Immunological and viral factors associated with the response of vulval intraepithelial neoplasia to photodynamic therapy.

Topical 5-aminolevulinic acid-based photodynamic therapy (PDT) has produced complete response rates of >90% for nonmelanoma skin carcinomas, which are mostly human papillomavirus (HPV) negative. Using a similar treatment protocol, we observed a short-term response in only one third (10 of 32) of high-grade vulval intraepithelial neoplasia (VIN 2-3) lesions. Unifocal lesions were found more responsive than multifocal and pigmented lesions. Animal model studies have suggested that long-term PDT response involves an immune reaction in which CTLs play a crucial role. In this study, we have assessed: (a) HPV infection; (b) HLA expression; and (c) immune infiltrating cells in VIN biopsies from responders and nonresponders to determine whether these factors may limit response to topical 5-aminolevulinic acid-based PDT. Tissues from normal vulva (n = 9), vulval carcinoma (n = 11), and VIN (32 patients from which 19 pre- and 43 post-PDT biopsies were taken) were investigated for immune cell infiltration and HLA class I expression by immunohistochemistry and HPV infection by PCR. There was a greater likelihood of HPV positivity associated with a lack of response of VIN to PDT (P = 0.002), and VIN nonresponders were more likely to show HLA class I loss compared with responders (P = 0.030). HLA class I down-regulation was significantly greater in the carcinomas (82%, total loss) than the VIN (28%, 19%, total loss; and 9%, allele loss; P = 0.004). None of the cases with class I down-regulation responded to PDT, whereas 3 of 6 (50%) of cases that showed total class I loss subsequently developed superficial invasion. Compared with normal vulval skin, VIN lesions showed increased infiltration by CD4 (T-helper) and CD68 (macrophages) but not CD1a (Langerhans cells) or CD8 (CTLs). There was, however, a significant increase of CD8 infiltration in posttreatment VIN responders compared with nonresponders (P = 0.0001). These data clearly support the contention that high-risk HPV infection and lack of cell-mediated immunity may play a role in the observed poor response of lower genital lesions to topical PDT.

Graft failure in human donor corneas due to transmission of herpes simplex virus.

AIM: To report the clinical consequences of contamination of human donor corneas by herpes simplex virus (HSV) in organ culture. METHODS: Two patients without previous history of ocular HSV infection underwent penetrating keratoplasty (PK), one for keratoconus and the other for Fuchs' endothelial dystrophy. One patient suffered primary graft failure while the other developed a persistent epithelial defect, ultimately resulting in graft failure. Viral culture of swabs taken from both corneas during the early postoperative period was undertaken. The failed donor corneas were examined histopathologically by immunohistochemistry (IHC) for HSV-1 antigens, transmission electron microscopy (TEM), and by polymerase chain reaction (PCR) for HSV DNA. Both failed corneas were replaced within 6 weeks of the initial surgery. The records of the fellow donor corneas were also examined for evidence of infection. RESULTS: HSV was cultured from both corneas during the early postoperative period. Histology of both donor corneas demonstrated a thickened corneal stroma with widespread necrosis of keratocytes and loss of endothelial cells. IHC showed keratocytes positive with antibodies to HSV-1 antigens. TEM demonstrated HSV-like viral particles within degenerating keratocytes. PCR performed on the failed corneal grafts was positive for HSV-1 DNA, whereas PCR performed on the excised host corneal buttons was negative in both patients. Records of the fellow donor corneas showed that one cornea was successfully transplanted into another recipient after 18 days in organ culture, whilst the other was discarded because of extensive endothelial cell necrosis noted after 15 days in organ culture. CONCLUSION: HSV within a donor cornea may cause endothelial destruction in organ culture and both primary graft failure and ulcerative keratitis after transplantation. Endothelial necrosis of a donor cornea in culture also raises the possibility of HSV infection within the fellow cornea.

Immunisation against varicella in end stage and pre-end stage renal failure. Trans-Pennine Paediatric Nephrology Study Group.

OBJECTIVES: To investigate the seroconversion rate and duration of persistence of protective antibody titres after varicella immunisation in children with renal failure. DESIGN: 32 children (25 end stage and 7 pre-end stage renal failure) were immunised using 2 x 2,000 plaque forming unit doses of varicella vaccine 3 months apart. Varicella antibody titres were measured by enzyme linked immunosorbent assay. RESULTS: All children initially seroconverted after immunisation. At a mean follow up of 20.3 months, 23 of 28 had protective antibody titres, 4 children having died of unrelated causes. Two children required a third booster dose. 11 children underwent renal transplantation; 10 had protective titres at the time of transplantation and, at a mean of 23.4 months after immunisation, 6 currently have protective titres. Minor side effects occurred after 11 vaccine doses in 9 children. No child developed varicella, despite 10 clear episodes of exposure to the wild-type virus. CONCLUSIONS: Varicella immunisation in children with end stage and pre-end stage renal failure results in a high rate of seroconversion and persistence of protective antibody titres. More widespread use of the vaccine before renal transplantation is recommended.

Investigation of cytomegalovirus and human herpes viruses 6 and 7 as possible causative antigens in psoriasis.

Psoriasis is probably a T-cell-mediated autoimmune disease. Infectious models of autoimmune diseases have been proposed and in psoriasis, it has been suggested that there may be molecular mimicry between streptococcal antigens and epidermal keratins. The immunological profile of stable psoriasis plaques suggests, however, that viral antigens may be important. We investigated, using polymerase chain reaction techniques, whether DNA from either cytomegalovirus (CMV) or human herpes viruses (HHV) 6 and 7 is present in the skin of patients (n = 10) with chronic plaque psoriasis. We also investigated 29 patients for the presence of serum IgG to CMV. We found no evidence of CMV or HHV 7 DNA in psoriasis plaques although DNA for HHV 6 was detected in both involved and uninvolved skin in 1 out of 10 patients. There was no statistically significant increase in prior CMV infection, as assessed by the presence or absence of serum IgG to CMV, in psoriasis, compared to our local population. Although there is circumstantial evidence that viral antigens may be important in the pathogenesis of psoriasis we found no evidence to link infection with CMV or HHV 6 and 7 with subsequent development of chronic plaque psoriasis.

A study of anal intraepithelial neoplasia in HIV positive homosexual men.

OBJECTIVES: To determine the prevalence of high grade anal intraepithelial neoplasia (HGAIN), the value of anal cytology in screening for HGAIN, and the characterisation of epidemiological factors and human papillomavirus (HPV) types. METHODS: Prospective cohort study of HIV positive homosexual men. Subjects were interviewed, underwent STD, anal cytological, and HPV screening at enrolment and at subsequent follow up visits with anoscopy and biopsy at the final visit. 57 enrolled, average CD4 count 273 x 10(6)/l (10-588); 41 completed the cytological surveillance over the follow up period (181 visits, average follow up 17 months), 38 of these had anoscopy and anal biopsy. RESULTS: Oncogenic HPV types were detected in 84% and high grade dyskaryosis in 10.5% (6/57) at enrollment. There was a 70% incidence of high grade dyskaryosis during follow up in patients with negative/warty or low grade dyskaryosis at enrollment. Anoscopy correlated with histology in high grade AIN lesions (sensitivity 91%, specificity 54%) and cytology was 78% sensitive (18/23) for HGAIN on biopsy. CONCLUSIONS: AIN and infection with multiple oncogenic HPV types are very common among immunosuppressed HIV positive homosexual men. Apparent progression from low to high grade cytological changes occurred over a short follow up period, with no cases of carcinoma. All 23 cases of HGAIN were predicted by cytology and/or anoscopy. Future studies focusing on the risk of progression to carcinoma are needed before applying anal cytology as a screening tool for AIN in this population.

Influenza-like episodes in HIV-positive patients: the role of viral and 'atypical' infections.

OBJECTIVES: To document viral and 'atypical' infections in HIV-positive patients and association with influenza-like symptoms. PATIENTS AND METHODS: Monthly culture of urine, faeces and throat swabs in 63 HIV-positive patients (30 asymptomatic and 33 with AIDS-related complex/AIDS) over 5-27 months (with 1125 patient-months of follow-up), with further sample collections during influenza-like episodes. Standard viral detection methods were used. Throat swabs were assessed for Chlamydia sp. by culture and immunoblotting, and for Mycoplasma pneumoniae by polymerase chain reaction. RESULTS: Viruses were detected in 15 (50%) and M. pneumoniae in nine (30%) out of 30 HIV-positive patients during an influenza-like illness. A close temporal relationship with symptoms was observed in 12 (40%) patients: cytomegalovirus in six (20%), M. pneumoniae in three (10%), herpes simplex virus in three (10%), and enterovirus in one (4%). Influenza-like symptoms were more frequent in asymptomatic HIV infection than in AIDS-related complex/AIDS patients (actuarial risk at 1 year, 63 versus 26%; P=0.002), particularly in those with CD4 cell counts >300 x 10(6)/l at enrolment (P=0.002). At least 44% (four out of nine) M. pneumoniae infections were asymptomatic and 78% (seven out of nine) were associated with prolonged excretion (2-17 months). Chlamydia sp. were not detected. CONCLUSIONS: Influenza-like symptoms were more likely to be reported by HIV-positive patients at early stages of disease, possibly as a result of differences in immune responses to viral infection. There was a close association in 40% of cases between the development of symptoms and detection of cytomegalovirus, herpes simplex virus, enterovirus and M. pneumoniae (a previously unrecognized association).

Detection of male genital infection with Chlamydia trachomatis and Neisseria gonorrhoeae using an automated multiplex PCR system (Cobas Amplicor).

We evaluated Cobas Amplicor, a highly automated polymerase chain reaction (PCR) system, to test first-void urine (FVU) and urethral swab specimens for Chlamydia trachomatis and Neisseria gonorrhoeae in men attending a sexually transmitted infection (STI) clinic. Results were compared against an in-house radioimmune dot blot (DB) test for C. trachomatis and selective culture for N. gonorrhoeae. Three hundred and ninety sets of specimens were obtained from 378 consecutive new and returned-new patients. Gonorrhoea prevalence was 9.49%, with no significant difference in sensitivity or specificity between culture and PCR. Chlamydia prevalence was 15.4%, with sensitivities of: DB 55%, PCR of FVU 86.7%, urethral swab PCR 90%. The specificity of PCR on FVU and urethral swabs was 100%. We have shown that Cobas Amplicor PCR is highly sensitive and specific in the diagnosis of chlamydia and gonorrhoea in men attending an STI clinic. Further economic and scientific studies are needed to determine the cost-effectiveness of this technique for screening in primary care settings.

Low HIV and high STD among commercial sex workers in a brothel in Bangladesh: scope for prevention of larger epidemic.

The present study documents the first systematic assessment of a brothel in Bangladesh in terms of sexually transmitted disease (STD) and human immunodeficiency virus (HIV). A cross sectional study was undertaken on brothel-based commercial sex workers (CSWs) selected systematic random sampling to assess the prevalence of STDs and HIV among CSWs in a brothel setting. Two hundred and ninety-six CSWs were selected from a brothel with a population of 593 women. Following informed consent, endocervical and blood samples were obtained for the diagnosis of genital chlamydia, gonorrhoea, HIV and syphilis respectively. In addition, another 170 consecutive blood samples were collected from the total CSW population for HIV tests. All blood samples for HIV testing were made anonymous by removing patient identifiers before testing. Endocervical specimens were tested by polymerase chain reaction (PCR) for the diagnosis of genital chlamydia and gonorrhoea. Syphilis and HIV infections were diagnosed by serology. One hundred and sixty-nine (57.1%) of the women were Treponema pallidum haemagglutination (TPHA)-positive, 20 (6.8%) of the women were Venereal Disease Research Laboratory (VDRL)-positive at a greater than 1:8 dilution. Eighty-two (28%) of the women were found to be infected either by gonorrhoea or chlamydia. No HIV antibody was found in any of the 466 blood samples. A high prevalence of STDs and low prevalence of HIV in the CSWs in Bangladesh suggest potential for the rapid spread of HIV once it is introduced in this high-risk population. The opportunity to control STD and HIV infection in this population should not be missed, in order to prevent a large epidemic in the future.

PIP: While the incidence of new HIV infections or HIV prevalence appears to be declining in North America, Australasia, and Western Europe, and beginning to either plateau or decline in sub-Saharan Africa, levels of HIV infection are rapidly increasing in southeast Asia. Bangladesh is surrounded by Myanmar and India, two countries experiencing major HIV epidemics. Findings are presented from a study conducted to assess the prevalence of HIV antibody and selected STDs in a resident population of 593 brothel-based female prostitutes in a business town 100 km from Dhaka. Endocervical and blood samples from 296 of the women were tested for the presence of genital chlamydia, gonorrhea, HIV, and syphilis, while consecutive blood samples were taken from another 170 of the subjects for HIV testing. No HIV antibody was found in any of the 466 blood samples. 169 (57.1%) women, however, had evidence of either past or present infection with syphilis as measured by Treponema pallidum hemagglutination (TPHA) testing, 20 (6.8%) women were Venereal Disease Research Laboratory (VDRL)-positive at a more than 1:8 dilution, and 82 (28%) women were infected with either gonorrhea or chlamydia. The high prevalence of gonorrhea or chlamydia and TPHA detected in this study suggest that HIV infection would spread rap[idly if introduced in this population, and later expand into the general population.

Differences in serological IgA responses to recombinant baculovirus-derived human papillomavirus E2 protein in the natural history of cervical neoplasia.

Infection with certain types of human papillomavirus (HPV) presents a high risk for the subsequent development of cervical intraepithelial neoplasia (CIN) and cervical carcinoma. Immunological mechanisms are likely to play a role in control of cervical HPV lesions. The HPV E2 protein has roles in virus replication and transcription, and loss of E2 functions may be associated with progression of cervical neoplasia. Accordingly, it is of interest to monitor immune responses to the E2 protein, and previous studies have reported associations between serological reactivity to E2 peptide antigens and cervical neoplasia. In order to investigate serological responses to native, full-length E2 protein, we expressed HPV-16 E2 proteins with and without an N-terminal polyhistidine tag using the baculovirus system. Purified HPV-16 E2 protein was used to develop enzyme-linked immunosorbent assays to detect serological IgG and IgA responses in cervical neoplasia patients and controls. We found that serum IgA levels against the E2 protein were elevated in CIN patients relative to normal control subjects but were not elevated in cervical cancer patients. Moreover, there appeared to be a gradient of response within cervical neoplasia such that the highest antibody levels were seen in lower grades of neoplasia up to CIN 2, whereas lower levels were observed in CIN 3 and still lower levels in cervical carcinoma. These findings suggest that the IgA antibody response to E2 may associate with stage and progression in cervical neoplasia.

Comparison of primer sets for detection of fecal and ocular adenovirus infection using the polymerase chain reaction.

Adenoviruses of subgenus F (types 40 and 41) cause infantile gastroenteritis and adenoviruses principally of types 1-7 are found in feces during respiratory or generalized infections. Adenoviruses (mostly types 3, 4, 8, 19, or 37) are also linked with follicular or epidemic conjunctivitis. The diagnostic efficiency of the polymerase chain reaction (PCR) for adenoviruses was assessed using genus-reactive primers H1 and H2 or JCH1 and JCH2 or subgenus F-specific primers F1a and F2a. With diarrheal stool specimens containing subgenus F adenoviruses, F1a/F2a PCR achieved at least as high a positivity rate (75/76 [99%]) as electron microscopy (72/76 [95%]) and was more sensitive than polyclonal antibody-based immune electron microscopy (IEM) for subgenus identification (75/76 [99%] vs. 66/76 [87%], P = 0.008). Twenty-three subgenus F strains untypeable by monoclonal antibody-based IEM were typed as 40 (n = 4) or 41 (n = 19) by Hha I digestion of the PCR product. The genus-reactive primer pairs provided DNA amplification assays of generally equal efficiency on conjunctival swab specimens though possible nucleic acid degradation in DNA extracts during storage could have meant that JCH1/JCH2 PCR was truly the more sensitive. The use of either genus-reactive primer set on fecal specimens cannot be recommended because, although the positivity rates with subgenus F PCR positive specimens were high (70/75 [93%] for H1 and H2, 14/15 [93%] for JCH1 and JCH2), the detection rates were disappointing with similar specimens yielding nonsubgenus F adenoviruses.

Polymerase chain reaction for rapid detection of ocular adenovirus infection.

Adenoviruses are associated with endemic and epidemic acute conjunctivitis, large nosocomial outbreaks reflecting virus transmission on unwashed hands or inadequately sterilised ophthalmic instruments. The polymerase chain reaction (PCR) proved more sensitive than antigen detection by immune dot-blot test for the rapid diagnosis of ocular adenovirus infection (sensitivities in a retrospective study 112/123 (91%) versus 72/123 (59%), P < 0.001). Indeed, in a prospective comparison, DNA amplification and virus isolation generated similar numbers of positive results (34 versus 32), though five PCR positive results were possibly false positives. The sensitivity of the PCR was largely independent of adenovirus subgenus or serotype, though reduced sensitivity with subgenus B strains could not be excluded. Specimen preparation for DNA amplification using a simple lysis buffer proved more effective than phenol-chloroform extraction. The immune dot-blot test gave unavoidable false positive results, but with the PCR this problem could be minimized by technical modifications. The PCR could replace antigen detection and virus isolation as the initial test for adenoviruses in conjunctival swabs, with cell culture only being retained for adenovirus serotyping in PCR positive specimens and for other viruses such as herpes simplex.

A single tube nested PCR for the detection of hepatitis C virus RNA.

An assay was developed for the detection of hepatitis C virus RNA in serum which combined cDNA synthesis and a hot start nested polymerase chain reaction (PCR) in a single tube. This was made possible by separation of the reagents necessary for cDNA synthesis and PCR during cDNA synthesis with a high melting temperature wax interface and by the use of 'drop in-drop out' nested primers which enabled each primer set to be selectively extended within the same reaction tube. The increase in sensitivity following amplification with the internal primer pair was comparable to that achieved when nested reactions are carried out separately.

Same-day testing for human immunodeficiency virus antibodies.

We analysed the specificity of a screening enzyme-linked immunosorbent assay for human immunodeficiency virus antibodies, and found that 18 of 1878 serum specimens (1.0%) from an open access clinic gave false reactive results. Introduction of true same-day testing therefore required the use of more than one assay before a reactive result could be reported. A testing strategy was devised with a 4 h turn-around time which gave accurate results for 11 positive and 119 negative specimens.

Gastroenteritis due to adenovirus type 41 in an adult with chronic lymphocytic leukemia.

We describe a case of gastroenteritis due to adenovirus type 41 that lasted for 46 days (until the patient's death) in a 56-year-old woman with terminal B cell chronic lymphocytic leukemia. Adenovirus particles were detected by electron microscopy in five stool specimens collected between 5 and 39 days after the onset of the diarrheal illness. The virus was identified as adenovirus type 41 by viral neutralization tests and other assays. We discuss the potential pathogenicity of subgenus F adenoviruses in immunocompromised patients.

Computer network for a diagnostic virology laboratory.

A data base for a large diagnostic virology laboratory is described. The system uses a network of personal computers. It allows the entry, long-term storage, and subsequent retrieval of specimen and patient records (comprising personal identifiers and specimen and result information), and hard-copy results reporting. Sited entirely within the laboratory, the network is not connected to a modem. Within the laboratory there is restricted access to human immunodeficiency virus test results to guarantee patient confidentiality. Retention of a hard-copy of specimen request cards ensures the availability of the original clinical information. The data base is copied on a second file server to facilitate searches, and daily streaming onto magnetic tape provides system protection in the event of hard disc failure. Matching of old and new patient records is done by surname, date of birth, and sex, and therefore duplicate records accumulate when patient names are misspelt on specimen request forms. The system requires further development to speed searches of the data base and to achieve automatic generation of laboratory worksheets. Future goals are the replacement of hard-copy records of clinical information and hard-copy reporting with on-line access to hospital data bases and on-line requesting by and reporting to the clinician.

Detection of hepatitis C virus RNA in serum, by combining reverse transcription and polymerase chain reaction in one tube.

Reverse transcription of viral RNA into cDNA and its subsequent amplification by polymerase chain reaction (PCR) are generally carried out as two separate reactions. Here we describe a novel method for the detection of HCV RNA in serum combining both steps in one reaction tube using a wax interface to separate the two sets of reactants during initial cDNA synthesis. This enabled optimisation of both reactions, simplified the test and thereby reduced the risk of cross-contamination.