Influence of HPV type on prognosis in patients diagnosed with invasive cervical cancer.

While much is known about the influence of HPV type on the progression of pre-invasive cervical lesions, the impact of HPV type on cervical cancer prognosis is less evidenced. Thus, we assessed the impact of HPV type on the survival of women diagnosed with cervical cancer. A total of 370 cases of cervical cancer were assessed. Univariate analysis is presented using Kaplan-Meier survival curves and log-rank statistics and multivariable Cox proportional hazard models were generated using age group, socio-economic deprivation, FIGO stage, differentiation and HPV type. HPV grouping was considered in a number of ways with particular reference to the presence or absence of HPV 16 and/or 18. In the univariate analysis, FIGO, age at diagnosis and treatment were associated with poorer survival (p < 0.0001) as was absence of HPV 16 and/or 18 (p = 0.0460). The 25% mortality time in the non-HPV 16/18 vs. HPV16/18 positive group was 615 days and 1,307 days respectively. An unadjusted Cox PH model based HPV16/18 vs. no HPV 16/18 resulted in a hazard ratio of 0.669 (0.450, 0.995). Adjusting for deprivation, FIGO and age group resulted in a hazard ratio of 0.609 (0.395, 0.941) p = 0.025. These data indicate that cancers associated with HPV 16 and/or 18 do not confer worse survival compared to cancers associated with other types, and may indicate improved survival. Consequently, although HPV vaccine is likely to reduce the incidence of cervical cancer it may not indirectly improve cervical cancer survival by reducing the burden of those cancers caused by HPV16/18.

Evaluation of commercial HPV assays in the context of post-treatment follow-up: Scottish Test of Cure Study (STOCS-H).

AIMS: Human papillomavirus (HPV) testing is more sensitive than cytology for detection of residual/recurrent cervical disease after lesion treatment. Several HPV test comparison studies have been performed within triage and screening populations, but data on their comparative performance in a test of cure context is lacking. This study aims to address this gap. METHODS: We compared the technical and clinical performance of Abbott RealTime High risk (HR)-HPV, Genprobe Aptima PV, Hologic Cervista HPV-HR, Qiagen Hybrid Capture 2 and Roche cobas HPV in the Early Implementation phase of a 'test of cure' service within the Scottish Cervical Screening Programme. RESULTS: Valid results with all five HPV Tests from 1020 first samples taken ∼6 months post-treatment showed HPV positivity ranging from 17.84% to 26.96%. There was perfect agreement in 74%, and greatest variation between assays was observed in cytologically negative samples. Clinical performance was judged on cumulative incidence of cervical intraepithelial neoplasia 2+ (CIN2+) during follow-up (mean: 13.2 months). There were 23 cases of CIN2+ of which 14 were CIN3+. All assays, including cytology, were 100% sensitive for detection of CIN3+. Of the nine cases of residual CIN2, three assays detected all, one assay missed one and one assay missed two cases. Specificity ranged from 75% to 84% according to assay. CONCLUSIONS: All assays were sensitive for detection of CIN2+ at 6 months post-treatment. The range of positivity equated to a 50% increase between assays with the lowest and highest positivity rates. The relevance of HPV positivity in the absence of cytological abnormalities requires longer follow-up to determine whether additional tools for risk stratification are required.

Understanding HPV tests and their appropriate applications.

Greater understanding of the role played by human papillomavirus (HPV) in the causation of disease has led to the development of an increasing number of HPV tests with different characteristics. The bewildering choice facing healthcare professionals and providers is daunting. Clearly, HPV testing is no longer simply of research interest, but can provide information that can be used for individual patient management and at the population level for cervical screening and vaccine surveillance. This review aims to provide the background to the development of HPV tests, to explain the different technologies and to discuss the challenges of the application of these optimally in the varied contexts of disease management. Few HPV tests are approved for clinical use and it is important that clinicians understand which test can be utilized, in what circumstances, with what specimens and the meaning of the report issued. HPV testing is no longer applicable only to cervical disease, and we have suggested additional areas, such as the oropharynx, in which HPV testing services might be implemented in the near future. New tests will continue to emerge and we have identified some of the indirect measures of HPV activity, or biomarkers, that could help in the risk stratification of HPV infection and associated disease. The challenges relating to the optimal application of the various HPV technologies are compounded by the lack of evidence regarding their performance in vaccinated populations. Currently published work, including modelling studies, has been undertaken in non-immunized populations. We therefore end by addressing the issues regarding appropriate strategies and tests for immunized populations.

Effect of glacial acetic acid pre-treatment of cervical liquid-based cytology specimens on the molecular detection of human papillomavirus.

OBJECTIVE: Cytology laboratories routinely treat cervical liquid-based cytology (LBC) specimens that are heavily contaminated with blood with glacial acetic acid (GAA) in order to lyse red blood cells and facilitate assessment. However, the impact on downstream human papillomavirus (HPV) detection is not well understood. This study examines the effect of GAA pre-treatment of ThinPrep(®) Preservcyt(®) specimens on the molecular detection of HPV. METHODS: A panel of 150 routinely collected cervical LBC specimens was tested with two commercial HPV tests, the Abbott RealTime High Risk HPV test (rtHPV) and the Qiagen Hybrid Capture 2 High Risk HPV DNA test (HC2), as aliquots before and after GAA treatment. Statistical analysis was performed using McNemars test and Bland and Altman plots. RESULTS: Agreement between the results of the rtHPV test on GAA-untreated and GAA-treated specimens was 95.7%, with no evidence of a significant difference in the distribution of the discrepant results (P = 0.414). HC2 test agreement on GAA-untreated and GAA-treated specimens was 91% at a cut-off of 1 relative light unit index (RLUI) and 92% at a cut-off of 2 RLUI. There was no evidence of a difference in the distribution of discordant results at a cut-off of 1 (P = 0.405) and 2 RLUI (P = 0.564). CONCLUSIONS: GAA pre-treatment of cervical ThinPrep Preservcyt LBC specimens had little effect on the two commercial HPV tests used in this study. The impact of GAA treatment on HPV testing should, however, be validated for all HPV tests and all LBC collection media used in each particular diagnostic setting.

Characteristics of HPV infection over time in European women who are HIV-1 positive.

OBJECTIVE: The objectives of the study were to investigate high-risk human papillomavirus (HR-HPV) infection and type distribution in women infected with HIV-1, and to determine the relevance of HR-HPV positivity and persistence/loss to the development of high-grade cervical disease. DESIGN: A total of 518 European women infected with HIV attending for routine gynaecological care consented to 6-monthly follow-up visits over 3 years, with surveillance of cytology, colposcopy and histopathology, where relevant, and longer follow-up, where possible. SETTING: European women infected with HIV attending for routine gynaecological care. POPULATION OR SAMPLE: 518 European women infected with HIV attending for gynaecological care in 6 hospital-based European centres - Dublin, Edinburgh, London, Milan, Paris, and Warsaw. METHODS: Cervical screening was achieved by liquid-based cytology (LBC) of brush samples in PreservCyt® medium. The HPV testing of residual samples was performed by Hybrid-Capture II, with genotyping of positives using the HPV Line Blot Assay. Histology results were accessed where available. MAIN OUTCOME MEASURES: Description of high risk human papillomavirus (HR-HPV) infection and type distribution in HIV-1 infected women. RESULTS: The estimated prevalence at baseline of any HR-HPV type was 49.5% (46.3-52.8%): 10.2% for HPV 16 and 4.3% for HPV 18. The prevalence increased with increasing immunosuppression. Multiple infections were detected in 26.8%. HR-HPV genotypes were detected in 34.9% of cases with normal cytology, in 77.2% of cases with atypical squamous cells of undetermined significance/low-grade squamous intraepithelial lesion (ASCUS/LSIL) and in 90.8% of cases with high-grade SIL (HSIL). The prevalence of HPV 16 in HSIL was 38.5%, with the three most common types thereafter having prevalence rates of 19.2% (HPV 58), 19.2% (HPV 53) and 16.6% (HPV 52). The overall persistence of any high-risk type was 55.8%. We found that 6 months persistence of HPV 16 occurred in 24 women. Seven cases of high-grade cervical disease developed, and all were associated with initial/persistent HR-HPV positivity. CONCLUSIONS: A wide diversity of HPV types was evident, and multiple infections were common. Detection or persistence of any HR-HPV was associated with a very low incidence of subsequent high-grade disease.

HPV type-specific prevalence using a urine assay in unvaccinated male and female 11- to 18-year olds in Scotland.

BACKGROUND: We conducted a baseline prevalence survey of unvaccinated 11- to 18-year olds to inform effectiveness studies for the new human papillomavirus (HPV) immunisation programme in Scotland. METHODS: Participants were recruited from schools and colleges and invited to provide demographic data and an anonymous urine sample for type-specific HPV testing. RESULTS: Among females aged 11-14 years, the weighted prevalence was 1.1% overall; 0.9% for high-risk types and no infections were associated with types 16 and 18. Among 15- to 18-year old females, the weighted prevalence was 15.2% overall; 12.6% for high-risk types and 6.5% for types 16 and 18. Among females aged 16-18 years, infection was more frequently associated with attending college and rural schools, and showed a trend towards increasing prevalence with increasing social deprivation (P=0.045). Among males aged 11-14 years, the weighted prevalence was 1.4% overall; 1.0% for high-risk types and 0.7% for types 16 and 18. Among 15- to 18-year old males, the weighted prevalence was 3.9% overall; 2.4% for high-risk types and 0.7% for types 16 and 18. CONCLUSIONS: Human Papillomavirus prevalence is low among 11- to 14-year olds, which includes the age group targeted for routine vaccination. The prevalence in males and correlation with deprivation require further investigation.

Distribution of HPV types associated with cervical cancers in Scotland and implications for the impact of HPV vaccines.

BACKGROUND/METHODS: This study evaluated human papillomavirus (HPV) type prevalence in 370 Scottish invasive cervical cancers (ICCs) using HPV genotyping and HPV mRNA detection. RESULTS: HPV 16 and/or 18 was detected in 72% of cancers overall and in 82% of HPV-positive cancers. HPV 45 and 16 were the most frequently transcribed types. CONCLUSION: A significant reduction in ICC in Scotland should be achieved through the HPV immunisation programme.

High-risk HPV detection in specimens collected in SurePath preservative fluid: comparison of ambient and refrigerated storage.

OBJECTIVE: With moves to introduce human papillomavirus (HPV) triage at sentinel sites in England, it is essential that optimal storage and transport conditions are determined for efficient HPV detection using residual liquid-based cytology specimens. METHODS: Two cytology laboratories with comparable workloads sent residual cervical cytology specimens collected in BD Surepath Preservative Fluid to the Specialist Virology Centre for HPV testing. Storage and transport of specimens was at ambient (site A) or refrigerated (site R) temperatures. The effect of temperature on the ability to detect high-risk human papillomavirus (HR-HPV) using Digene Hybrid Capture 2 High-Risk HPV DNA Test (hc2) and Roche AMPLICOR HPV Test (AMPLICOR) was assessed. All specimens with discordant results were tested using Roche Linear Array HPV Genotyping test. RESULTS: A total of 796 residual cytology specimens, with cytology ranging from normal to severe dyskaryosis, were provided (399 from site A and 397 from site R). Ambient storage and transit of cervical specimens in SurePath medium did not appear to affect significantly the suitability of the specimen for HPV testing, as measured by the concordance of the HR-HPV screening assays for ambient versus refrigerated specimens and by the proportion of specimens which tested invalid. CONCLUSION: Residual cytology specimens in SurePath medium, stored and transported at ambient temperature, appear suitable for HR-HPV detection by AMPLICOR beyond the manufacturer's recommended time and potentially up to four weeks.

Human Papillomavirus testing for the management of low-grade cervical abnormalities in the UK--Influence of age and testing strategy.

BACKGROUND: The results of the UK pilot studies were encouraging with respect to the introduction of Human Papillomavirus (HPV) testing as a means to improve the management of low-grade cytological abnormalities. However, several important unresolved issues related to HPV triage remain, two of which are: what type of HPV test should be used and what age group should be targeted. OBJECTIVES: To perform an evaluation of two commercial HPV detection tests and to correlate disease persistence and clearance with age and HPV status by the two tests. STUDY DESIGN: We performed an evaluation of two commercial HPV tests in a cross-sectional analysis of 322 cervical cytology specimens with low-grade abnormalities. A subset of these specimens were then examined longitudinally, in order to correlate disease persistence and clearance with age and HPV status by the two detection tests. RESULTS: The two tests performed similarly with respect to the longitudinal identification/prediction of high-grade cervical disease. Age did not appear to be a factor in determining which cases went on to manifest high-grade disease within 3 years of a low-grade result (p=0.678). CONCLUSIONS: This study weakens the case for age-adjusted HPV triage of low-grade cervical abnormalities.

Public awareness of human papillomavirus.

OBJECTIVES: The main objective of this study was to review the evidence relating to the level of awareness of human papillomavirus (HPV) in the general population and the implications for the potential introduction of HPV vaccination and HPV testing as part of screening. METHODS: PubMed search performed on terms: 'HPV education', 'HPV awareness' 'Genital Warts Awareness' Results: Public awareness of HPV is generally very low, particularly with respect to its relation to abnormal smears and cervical cancer although knowledge levels vary to some extent according to sociodemographic characteristics. There is also much confusion around which types cause warts and the types that can cause cancer. The sexually transmissible nature of the infection is of major concern and confusion to women. CONCLUSIONS: Due to the lack of current awareness of HPV, significant education initiatives will be necessary should HPV vaccination and/or HPV testing be introduced. Organized edification of health-care workers and the media, who constitute the two most preferred sources of information, will be crucial.

Persistent high risk HPV infection associated with development of cervical neoplasia in a prospective population study.

AIMS: To monitor the association between the course of high risk human papillomavirus (HR-HPV) infection and the development of cervical neoplasia over time, from a baseline of normal cervical cytology. METHODS: This paper presents the follow up data from a previous cross sectional analysis. Women from a screening population who had normal cytology and who were HR-HPV positive were recalled after two to three years for cytology and HPV genotyping. The development of cervical neoplasia at follow up was related to the course of HPV infection (clearance, persistence, or sequential infection) and the presence of single or multiple HPV infections at baseline. A comparator control group of women who were HPV and cytologically negative at baseline were selected from the same population. RESULTS: Twelve cases of dyskaryosis were found in women who were HPV positive at baseline; four were high grade. Only three cases of low grade dyskaryosis were found in the control group. Women with type specific persistent infections were significantly more likely to develop cervical neoplasia than women who cleared the infection (p = 0.0001) or were sequentially infected with different types (p = 0.001). Women with multiple HPV infections at baseline were no more likely to develop cervical dyskaryosis than those with a single infection. CONCLUSIONS: Type specific persistent HR-HPV infection as monitored by genotyping can identify women at increased risk of cervical neoplasia more accurately than a single or repeated presence/absence HPV test. The cost effectiveness of such an approach should be investigated by an appropriate, large scale cost-benefit analysis.

Multiple high risk HPV infections are common in cervical neoplasia and young women in a cervical screening population.

AIMS: If human papillomavirus (HPV) testing is to be included within cervical screening programmes, the importance of multiple HPV infections in cervical neoplasia needs to be determined. This study investigated the diversity of multiple HPV types in a routine cervical screening population, and assessed associations with cervical neoplasia. METHODS: Overall HPV prevalence, type specific prevalence, and extent of multiple infection were assessed in residual material from 3444 liquid based cytology samples, using real time GP5+/GP6+ polymerase chain reaction for screening and linear array assay for genotyping. HPV status was studied in relation to age and concurrent cytological evidence of dyskaryosis. RESULTS: Twenty per cent of samples were HPV positive. HPV type diversity was broad, and multiple HPV infections occurred in half of the HPV positive samples. Younger women were significantly more likely to harbour multiple high risk HPV (HR-HPV) infections. Infections with multiple HR-HPV types were found in 3.4% of samples negative for neoplasia and in 33.3%, 41.8%, and 40.4% of samples with borderline, mild, or high grade dyskaryosis, respectively. Single HR-HPV infections were found in 4.9%, 38.6%, 45.0%, and 51.1% of negative, borderline, mild, or high grade dyskaryosis samples, respectively. CONCLUSIONS: Multiple HR-HPV infections were most prevalent in young women. Multiple HR-HPV infections were not more frequent in high grade than in low grade cervical neoplasia, reflecting common sexual transmission of multiple HR-HPV. Prospective cohort studies linking sequential loss or gain of HPV types with cytological analysis are required to assess the impact of multiple HR-HPV infections on neoplastic progression.

Detection of herpes viruses in clinical samples using real-time PCR.

Herpes viruses including cytomegalovirus, varicella zoster and herpes simplex are an important cause of morbidity and mortality, especially in immunocompromised patients. Real-time PCR assays were developed with the aim of introducing a rapid and sensitive test to replace culture, and as a surveillance system for high-risk patients. The assays were optimised using cell culture derived material, and the sensitivity ascertained using cloned product before applying to extracted and non-extracted clinical samples. The sensitivity was between 1--100 virus copies with increased sensitivity to detect less than 10 copies possible when an initial round of amplification was carried out using external primers. Results were available within four hours of receipt compared with a median of 4.4 days for culture and immunofluorescence. Real-time PCR was found to be a sensitive and rapid method of detecting these viruses and will be a valuable tool for the surveillance of immunosuppressed patients.

Rapid real time PCR to distinguish between high risk human papillomavirus types 16 and 18.

AIMS: To assess the validity and practicality of real time polymerase chain reaction (PCR) for human papillomavirus (HPV) testing in combination with liquid based cytology samples for cervical screening. METHODS: Real time PCR using consensus (GPS+/6+) and type specific primers was developed to detect genital HPV types. This provides rapid, efficient amplification followed by denaturation of the product and computer analysis of the kinetics data that are generated. Liquid based cytology samples were obtained from patients attending routine cervical screening clinics. DNA was extracted from the residual cellular suspension after cytology using spin columns. RESULTS: Real time PCR successfully distinguished between HPV-16 and HPV-18 on the basis of amplification with consensus primers followed by DNA melting temperature (Tm) analysis. Sensitivities of one to 10 copies of HPV-16 (mean Tm = 79.4 degrees C; 2 SD, 0.8) and four to 40 copies of HPV-18 (mean Tm = 80.4 degrees C; 2 SD, 0.4) were obtained. In a mixed population of SiHa and HeLa cells containing known copy numbers of HPV-16 and HPV-18 genomes, HPV-16 and HPV-18 products were clearly separated by Tm analysis in mixtures varying from equivalence to 111000. Together with detailed melt analysis, type specific primers from the same region of the L1 gene confirmed the differential ability of this system. The method was applied to 100 liquid based cytology samples where HPV status using conventional GP5+/6+ PCR was already known. There was 95% agreement between the methods, with 55 positives detected by conventional PCR and 59 with real time PCR. The method was then tested on 200 routine liquid based cytology samples. Approximately 10% were positive by real time PCR, most of which were classified as HPV-16 by detailed melt analysis. Thirteen (6.8%) HPV positives were identified in 189 samples showing no evidence of cervical cytological abnormality. CONCLUSIONS: Real time PCR is a rapid, efficient method for the detection of HPV with the separation of HPV-16 and HPV-18 on the basis of differential Tm. Preliminary results suggest it could prove

A longitudinal study of HPV detection and cervical pathology in HIV infected women.

OBJECTIVE: To monitor the presence and persistence of high risk (HR) human papillomavirus (HPV) in cervical brushings from HIV infected women. METHODS: Prospective observational cohort study of HIV infected women. Women were enrolled from the cohort of 164 HIV infected women who attend the colposcopy clinic at the Edinburgh Regional Infectious Diseases Unit. A single cervical brush scrape was obtained from 39 women and two or more samples from 63 women who attended regularly at approximately 6 monthly intervals. HPV typing was carried out using a commercial hybrid capture assay (HCA). Details of antiretroviral therapy, cytological assessment, and histological evaluation were made available and the interrelation with HR-HPV detection analysed. RESULTS: Abnormal cervical cytology, particularly of low grade, was common in these HIV infected women. HR-HPV types were detected in 25% of the women with normal cytology, while over 80% of those with abnormal cytology of any grade were HR-HPV positive. Persistent HR-HPV, as defined by two or more consecutive HPV positive results, was common and found in 27/63 women from whom multiple samples were obtained. HR-HPV was detected at high levels whether or not patients were receiving antiretroviral therapy. Profound immunosuppression was not necessarily associated with progression of cervical disease and no cases of invasive cervical disease were seen. CONCLUSION: While mild dyskaryosis (low grade squamous intraepithelial lesion (LSIL)) and persistence of HR-HPV are common in HIV infected women in Edinburgh, regular cytological and colposcopic evaluation with appropriate intervention and treatment appears to limit the progression of cervical disease.

Presence of antibodies to human papillomavirus virus-like particles (VLPs) in 11-13-year-old schoolgirls.

To allow meaningful approaches to vaccine development, it is important to know the extent of exposure to human papillomavirus (HPV) within the general population, and particularly the age at which the at risk population is infected. The humoral response to human papillomavirus is directed largely to conformationally-dependent epitopes on the whole virion. Virus-like particles (VLPs) of HPV types 1, 2, and 16 were produced using a baculovirus expression system, and were used in the intact state as antigen in an indirect ELISA. Anonymised serum samples from a cohort of Edinburgh schoolgirls were tested for the presence of IgG antibodies directed against the VLPs. The reproducibility of the ELISA was assured by repeated testing of control samples, and by testing all samples in duplicate and, where possible, on several occasions. Of 1,192 tested with the HPV16 VLPs, 90 (7.6%) were classified as clearly positive, and a further 87 (7.3%) were positive but close to the cutoff calculated by comparison with a group of consistently negative sera. Antibodies to HPV2 were detected in 37.5% (407/1,139) and antibodies to HPV 1 in 51.9% (558/1,076) of the schoolgirls. Antibodies to both HPV1 and HPV2 were found frequently, being present in 29.7% (295/ 993) of samples tested; 40 samples had antibodies to all three types. The significance of these results is discussed.

Detection of high-risk human papillomavirus nucleic acid in archival cervical smears.

OBJECTIVE: To test the usefulness of a commercial DNA hybridization assay for the detection of high-risk (HR) human papillomavirus (HPV) types in archival cervical smears and to compare the sensitivity with that of polymerase chain reaction (PCR) using consensus primers. STUDY DESIGN: Stained material was scraped from archival slides and the pellet volume noted. DNA was extracted using silica/guanidinium isothiocyanate and the quality checked by amplification of the beta-globin gene. HR-HPV DNA was detected using a commercial hybrid capture assay (HCA) and the results compared with an in-house amplification system with consensus primers. RESULTS: Of 156 archival smears stored for 12-13 years, 20 were positive by HCA using an HR probe cocktail. Ninety-eight were also tested by PCR, and 35 were positive. The percentage of HPV-positive samples increased with the increasing size of the pellet. HR-HCA detected more positives in samples with high grade squamous intraepithelial lesion (moderate/severe dyskaryosis). CONCLUSION: Both hybridization by HCA and amplification by PCR could be used to detect genital HPV in archival smears. The general primers PCR detected more positives than HR-HCA but included HPV 6/11. While variation in sample size and prolonged storage may reduce the quality of DNA, the use of archival material for longitudinal studies of HPV presence is potentially worthwhile.

Detection of respiratory syncytial virus nucleic acid in archival postmortem tissue from infants.

Archival lung tissue from 99 cases of sudden infant death syndrome (SIDS) and from 58 matched comparison cases with known causes of death was studied. Sections were examined by in situ hybridization (ISH) using a cocktail of three synthetic oligonucleotides with sequences chosen from the published sequence of the nucleoprotein gene of respiratory syncytial virus (RS virus). The oligonucleotides were end-labelled with dinitrophenyl (DNP) or digoxigenin (DIG) and hybrids were detected immunocytochemically. RS virus nucleic acid was detected in 24 cases of SIDS (24%) and in 11 (19%) of the comparison group. Specificity was confirmed using a DIG-labeled cloned probe covering the whole of the nucleoprotein gene sequence. With one exception, the same results were obtained. Reverse transcriptase-polymerase chain reaction (RT-PCR) was used to confirm the specificity of these results. When matched for age and month and year of death, 76 SIDS cases and 38 controls could be compared. Twenty-one SIDS cases (27.6%) and seven comparison cases (18.4%) contained detectable RS virus sequences by ISH, with a higher detection rate in winter in both groups. The differences were not significant and reflected the seasonal pattern of RS virus infection in the community rather than a causal relationship of RS virus with SIDS. Detection of RS viral mRNA through the summer months suggests that persistence is possible.

Human herpesvirus-6 DNA in the saliva of paediatric oncology patients and controls.

Children with malignancy are immunosuppressed and susceptible to serious infections with herpesviruses. The majority of children on chemotherapy for malignancy are seropositive for human herpesvirus-6 (HHV-6), and although HHV-6 has been demonstrated to be a pathogen in severely immunocompromised patients, whether this is the case for paediatric oncology patients is unknown. HHV-6 is secreted in saliva and in this study samples were examined prospectively for HHV-6 DNA in healthy children and those with malignancy. In a nested polymerase chain reaction (PCR), a 287 bp outer fragment and 163 inner fragment of HHV-6 DNA were amplified. The resulting amplimer contained a Hind III restriction site present only in "B" type HHV-6 and this was used to identify the type of HHV-6 amplified. In saliva from healthy control children, 74% (28/38) of samples were HHV-6 DNA-positive in either the supernate, pellet or both. In the patients, 58% (45/77) of all samples were HHV-6 DNA-positive. When sequential samples from twelve patients were examined the children appeared to fall into two groups: those who were frequently HHV-6 DNA-positive (60% of samples or more) and those who were rarely HHV-6 DNA-positive (33% of samples or less) (P < 0.0001). The only apparent difference between these two groups was that the less frequently HHV-6-positive group was more often febrile and unwell with neutropaenia. Hind III digestion demonstrated all the positive samples to be "B" type HHV-6. Possible explanations for this difference in HHV-6 secretion between the patient groups are discussed.