Human papillomavirus modifies the prognostic significance of T stage and possibly N stage in tonsillar cancer.

BACKGROUND: Despite the association with more advanced nodal stage, patients with human papillomavirus (HPV) positive oropharyngeal cancers have better outcomes. We examined whether the HPV can modify the effect of known prognostic factors in tonsillar cancer. PATIENTS AND METHODS: A total of 489 patients from 10 centres were followed up for recurrence or death for a median of 3.2 years. Determinants of the rate of locoregional recurrence, death from tonsillar cancer and overall survival were modelled using Cox regression. RESULTS: The prognostic value of T and N stages were modified by HPV as indicated by statistically significant interaction terms. After adjusting for age, gender and treatment, T stage appeared relevant only for HPV-positive cancers (where a higher T stage was associated with worse outcomes). There was some evidence that N stage was a more relevant prognostic factor for HPV-negative than -positive cancers. There was no evidence that the HPV modifies the effect of age, gender or grade on outcomes. CONCLUSIONS: This study suggests that the prognostic significance of the conventional staging system in tonsillar cancer is modified by HPV.

Human papillomavirus predicts outcome in oropharyngeal cancer in patients treated primarily with surgery or radiation therapy.

OBJECTIVE: This study examines the prognostic significance of human papillomavirus (HPV) in patients with locally advanced oropharyngeal squamous cell carcinoma (SCC) treated primarily with surgery or definitive radiotherapy. METHODS: One hundred and ninety-eight patients with Stage 3/4 SCC were followed up for recurrence in any form or death from any cause for between 1 and 235 months after diagnosis. HPV status was determined using HPV E6-targeted multiplex real-time PCR/p16 immunohistochemistry. Determinants of recurrence and mortality hazards were modelled using Cox's regression with censoring at follow-up dates. RESULTS: Forty-two per cent of cancers were HPV-positive (87% type 16). HPV predicted loco-regional control, event-free survival and overall survival in multivariable analysis. Within the surgery with adjuvant radiotherapy (n=110), definitive radiotherapy-alone (n=24) and definitive radiotherapy with chemotherapy (n=47) groups, patients with HPV-positive cancers were one-third or less as likely to have loco-regional recurrence, an event or to die of any cause as those with HPV-negative cancers after adjusting for age, gender, tumour grade, AJCC stage and primary site. The 14 patients treated with surgery alone were considered too few for multivariable analysis. CONCLUSION: HPV status predicts better outcome in oropharyngeal cancer treated with surgery plus adjuvant radiotherapy as well as with definitive radiation therapy±chemotherapy.

Replication-restricted vaccinia as a cytokine gene therapy vector in cancer: persistent transgene expression despite antibody generation.

BACKGROUND: As antitumoral immunity requires the generation of local immunity directed against tissue proteins, we attempted to recreate within tumors the same environment found within tissues affected by autoimmune diseases (i.e., prolonged cytokine expression). Vaccinia virus (VV) has not been widely used as a cytokine gene therapy vector because of presumed high immunogenicity that would likely make repeated injections impossible; therefore, we modified it by inserting the cytokine gene into the thymidine kinase region, rendering it replication-restricted. The cytokine chosen was human interleukin-2 (IL-2); a molecule with powerful antitumoral effects. METHODS: Six patients with the treatment-resistant tumor malignant mesothelioma received intratumoral (i.t.) VV-IL-2 therapy for 12 weeks by injection of 10(7) plaque-forming units of VV-IL-2 per dose. Serial tumor biopsies, sputum, urine, and blood samples were tested for VV-IL-2 mRNA expression; VV culture and T-cell infiltrates were evaluated by immunohistochemistry. Patients and contacts of patients were monitored for changes in VV immunoglobulin G (IgG) levels and clinical evidence of VV infection. RESULTS: VV-IL-2 was not excreted and was only cultured in one patient from tumor biopsies. A T-cell infiltrate was detected in 50% of tumor biopsies. VV-IL-2 mRNA expression was highest on days 1-3 postinjection and was detected for up to 3 weeks after each injection even though VV IgG levels rose in all patients. No significant toxicities, infection of patient contacts, or tumor regressions were observed. CONCLUSIONS: I.t. VV-IL-2 administration is safe, is associated with minimal toxicity, and results in i.t. expression of VV-IL-2 for up to 3 weeks postinjection regardless of the level of anti-VV IgG titers generated. This suggests that VV may be a good vector for repeated cytokine gene therapy of solid human cancer.

Comparison of immunodiffusion and enzyme linked immunosorbent assay for antibodies to four Aspergillus species.

Antigenic extracts were prepared from Aspergillus fumigatus, A niger, A flavus and A terreus for use in enzyme linked immunosorbent assay (ELISA) and immunodiffusion (ID) tests for Aspergillus antibodies to determine whether the use of antigenic extracts from species other than A fumigatus increased the sensitivity of the ELISA. ELISA titres correlated well with positive ID tests. Patient titres by ELISA were significantly higher than control titres for all species. Patient titres to A niger were also significantly higher than titres to the other species. Total number of ID bands to A fumigatus correlated significantly with anti-A fumigatus ELISA titres. It is concluded that the use of antigenic extracts from species other than A fumigatus improves the sensitivity of the ELISA.

A simple method for detecting mycoplasma infection of cell cultures.

This paper describes the overnight Giemsa staining method for detecting mycoplasma infection of cell cultures. This simple method requires no special equipment and has practical application in most cell culture laboratories. This technique has been used conveniently to monitor stock cell cultures for mycoplasma infection over a 12-month period avoiding any contamination.

Human herpes virus type 6 (HHV-6) and its in vitro effect on human immunodeficiency virus (HIV).

Human herpes virus type 6 (HHV-6) was isolated from the peripheral blood lymphocytes of a patient infected with human immunodeficiency virus (HIV). Antibodies to this herpes virus were found to be widespread among adults and children in Western Australia. Co-infection studies indicated that HIV replication was inhibited by the presence of HHV-6.

Diagnosis of Chlamydia trachomatis and Neisseria gonorrhoeae. Genitourinary infections in males by the Amplicor PCR assay of urine.

The Amplicor CT/NG polymerase chain reaction (PCR) test on urine specimens from males was prospectively evaluated against established specimens and laboratory methods for diagnosing Chlamydia trachomatis and Neisseria gonorrhoeae genitourinary infections, in patients from a remote region of Western Australia. Seventy-three males who were tested for both C. trachomatis and N. gonorrhoeae by both conventional methodology and Amplicor PCR on urine were enrolled in the study. Established testing comprised enzyme immunoassay/immunofluorescence antigen testing (EIA/IF) for C. trachomatis and microscopy and/or culture for N. gonorrhoeae on urethral swabs. Positive test results were confirmed using a set of criteria that included supplemental PCR testing and clinical history. Overall, 13.7% of patients were resolved as positive for C. trachomatis and 52.1% as positive for N. gonorrhoeae. The sensitivity and specificity of the Amplicor CT/NG PCR on male urine specimens for C. trachomatis were 80.0% (8/10) and 95.2% (60/63), compared with 60.0% (6/10) and 100.0% (63/63) for EIA/IF on urethral swabs. For N. gonorrhoeae, the sensitivity and specificity of the Amplicor CT/NG PCR on male urine specimens were both 100% (38/38 and 35/35, respectively) compared with 86.8% (33/38) and 100% (35/35) for microscopy and/or culture on urethral swabs. The results of this study indicate that the Amplicor CT/NG multiplex PCR test for C. trachomatis and N. gonorrhoeae performed on urine in males provides a highly sensitive, specific, and robust method for the diagnosis of both C. trachomatis and N. gonorrhoeae, for the early detection of both symptomatic and asymptomatic infected individuals.

A modified immunofluorescence test for Epstein-Barr virus-specific IgM antibody.

The fluorescent antibody (FA) test for Epstein-Barr virus (EBV)-specific IgM antibody was improved by the use of sodium butyrate to induce a higher level of EBV antigen expression in P3HR-1 slide preparations and by removal of rheumatoid factor (RF) and IgG antibodies from test sera by means of adsorption with suspensions of Sepharose-IgG and Streptococcus pyogenes strain AR1. This method was compared with the Paul-Bunnell test (PB) on 1106 sera submitted to a routine virus diagnostic laboratory for infectious mononucleosis serology and 96.4% of sera showed concordant results. Thus the EBV-IgM-FA method was suitable for routine diagnostic use. However, it proved helpful to test EBV-IgM positive sera by PB to assist in the detection of cross-reacting IgM antibodies sometimes present.

False positive results occurring in a radioimmunoassay for hepatitis A IgM antibody of the IgM class.

The diagnosis of hepatitis A infection is usually based on the presence of hepatitis A specific IgM in a single serum sample. The fortuitous observation in one patient that this reactivity was apparently still present 19 mth after her original illness led to the discovery that the ABBOTT HAVAB-M kit method may produce false positive results. A series of patients who had previously had hepatitis A was retested and false positive results were found in 6% of this group. Control groups consisted of patients with other acute and chronic liver disorders and other acute viral diseases. No reactivity was detected in the control sera. Sucrose gradient fractionation revealed that the factor responsible for the false positive results was associated only with serum fractions containing IgA and IgG and that it could be removed by absorption of sera with staphylococcal protein A but not by absorption with streptococcus AR1 or by 2-mercaptoethanol treatment. It was concluded that following hepatitis A infection some patients produce a rheumatoid factor-like substance (not of IgM class) which is cleared from the serum in 2-3 yr. The presence of this factor may lead to a misdiagnosis in patients presenting with jaundice.

Centrifugal enhancement of human immunodeficiency virus (HIV) and human herpesvirus type 6 (HHV-6) infection in vitro.

The effect of centrifugal inoculation of human immunodeficiency virus (HIV) and human herpesvirus-6 (HHV-6) on the infectivity of the viruses for cell cultures was examined. Three HIV-1 strains, ARV-2, HTLV-IIIb and a local isolate, WA-46c, were tested in peripheral blood lymphocytes, HUT-78, H9 and MT-2 cells. The HHV-6 strain was a local isolate and was studied only in peripheral blood lymphocyte cultures. Centrifugal inoculation of the viruses at a force of 2500 x g for 60 min, enhanced HIV-1 infectivity by a factor of about 10-fold in all cell cultures tested. Infectivity was increased about 100-fold for HHV-6.

Electron microscopy after direct ultracentrifugation.

A direct ultracentrifugation technique was used in the preparation of skin lesion specimens for examination by electron microscopy. The concentration factor of centrifuged specimens was estimated to be in excess of 1,000-fold compared to conventional adsorption techniques. This resulted in an increase of over 300% in the detection rate of herpesviruses and poxviruses from skin lesion specimens.

Variation in murine cytomegalovirus replication in fibroblasts from different mouse strains in vitro: correlation with in vivo resistance.

The in vitro growth of murine cytomegalovirus (MCMV) in mouse embryo fibroblasts (MEF) from Balb/c, C57BL, C3H and CBA mouse strains has been shown to correlate with the genetically determined in vivo susceptibility of these strains to MCMV infection. MEF from Balb/c and C57BL mice were more susceptible to MCMV than those from C3H and CBA mice. This was evident regardless of whether replication was measured by cytopathic effect (c.p.e.) score, virus yield, plaque count, plaque size or time of onset of c.p.e. The growth of MCMV in tracheal organ cultures from different mouse strains was similar to that observed in MEF from these strains. The replication in MEF when measured by c.p.e. score and virus yield was affected by the density of the cell cultures. The strain of mouse used to produce MCMV also affected the comparative sensitivity of MEF to the virus. This appeared to be due to reduced growth of MCMV in its homologous MEF type, an unexpected result. In contrast, cell density had little effect on the replication of herpes simplex virus type 1 (HSV-1), but again the in vitro susceptibility of MEF reflected the in vivo susceptibility of mouse strains to infection with this virus.

Isolation of adenovirus type 19 from the male and female genital tracts.

During routine screening for genital herpes simplex virus infection in patients attending a sexually transmitted diseases clinic adenovirus type 19 was isolated from both men and women. Peak incidences of genital infection with adenovirus type 19 corresponded with those of eye infection with the same virus in the general community. Thus, the relationship between genital and eye infection with adenovirus, the part played by genital infection in its dissemination, and the clinical symptoms it may produce need further study.

The diagnosis of chlamydia, gonorrhoea, and trichomonas infections by self obtained low vaginal swabs, in remote northern Australian clinical practice.

OBJECTIVE: To examine the diagnostic performance of self obtained low vaginal swabs (SOLVS) and polymerase chain reaction (PCR) techniques in the diagnosis of Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG), and Trichomonas vaginalis (TV) infection in a variety of clinical practice settings in remote north western Australia. DESIGN: A cross sectional field study of microbiological collection techniques in women undergoing gynaecological investigation in remote settings performed by a variety of practitioner types over 10 months. PARTICIPANTS AND SETTING: 349 women from remote towns and communities in the Kimberley region of north west Western Australia having gynaecological examinations for clinical reasons, well women screening, antenatal screening, and sexual health examinations. RESULTS: The overall prevalence of infection in the study population based on any positive conventional sample was 9.2%, 7.6%, and 16.1% for CT, NG, and TV respectively. The detection rates for CT and NG by SOLVS were 89% and 96% respectively, compared with 79% and 91% for endocervical swabs and 79% and 83% for first void urine. SOLVS had a sensitivity of 93% for TV detection, equal to that of clinician obtained low vaginal swabs. None of these differences reached statistical significance. A combination of SOLVS and first void urine detected 96% of the CT cases, 100% of the NG cases, and 96% of TV cases. CONCLUSIONS: Self obtained low vaginal swabs are an acceptable, simple and sensitive diagnostic sample for the detection of CT, NG, and TV, and have particular applications in remote clinical practice and as a screening technique.

Diagnosis of malaria aided by polymerase chain reaction in two cases with low-level parasitaemia.

Light microscopy of thick and thin blood smears is the mainstay of malaria diagnosis. In situations of low-level parasitaemia such as drug-modified disease, however, this may be difficult making clinical management problematic. Polymerase chain reaction (PCR) methods have shown high sensitivity for the diagnosis of malaria and are able to differentiate the Plasmodium species involved. Two cases are presented in the present study, which illustrate how a PCR method can aid light microscopic malaria diagnosis and species differentiation in returned travellers with low-level parasitaemia. Plasmodium vivax was detected by PCR prior to the light microscopy becoming positive in one case, and in the second case Plasmodium malariae was detected when light microscopy was unable to speciate the causative Plasmodium species.

Importance of virus passage history on the growth of Coxsackie B4 in human skin fibroblasts.

Human skin fibroblasts have previously been reported to display an age-dependent resistance to infection with coxsackie B4 virus. We have shown that the virus will replicate and produce CPE in human skin fibroblasts regardless of the age of the donor of the cells. The passage history of the virus was found to influence the titre of the virus in these cells.

Efficient primary isolation of human immunodeficiency virus.

A highly-sensitive and efficient culture technique for human immunodeficiency virus type-1 (HIV-1) is described; HIV-1 was recovered from the lymphocytes of 44 (94%) antibody-seropositive healthy or symptomatic individuals. The reductions in the requirements for both the reagent volume and the number of patients' lymphocytes, together with an increased efficiency, has made this HIV-1 culture system more practical for diagnostic virology laboratories.

Association of genital adenovirus infection with urethritis in men.

Adenoviruses were isolated from the urethral swabs of 129 male patients in an STD clinic. After exclusion of patients with Chlamydia trachomatis or Neisseria gonorrhoea infections, 85 of the remaining 120 patients had urethritis, compared with 28 men with urethritis detected in a control group which was closely matched for age, sex, and date of specimen collection. This statistically significant difference suggests that genital adenovirus infection may be a cause of urethritis in some male patients.