Assessment of a New Lower-Cost Real-Time PCR Assay for Detection of High-Risk Human Papillomavirus: Useful for Cervical Screening in Limited-Resource Settings?

Inexpensive and easy-to-perform human papillomavirus (HPV) tests are needed for primary cervical cancer screening in lower-resource regions. In a convenience sample of 516 residual exfoliative cervical specimens from the Kaiser Permanente Northern California and U.S. National Cancer Institute Persistence and Progression Study, we assessed the agreement and clinical performance of a simple, inexpensive real-time PCR assay for the detection of 13 carcinogenic HPV types (the H13 assay; Hybribio, Hong Kong) that is marketed in limited-resource settings compared to previous testing by the Hybrid Capture 2 assay (HC2; Qiagen, Germantown, MD) and the Onclarity assay (BD Diagnostics, Sparks, MD). The test set was chosen to include many HPV-positive specimens. The reference standard was a combination of HC2 and Onclarity results for HPV detection and histologic diagnosis of controls (less than cervical intraepithelial neoplasia grade 2 [

Cost-Effectiveness Analysis of Different Management Strategies for Detection CIN2+ of Women with Atypical Squamous Cells of Undetermined Significance (ASC-US) Pap Smear in Thailand.

BACKGROUND: To identify the optimal cost effective strategy for the management of women having ASC-US who attended at King Chulalongkorn Memorial Hospital (KMCH). DESIGN: An Economical Analysis based on a retrospective study. SUBJECT: The women who were referred to the gynecological department due to screening result of ASC-US at King Chulalongkorn Memorial Hospital, a general and tertiary referral center in Bangkok Thailand, from Jan 2008 - Dec 2012. MATERIALS AND METHODS: A decision tree-based was constructed to evaluate the cost effectiveness of three follow up strategies in the management of ASC-US results: repeat cytology, triage with HPV testing and immediate colposcopy. Each ASC-US woman made the decision of each strategy after receiving all details about this algorithm, advantages and disadvantages of each strategy from a doctor. The model compared the incremental costs per case of high-grade cervical intraepithelial neoplasia (CIN2+) detected as measured by incremental cost-effectiveness ratio (ICER). RESULTS: From the provider's perspective, immediate colposcopy is the least costly strategy and also the most effective option among the three follow up strategies. Compared with HPV triage, repeat cytology triage is less costly than HPV triage, whereas the latter provides a more effective option at an incremental cost-effectiveness ratio (ICER) of 56,048 Baht per additional case of CIN 2+ detected. From the patient's perspective, the least costly and least effective is repeat cytology triage. Repeat colposcopy has an incremental cost-effectiveness (ICER) of 2,500 Baht per additional case of CIN2+ detected when compared to colposcopy. From the sensitivity analysis, immediate colposcopy triage is no longer cost effective when the cost exceeds 2,250 Baht or the cost of cytology is less than 50 Baht (1USD = 31.58 THB). CONCLUSIONS: In women with ASC-US cytology, colposcopy is more cost-effective than repeat cytology or triage with HPV testing for both provider and patient perspectives.

A real time genotyping PCR assay for polyomavirus BK.

BACKGROUND: Polyomavirus BK (BKV) may cause nephropathy in renal transplant recipients and hemorrhagic cystitis in bone marrow recipients. We developed real-time PCRs (RT-PCR) to determine easily and rapidly the different BKV genotypes (BKGT) (I-IV). METHODS: On the VP1 gene a duplex of RT-PCRs was developed and validated to differentiate the four main BKGT. 212 BKV positive samples (21 plasma, 191 urine) were tested with these specific PCRs. Of these 212 samples, 55 PCR results were additionally confirmed by sequencing a VP1 gene fragment (nucleotide 1630-1956). RESULTS: For every genotype, a highly specific, precise and internally controlled assay was developed with a limit of detection of log 3 copies per ml. In 18 (8.5%) of these samples genotyping was not successful due to a low viral load. By sequence analysis, the genotype of 46 out of 55 and 2 out of 4 samples with double infection could be confirmed. CONCLUSIONS: This study describes RT-PCRs for detection of the main BKGT. It proved to be rapid, cheap and sensitive compared to sequencing. Double infections can also be detected. This method will be of value to investigate the role of BKV infection in relation to the genotype.

Cost-effectiveness analysis of 2 surveillance options for cervical intraepithelial neoplasia 1.

OBJECTIVE: We aim to determine the difference in cost between 2 accepted surveillance strategies for women diagnosed with cervical intraepithelial neoplasia 1 (CIN 1): repeat cytology at 6 and 12 months versus human papillomavirus (HPV) DNA testing at 12 months. MATERIALS AND METHODS: Extracting data from the literature regarding the natural history of HPV infection and CIN 1, we estimated regression, persistence, and progression rates during a 2-year interval. Costs were based on 2011 Medicaid reimbursements for cytology, biopsy interpretation, HPV testing, and the associated office visit or procedure fee. We constructed a decision tree model to estimate the potential cost benefits of using HPV testing, and sensitivity analyses were performed. Treatment costs for high-grade disease were not included because of equal occurrence in both groups. RESULTS: In a hypothetical cohort of 100 women with CIN 1 (assumed compliant with 2 y of follow-up), the total cost for cytology-based follow-up was $89,969, whereas the total cost for HPV-based follow-up was $37,357. This indicates an average cost savings of $526 per patient in favor of HPV testing. If we then consider the 234,603 incident cases of CIN 1 in the United Sates per year, preferential use of HPV-based follow-up would save $123,429,305. CONCLUSIONS: Although both cytology and HPV testing are sound methods for surveillance of CIN 1, it is more cost-effective to use HPV testing.

Development of a loop-mediated isothermal amplification assay for rapid detection of iridovirus in the Chinese giant salamander.

The Chinese giant salamander (Andrias davidianus) iridovirus (GSIV) is an emerging infectious pathogen responsible for severe hemorrhagic disease and high mortality in cultured Chinese giant salamanders. A loop-mediated isothermal amplification (LAMP) assay based on the major caspid protein (MCP) gene has been developed to detect this virus. Primer pairs for the LAMP assay were designed based on the GSIV MCP gene sequence. Amplification results indicate that under optimized conditions the LAMP assay has the ability to specifically detect the virus in both diseased animals and infected epithelioma papilloma cyprinid (EPC) cells. The assay was shown to be 10-fold more sensitive than nested PCR and was able to detect concentrations of 10(-9) (approximately 0.01 pg/µL). The LAMP assay is relatively easy to perform in situ and the amplification products can be observed directly under UV light or via staining with SYBR Green I. The LAMP assay is also rapid and cost-effective. This study establishes the use of a LAMP assay for rapid detection of GSIV, which is a novel and important tool for the diagnosis of GSIV infection in laboratory or farmed Chinese giant salamanders.

Allele-specific polymerase chain reaction for detection of a mutation in the relax circular DNA and the covalently closed circular DNA of hepatitis B virus.

The relax circle DNA (rcDNA) sequence and the covalently closed circle DNA (cccDNA) sequence in hepatitis B virus (HBV) are crucial regions for HBV infections. To analyze mutations in rcDNA and cccDNA, DNA sequencing is often used, although it is time-consuming and expensive. Herein, we report a simple, economic, albeit accurate allele-specific polymerase chain reaction (AS-PCR) to detect mutations in these regions of HBV. This method can be extensively used to screen for mutations at specific positions of HBV genome.

A cost effective real-time PCR for the detection of adenovirus from viral swabs.

Compared to traditional testing strategies, nucleic acid amplification tests such as real-time PCR offer many advantages for the detection of human adenoviruses. However, commercial assays are expensive and cost prohibitive for many clinical laboratories. To overcome fiscal challenges, a cost effective strategy was developed using a combination of homogenization and heat treatment with an "in-house" real-time PCR. In 196 swabs submitted for adenovirus detection, this crude extraction method showed performance characteristics equivalent to viral DNA obtained from a commercial nucleic acid extraction. In addition, the in-house real-time PCR outperformed traditional testing strategies using virus culture, with sensitivities of 100% and 69.2%, respectively. Overall, the combination of homogenization and heat treatment with a sensitive in-house real-time PCR provides accurate results at a cost comparable to viral culture.

Detection and genotyping of human papillomavirus by real-time PCR assay.

BACKGROUND: Diagnosis of human papillomavirus (HPV) disease remains a challenge due to several factors related to the cost, the workload of available commercial assays to detect and genotype HPV, and to the low prevalence of infected patients. OBJECTIVE: Our study aimed to develop a real-time PCR, based on SPF10 primers, in order to combine HPV-DNA detection and genotype identification avoiding the negative samples. STUDY DESIGN: Validation of SYBR-green based SPF10 real-time PCR on HPV-DNA plasmids followed by the investigation of the viral status in 92 samples from oropharyngeal (94%) cutaneous biopsies (3%) and anal smears (3%) which had previously been HPV-genotyped by LiPA hybridization. In-house HPV viral loads were performed to evaluate the SPF10 real-time PCR sensitivity. RESULTS: Data showed that 100% of HPV plasmids, assessable by LiPA hybridization, were detected and genotyped appropriately after SPF10 real-time PCR assays. These results defined a range of melting temperature peaks for HPV positivity by real-time PCR. The efficient determination of the presence of HPV-DNA by SPF10 real-time PCR was validated for 98% of clinical samples compared to commercial method. Discordant results were due to a low HPV-DNA amount and to a supplementary HPV genotype identified. The SPF10 real-time PCR sensitivity was evaluated between 1 and 10 copies/10(3)cells using in-house HPV (6, 11 and 16) viral load assays. CONCLUSION: The real-time PCR method was efficient in combining screening and genotyping of HPV-DNA. Cost and workload reduction by SPF10 real-time PCR approach may facilitate earlier diagnosis and clinical management of HPV infected patients.

Modeling preventative strategies against human papillomavirus-related disease in developed countries.

Over the last 5 years, prophylactic vaccination against human papillomavirus (HPV) in pre-adolescent females has been introduced in most developed countries, supported by modeled evaluations that have almost universally found vaccination of pre-adolescent females to be cost-effective. Studies to date suggest that vaccination of pre-adolescent males may also be cost-effective at a cost per vaccinated individual of ~US$400-500 if vaccination coverage in females cannot be increased above ~50%; but if it is possible, increasing coverage in females appears to be a better return on investment. Comparative evaluation of the quadrivalent (HPV16,18,6,11) and bivalent (HPV16,18) vaccines centers around the potential trade-off between protection against anogenital warts and vaccine-specific levels of cross-protection against infections not targeted by the vaccines. Future evaluations will also need to consider the cost-effectiveness of a next generation nonavalent vaccine designed to protect against ~90% of cervical cancers. The timing of the effect of vaccination on cervical screening programs will be country-specific and will depend on vaccination catch-up age range and coverage and the age at which screening starts. Initial evaluations suggest that if screening remains unchanged, it will be less cost-effective in vaccinated compared to unvaccinated women but, in the context of current vaccines, will remain an important prevention method. Comprehensive evaluation of new approaches to screening will need to consider the population-level effects of vaccination over time. New screening strategies of particular interest include delaying the start age of screening, increasing the screening interval and switching to primary HPV screening. Future evaluations of screening will also need to focus on the effects of disparities in screening and vaccination uptake, the potential effects of vaccination on screening participation, and the effects of imperfect compliance with screening recommendations. This article forms part of a special supplement entitled "Comprehensive Control of HPV Infections and Related Diseases" Vaccine Volume 30, Supplement 5, 2012.

Cost savings associated with testing of antibodies, antigens, and nucleic acids for diagnosis of acute HIV infection.

Efforts to identify all persons infected with HIV in the United States are driven by the hope that early diagnosis will lower risk behaviors and decrease HIV transmission. Identification of HIV-infected people earlier in the course of their infection with HIV antigen/antibody (Ag/Ab) combination assays (4th-generation HIV assays) should help achieve this goal. We compared HIV RNA nucleic acid test (NAT) results to the results of a 4th-generation Ag/Ab assay (Architect HIV Ag/Ab Combo [HIV Combo] assay; Abbott Diagnostics) in 2,744 HIV antibody-negative samples. Fourteen people with acute HIV infection (HIV antibody negative/NAT positive) were identified; the HIV Combo assay detected nine of these individuals and was falsely negative in the remaining five. All five persons missed by the HIV Combo assay were in the stage of exponential increase in plasma virus associated with acute HIV infection (3, 7, 20, 35, 48). In contrast, most acutely infected persons detected by the HIV Combo assay demonstrated either a plateauing or decreasing plasma viral load. The HIV Combo assay also classified as positive five other samples which were negative by NAT. Taken together, the HIV Combo assay had a sensitivity of 73.7% and a specificity of 99.8%. Using published data, we estimated secondary transmission events had HIV infection in these five individuals remained undiagnosed. Screening of our population with NAT cost more than screening with the HIV Combo assay but achieved new diagnoses that we predict resulted in health care savings that far exceed screening costs. These findings support the use of more sensitive assays, like NAT, in HIV screening of populations with a high prevalence of acute HIV infection.

Detection of white spot syndrome virus by polymerase chain reaction performed under insulated isothermal conditions.

Aiming to develop a rapid, low-cost, and user-friendly system for the diagnosis of white spot syndrome virus (WSSV), a PCR assay performed in capillary tubes under insulated isothermal conditions (iiPCR assay) was established on the basis of Rayleigh-Benard convection. WSSV amplicons were generated reproducibly within 30 min from a target sequence-containing plasmid in an iiPCR device, in which a special polycarbonate capillary tube (R-tube™) was heated isothermally by a copper ring attached to its bottom and shielded by a thermal baffle around its upper half. Furthermore, WSSV-specific amplicons were produced from nucleic acid extracts of WSSV-infected Penaeus vannamei in the WSSV iiPCR assay, with sensitivity comparable to that of an OIE-certified commercial nested PCR kit (IQ2000™ WSSV Detection and Prevention System). Specificity of the WSSV iiPCR assay was demonstrated as no amplicons were generated from shrimp genomic DNA, and IHHNV, MBV, and HPV DNA. iiPCR has a potential as a low-cost method for sensitive, specific and rapid detection of pathogens.

Cost-effective method for microbial source tracking using specific human and animal viruses.

Microbial contamination of the environment represents a significant health risk. Classical bacterial fecal indicators have shown to have significant limitations, viruses are more resistant to many inactivation processes and standard fecal indicators do not inform on the source of contamination. The development of cost-effective methods for the concentration of viruses from water and molecular assays facilitates the applicability of viruses as indicators of fecal contamination and as microbial source tracking (MST) tools. Adenoviruses and polyomaviruses are DNA viruses infecting specific vertebrate species including humans and are persistently excreted in feces and/or urine in all geographical areas studied. In previous studies, we suggested the quantification of human adenoviruses (HAdV) and JC polyomaviruses (JCPyV) by quantitative PCR (qPCR) as an index of human fecal contamination. Recently, we have developed qPCR assays for the specific quantification of porcine adenoviruses (PAdV) and bovine polyomaviruses (BPyV) as animal fecal markers of contamination with sensitivities of 1-10 genome copies per test tube. In this study, we present the procedure to be followed to identify the source of contamination in water samples using these tools. As example of representative results, analysis of viruses in ground water presenting high levels of nitrates is shown. Detection of viruses in low or moderately polluted waters requires the concentration of the viruses from at least several liters of water into a much smaller volume, a procedure that usually includes two concentration steps in series. This somewhat cumbersome procedure and the variability observed in viral recoveries significantly hamper the simultaneous processing of a large number of water samples. In order to eliminate the bottleneck caused by the two-step procedures we have applied a one-step protocol developed in previous studies and applicable to a diversity of water matrices. The procedure includes: acidification of ten-liter water samples, flocculation by skimmed milk, gravity sedimentation of the flocculated materials, collection of the precipitate and centrifugation, resuspension of the precipitate in 10 ml phosphate buffer. The viral concentrate is used for the extraction of viral nucleic acids and the specific adenoviruses and polyomaviruses of interest are quantified by qPCR. High number of samples may be simultaneously analyzed using this low-cost concentration method. The procedure has been applied to the analysis of bathing waters, seawater and river water and in this study, we present results analyzing groundwater samples. This high-throughput quantitative method is reliable, straightforward, and cost-effective.

Validation of a low-cost human papillomavirus genotyping assay based on PGMY PCR and reverse blotting hybridization with reusable membranes.

The genotyping of human papillomaviruses (HPV) is essential for the surveillance of HPV vaccines. We describe and validate a low-cost PGMY-based PCR assay (PGMY-CHUV) for the genotyping of 31 HPV by reverse blotting hybridization (RBH). Genotype-specific detection limits were 50 to 500 genome equivalents per reaction. RBH was 100% specific and 98.61% sensitive using DNA sequencing as the gold standard (n = 1,024 samples). PGMY-CHUV was compared to the validated and commercially available linear array (Roche) on 200 samples. Both assays identified the same positive (n = 182) and negative samples (n = 18). Seventy-six percent of the positives were fully concordant after restricting the comparison to the 28 genotypes shared by both assays. At the genotypic level, agreement was 83% (285/344 genotype-sample combinations; κ of 0.987 for single infections and 0.853 for multiple infections). Fifty-seven of the 59 discordant cases were associated with multiple infections and with the weakest genotypes within each sample (P < 0.0001). PGMY-CHUV was significantly more sensitive for HPV56 (P = 0.0026) and could unambiguously identify HPV52 in mixed infections. PGMY-CHUV was reproducible on repeat testing (n = 275 samples; 392 genotype-sample combinations; κ of 0.933) involving different reagents lots and different technicians. Discordant results (n = 47) were significantly associated with the weakest genotypes in samples with multiple infections (P < 0.0001). Successful participation in proficiency testing also supported the robustness of this assay. The PGMY-CHUV reagent costs were estimated at $2.40 per sample using the least expensive yet proficient genotyping algorithm that also included quality control. This assay may be used in low-resource laboratories that have sufficient manpower and PCR expertise.

Development of an optimized method for the detection of airborne viruses with real-time PCR analysis.

BACKGROUND: Airborne viruses remain one of the major public health issues worldwide. Detection and quantification of airborne viruses is essential in order to provide information regarding public health risk assessment. FINDINGS: In this study, an optimized new, simple, low cost method for sampling of airborne viruses using Low Melting Agarose (LMA) plates and a conventional microbial air sampling device has been developed. The use of LMA plates permits the direct nucleic acids extraction of the captured viruses without the need of any preliminary elution step. Molecular detection and quantification of airborne viruses is performed using real-time quantitative (RT-)PCR (Q(RT-)PCR) technique. The method has been tested using Adenoviruses (AdVs) and Noroviruses (NoVs) GII, as representative DNA and RNA viruses, respectively. Moreover, the method has been tested successfully in outdoor experiments, by detecting and quantifying human adenoviruses (HAdVs) in the airborne environment of a wastewater treatment plant. CONCLUSIONS: The great advantage of LMA is that nucleic acids extraction is performed directly on the LMA plates, while the eluted nucleic acids are totally free of inhibitory substances. Coupled with QPCR the whole procedure can be completed in less than three (3) hours.

A single-tube multiplex PCR for rapid detection in feces of 10 viruses causing diarrhea.

A novel multiplex polymerase chain reaction assay was developed to identify 10 viruses in a single tube. The assay was targeted to detect group A and C rotaviruses, adenovirus, norovirus GI, norovirus GII, sapovirus, astrovirus, Aichi virus, parechovirus, and enterovirus. A total of 235 stool samples were collected from infants and children with acute gastroenteritis in Kyoto, Japan, from 2008 to 2009, then tested by this novel multiplex PCR and compared with a multiplex PCR described previously, which used 3 primer sets. The novel multiplex PCR could detect the targeted viruses in 111 of the 235 (47.2%) stool samples. Of these, 9 out of 10 types of viruses were identified, including group A rotavirus, norovirus GII, enterovirus, sapovirus, adenovirus, parechovirus, group C rotavirus, astrovirus, and norovirus GI. In contrast, the multiplex PCR that used 3 sets of primers could detect the targeted viruses in 109 of the 235 (46.4%) stool samples. Among these, 8 types of viruses were identified, including group A rotavirus, norovirus GII, enterovirus, adenovirus, parechovirus, group C rotavirus, sapovirus, and astrovirus. The results suggested that the new multiplex PCR is useful as a rapid and cost effective diagnostic tool for the detection of major pathogenic viruses causing diarrhea.

Twenty-four mini-pool HCV RNA screening in a routine clinical virology laboratory setting: a six-year prospective study.

The usefulness of combined anti-HCV and 24 mini-pool HCV RNA screening strategy was re-evaluated after a six-year continuous routine use in a clinical virology laboratory, at which more than half of newly diagnosed hepatitis C patients are intravenous drug users. Pools of 24 samples were prepared from 20,448 anti-HCV negative serum samples and tested using an automated commercial PCR assay with a lower limit of detection of 50 IU/ml. After detection of anti-HCV negative/HCV RNA positive patients, responsible physicians provided follow-up samples. Thirty-eight (0.19%) anti-HCV negative/HCV RNA positive samples from 30 patients (28 intravenous drug users) were detected. Follow-up samples were available for 27/30 patients. Twenty, six and one patient seroconverted in the second, third and fourth available samples, respectively. The interval between the first HCV RNA positive and the first available anti-HCV positive sample was 17-517 days. The costs of detecting a single anti-HCV negative/HCV RNA positive patient were 1227 Euros. Combined anti-HCV and 24 mini-pool HCV RNA screening is a useful and cost effective strategy, not only in blood-transfusion settings but also in a routine clinical virology laboratory, at which a significant proportion of the tested population belongs to a high-risk population.

Detection of poliovirus by ICC/qPCR in concentrated water samples has greater sensitivity and is less costly using BGM cells in suspension as compared to monolayers.

The integrated cell culture quantitative reverse transcriptase PCR (ICC/qRT-PCR) method is used in our lab to detect enteroviruses in environmental waters. Typically we utilize monolayers of 3 cell lines; buffalo green monkey kidney (BGM), human colonic carcinoma (CACO-2) and African rhesus monkey kidney (MA104) with the intent of providing one or more permissive hosts to a wide range of enteroviruses. In this study the BGM cell line was used to compare poliovirus infectivity in conventional monolayer cultures to BGM cells in suspensions. Propagated virus was subsequently amplified by qRT-PCR. Our PCR data showed lower cycle threshold (Ct) values in the suspensions which corresponded to a higher rate of infectivity than that observed in the monolayers. The difference in Ct values was determined statistically significant by One-way ANOVA (0.000). Infecting BGM cells in suspensions required less hands-on time, less chance of contamination and was more cost effective than utilizing the conventional monolayer technique.

Development and application of a one-step low cost procedure to concentrate viruses from seawater samples.

A novel and simple procedure for concentrating adenoviruses from seawater samples is described. The technique entails the adsorption of viruses to pre-flocculated skimmed milk proteins, allowing the flocs to sediment by gravity, and dissolving the separated sediment in phosphate buffer. Concentrated virus may be detected by PCR techniques following nucleic acid extraction. The method requires no specialized equipment other than that usually available in routine public health laboratories, and due to its straightforwardness it allows the processing of a larger number of water samples simultaneously. The usefulness of the method was demonstrated in concentration of virus in multiple seawater samples during a survey of adenoviruses in coastal waters.

Cost-effectiveness analysis of liquid-based cytology and human papillomavirus testing in cervical cancer screening.

OBJECTIVE: To compare the outcomes of several cervix cancer screening strategies in a military population using a model that considers both direct and indirect costs of health care. METHODS: A Markov model of the natural history of cervical cancer was used to simulate an age-stratified cohort of 100,000 active duty women in the U.S. Army. Total costs and incremental cost-effectiveness ratios were estimated for different modalities of screening: liquid-based cytology with testing for human papillomavirus (HPV) irrespective of cytologic results compared with liquid-based cytology with HPV detection for cytologic results of atypical cells of undetermined significance (reflex HPV). The costs and outcomes of these screening methods were evaluated separately as well as in combination (liquid-based cytology and reflex HPV before age 30 years and DNA and Pap test every 3 years thereafter). Each of these screening methods was evaluated at 1-, 2-, and 3-year intervals. RESULTS: A screening strategy of liquid-based cytology and reflex HPV every 2 or 3 years is the least costly strategy among active duty women irrespective of age, especially when accounting for time costs associated with screening, diagnosis, and treatment of cervix cancer. A strategy of liquid-based cytology and HPV testing irrespective of cytology results is the most effective strategy; however, it is also the most costly of the strategies tested, even when performed in patients older than 30 years of age. CONCLUSION: In the U.S. Army, cervix cancer screening performed with liquid-based cytology and reflex HPV testing of atypical squamous cells of undetermined significance performed every 2 years is cost-effective, especially when indirect costs are considered.

PCR for HBV, HCV and HIV-1 experiences and first results from a routine screening programme in a large blood transfusion service.

We adapted the PCR method to screen up to 3000 blood donations per day for hepatitis B, hepatitis C and HIV-1 virus contamination. Up to 600 aliquots (average 418 donations) are pooled by using an automatic sample processor with disposable tips (validated to avoid contamination) taken from blood donations which are serologically negative and free for clinical usage according to federal regulations. In the case of a positive PCR pool result the viraemic donation is identified by two additional PCR pools testing steps with smaller pool sizes. All of the steps are supported by electronic data processing. After virus concentration by ultracentrifugation, and in the case of HCV and HIV-1 an additional reverse transcription step, PCR amplifications are performed. PCRs are done for each virus in two genomic regions. Laser-induced detection after PAGE and computer-analysis are used to identify the amplification products. Using this validated methodology routine we have checked 428 896 donations up to the end of August 1996. During this survey we found at least 24 viruses-containing donations which were negative in corresponding serological tests and would have been transfused (2 HBV-, 22 HCV-, 0 HIV-1 -containing donations). It seems possible for large transfusion centres to shorten the diagnostic window periods with our PCR-methodology with acceptable costs (15 DM per donation for all three viruses including logistics, developments and investments).