we investigated whether cross-reactive immune reactions between host smooth muscle/nuclear components and HCV antigens may contribute to the formation of SMA and ANA in chronic HCV infection. Computer-assisted protein database search methods were used to identify three smooth muscle (smoothelin698-717, myosin1035-1054, vimentin69-88) and three nuclear (matrin722-741, histone H2A11-30, replication protein A133-152) host antigens with the highest local sequence similarity to the HCV polyprotein and 20-mer peptides corresponding to these regions were constructed. Sera from 51 children with chronic HCV infection [median age: 8 (2-16); 27 boys], 26 SMA positive and five ANA positive, were tested for reactivity to the synthesized HCV peptides and their human homologues by enzyme linked immunosorbent assay (ELISA). Sera from patients with HBV infection and chronic liver disease of different aetiologies were used as controls. 'Double reactivity' to HCV peptides and smooth muscle/nuclear homologues was associated strongly with HCV infection (P < 0.001 for both). Humoral cross-reactivity was established as the basis for double recognition by competition ELISA. Double-reactivity to smooth muscle and HCV peptide antigens correlated with SMA positivity by indirect immunofluouresence (P = 0.05). Of 15 patients double-reactive to myosin1035-1054 and its HCV homologue, 13 recognized whole myosin by immunoblot. These results suggest that ANA and SMA in chronic HCV infection may arise, at least in part, as a consequence of cross-reactive immune responses to HCV and host smooth muscle/nuclear antigens.

Development and evaluation of an internally controlled semiautomated PCR assay for quantification of cell-free cytomegalovirus.

Quantification of circulating human cytomegalovirus (HCMV) is useful in clinical contexts such as virological surveillance of bone marrow transplant recipients and monitoring of antiviral therapy. This report describes an internally controlled, quantitative, semiautomated, HCMV genome assay that was developed primarily to measure HCMV DNA in the plasma of severely leucopaenic patients. It exhibits greater sensitivity, wider dynamic range and higher sample throughput than a number of previously described commercial and "in-house" assays. Viral DNA extraction from EDTA plasma samples was automated using a BioRobot 9604 (Qiagen). HCMV strain AD169 was used to prepare a calibration curve and murine cytomegalovirus (MCMV) strain Smith was added as internal control to all calibration standards and test samples. Amplification was performed using a set of primers based on the HCMV UL50 region, capable of amplifying both human and murine CMV. The yield of biotinylated polymerase chain reaction (PCR) products was estimated using HCMV-specific and MCMV-specific enzyme-labelled probes and automated chemiluminescence detection. Log-transformed HCMV-to-MCMV signal ratios were calculated and used for quantification of test samples against simultaneously extracted MCMV-spiked calibration standards. Evaluation of the assay sensitivity by Probit analysis demonstrated a 95% probability of detection at 100 HCMV genomes per ml of plasma; the dynamic range was shown to be > or = 4 log(10). A total of 315 samples from 61 bone marrow transplant patients were analysed by both the quantitative PCR (qPCR) and by a previously validated nested nonquantitative PCR (NQPCR). A high level of concordance (90%) was observed between the two assays, although the qPCR assay exhibited slightly greater sensitivity.

Evaluation of diagnostic methods for the detection of cytomegalovirus in recipients of allogeneic stem cell transplants.

BACKGROUND: Although several diagnostic methods are available for the surveillance of patients at risk of human cytomegalovirus (CMV) infection and disease, little data is available on their comparative performances in the diagnostic setting. OBJECTIVES: To compare different assays for CMV detection, especially assays based on (quantitative) DNA and mRNA detection. STUDY DESIGN: Eight allogeneic bone marrow and stem cell transplant recipients at high risk for developing CMV disease (donor CMV-negative, recipient positive) were regularly tested for 7-20 weeks post-transplant by spin-amplification rapid culture from urine (viruria), antigenemia (pp65 assay), pp67 mRNA in whole blood (NASBA), and CMV DNA both qualitatively (in-house PCR, whole blood) and quantitatively (in-house PCR, plasma; Cobas Amplicor CMV Monitor Test, plasma and whole blood; Hybrid Capture, whole blood). RESULTS: Four patients (50%) suffered CMV reactivation during follow-up. Out of 104 sample dates, 41 (39.4%) yielded a positive CMV result in at least one assay. Out of the 28 samples tested by all assays, the highest percentage of positive results was obtained with the in-house quantitative PCR (60.7%), followed by the Hybrid Capture system (39.3%), the Cobas Amplicor CMV Monitor Test, plasma version (35.7%), the Cobas Amplicor CMV Monitor Test, whole blood version (32.1%), in-house qualitative PCR (28.6%), and the mRNA assay (21.4%). Viruria was positive in one sample and pp65 antigenemia was found in two samples. CONCLUSIONS: Despite a considerable incidence of CMV reactivations, pre-emptive anti-CMV chemotherapy prevented the development of CMV disease with the exception of one case. The molecular assays had superior sensitivity to conventional ones. The antigenemia assay proved unsuitable for the surveillance of hematological transplant patients. However, none of the tests recognized all timepoints with CMV reactivation. Further comparative studies are needed to determine their respective diagnostic values.

Infrequency of detection of particle-associated MSRV/HERV-W RNA in the synovial fluid of patients with rheumatoid arthritis.

OBJECTIVES: To determine whether the recently identified multiple sclerosis-associated retrovirus, MSRV, is detectable in the serum and synovial fluid of patients with rheumatoid arthritis (RA). METHODS: A reverse transcription-polymerase chain reaction (RT-PCR) assay was used to seek evidence of particle-associated MSRV/HERV-W RNA in the plasma and synovial fluid of patients with RA and controls. Stringent precautions were taken to avoid detection of contaminating human genomic DNA and cellular RNA sequences. RESULTS: Thirty-seven plasma samples were tested (20 from RA patients and 17 from controls) but none had detectable MSRV/HERV-W RNA. Synovial fluid samples were available from nine patients with RA and 10 controls. Particle-associated MSRV/HERV-W RNA was reproducibly detected in two of nine synovial fluid samples from RA patients and in one control sample. The identity of RT-PCR products was confirmed by sequencing. CONCLUSION: MSRV/HERV-W RNA sequences are detectable in the synovial fluid of a small proportion of RA patients, but this phenomenon may not be specific to RA.

Histological structure of human nail as studied by synchrotron X-ray microdiffraction.

Three layers (characterized by different orientations of the keratin molecules) from the outer to the inner side of human nail were observed by synchrotron X-ray microdiffraction. These layers are associated with the histological dorsal, intermediate and ventral plates. The hair-like type alpha-keratin filaments (81 A in diameter), are only present in the intermediate layer (accounting for approximately 2/3 of the nail width) and are perfectly oriented perpendicular to the growth axis, in the nail plane. Keratin filaments of stratum corneum (epidermis) type, found in the dorsal and ventral cells, are oriented in two privileged directions; parallel and perpendicular to the growth axis. This "sandwich" structure in the corneocytes and the strong intercellular junctions, gives the nail high mechanical rigidity and hardness, both in the curvature direction and in the growth direction. Lipid bilayers (49 A thick) parallel to the nail surface fill certain ampullar dilations of the dorsal plate and intercellular spaces in the ventral plate. Using X-ray micro-diffraction, we show that onychomycosis disrupts the keratin structure, probably during the synthesis phase.

Study of the keratinization process in human hair follicle by X-ray microdiffraction.

Synchrotron X-ray micro-diffraction studies along the follicle and the hair fibre allowed us to follow the keratinization process and the progressive organization of the keratin: i) molecular organization appeared progressively in the follicle; the formation of alpha-helices was completed inside the follicle. ii) supramolecular organization appeared only outside the follicle, far from the bulb; filament structure was observed far from the follicle. Comparisons between structures observed for in vitro and in vivo grown hair, whatever in the bulb or in the fibre, indicate there is no evidence of any structural difference. More, no variation in the transition in vivo/in vitro zone has been observed. In vitro and in vivo fibres exhibited the same structure.

Combination therapy with interferon-alpha plus N-acetyl cysteine for chronic hepatitis C: a placebo controlled double-blind multicentre study.

A small pilot study in patients with chronic hepatitis C (HCV) infection suggested that antiviral treatment with interferon (IFN) plus N-acetyl cysteine (NAC) was more effective than treatment with interferon alone [Beloqui et al. (1993) Journal of Interferon Research 13:279-282]. An attempt was made to confirm this by performing a placebo-controlled double-blind study at 8 medical centres in Spain and Italy. One-hundred forty-seven patients with chronic HCV infection were investigated, 73 received 3MU IFN-alpha thrice weekly plus NAC 1800 mg daily and 74 received IFN alone. Treatment was continued for 6 months and patients were followed up for a further 6 months. Amongst patients receiving IFN plus NAC, sustained virological responses were observed in 5.5%, transient responses in 26% and non-response in 68.5%. The figures for patients receiving IFN only were 4.1%, 24.3% and 71.6% respectively. Sustained virological response was significantly associated with non-type 1 genotypes (P = 0.045) and with low pre-treatment viraemia levels (P = 0.034). Biochemical response (serum ALT concentrations) correlated with virological outcome in 97% (n = 139) of cases. Patients who experienced a sustained virological response also showed reduction in the Knodell histological activity index. It is concluded that patients with chronic HCV infection are very unlikely to benefit from the addition of N-acetyl cysteine to conventional therapy with interferon-alpha.

Detection of reverse transcriptase activity in the serum of patients with motor neurone disease.

The recognition that both human and murine retroviruses can cause motor neurone disease-like syndromes has raised the possibility that a retrovirus may be involved in the aetiology of motor neurone disease. This possibility was explored by looking for evidence of reverse transcriptase in the serum of motor neurone disease patients. Sera from 56 patients with motor neurone disease and 58 controls were tested by the product-enhanced reverse transcriptase assay, a technique that is approximately a million fold more sensitive than conventional reverse transcriptase assays and capable of detecting very low numbers of retroviral particles. Cell-free reverse transcriptase activity was detected in the serum of 33 of the 56 motor neurone disease patients (59%) but in only 3 of the controls (P < 0.00001). The reverse transcriptase activity was detectable in the presence of a large excess of an effective inhibitor of human cellular DNA polymerases and was therefore tentatively considered to be compatible with a retroviral origin. The reverse transcriptase activity, however, was not found to be due to the presence of known human exogenous retroviruses including HIV-1, HIV-2, HTLV-I, HTLV-II, HRV-5 or human foamy virus, as assessed by PCR-based assays. Further investigations will be required to determine the source of the reverse transcriptase activity observed in these motor neurone disease patient sera.

Effects of handling and storage of blood on the stability of hepatitis C virus RNA: implications for NAT testing in transfusion practice.

BACKGROUND AND OBJECTIVES: To determine the stability of hepatitis C virus (HCV) RNA during transport and storage of blood samples from donors, prior to screening for HCV by nucleic acid amplification technology. MATERIALS AND METHODS: Various blood and plasma sample types were stored for up to 120 h at different temperatures and the HCV RNA level was measured using an in house quantitative reverse transcription-polymerase chain reaction. RESULTS: No decline in HCV RNA level was observed after 72 h of storage of whole blood at 4 degrees C in EDTA tubes (Greiner) and Plasma Preparation Tubes (PPT; Becton Dickinson), while insignificant declines of 0.2 log10 and 0. 25 log10 occurred at 25 degrees C after 72 h in the EDTA tubes and PPT tubes, respectively. When whole blood was stored with mixed anticoagulants CPDA-1 and EDTA for up to 120 h, no decline in HCV RNA level was observed at 4 degrees C and 25 degrees C, while a significant decline of 0.37 log10 occurred at 37 degrees C after 120 h. The temperature during transportation was investigated with a 12-hour period at 25 degrees C and 37 degrees C before storage at 4 degrees C for 108 h. Neither temperature resulted in any loss of HCV RNA in comparison with 120 h of storage at 4 degrees C. CONCLUSION: Whole blood anticoagulated with EDTA or CPDA-1/EDTA may be stored at up to 25 degrees C (room temperature) for up to 5 days without any significant loss in plasma HCV RNA level.

Transmission rates of hepatitis C virus by different batches of a contaminated anti-D immunoglobulin preparation.

BACKGROUND AND OBJECTIVES: The aim of this study was to determine the hepatitis C virus (HCV) infection rate of recipients of different batches of anti-D immunoglobulin associated with an outbreak of HCV infection which occurred in 1977 and its relationship to the polymerase chain reaction (PCR) status of the implicated batches. This study was undertaken to determine the predictive value of HCV genome detection and quantification for subsequent infection in recipients of an HCV-contaminated anti-D immunoglobulin product for intravenous use. MATERIALS AND METHODS: Sera from recipients of anti-D were tested by HCV enzyme immunoassay and if found positive were subsequently tested by recombinant immunoblot assay and HCV PCR in a national HCV anti-D screening programme set up in 1994. The HCV status of 1,342 known recipients of infectious or potentially infectious batches has been compared to the amount of HCV RNA in the anti-D batch they received so as to determine the value of PCR in the prediction of infectivity in immunoglobulin preparations. RESULTS: It has been demonstrated that HCV-infected plasma derived from batches of anti-D showing levels of viral genome in excess of 10(4) genomes per millilitre led to infection of up to 60% of recipients. In contrast, batches with undetectable levels of HCV genome very rarely transmitted infection. CONCLUSIONS: The presence of HCV RNA in intravenous immunoglobulin preparations which have not undergone a specific viral inactivation step is a predictor of HCV infection in recipients.