Herpesvirus infection of eye and brain in HIV infected patients.

OBJECTIVES: To compare histological with genome detection methods for diagnosis of herpesvirus infection in eye and brain of HIV infected patients undergoing necropsy and to correlate these findings with both antemortem clinical findings and postmortem evidence of extraocular herpesvirus infection, especially in the CNS. METHODS: A prospective study of 31 consecutive HIV infected patients undergoing necropsy. In life 11 patients had been assessed by an ophthalmologist because of ocular symptoms. Ocular and brain samples were examined for herpesviruses by conventional histological methods and by nested polymerase chain reaction (nPCR) for all eight human herpesviruses; evidence of extraneural herpesvirus infection was sought by histological methods. RESULTS: Although only 12 out of 31 patients (39%) had antemortem clinical evidence of ocular or CNS herpesvirus associated disease, herpesviruses were detected by nPCR in eye and brain from 26 (84%) patients; six patients had more than one herpesvirus infection. There was concordance between ocular and CNS findings in 15 of 19 patients (79%) with CMV infection. 17 of 31 patients (55%) had extraocular or CNS CMV infection at necropsy. Genome detection using nPCR was superior to histological methods for diagnosis of ocular and CNS herpesvirus infection. CONCLUSION: Herpesvirus infection of eye and brain was a frequent finding at necropsy in this group of HIV infected patients; almost a fifth were co-infected by more than one herpesvirus. This was more than twice the incidence predicted from clinical evidence before death. Genome detection using nPCR was superior to histological methods for diagnosis of ocular and CNS herpesvirus infection.

Detection of polyomaviral DNA in clinical samples from immunocompromised patients: correlation with clinical disease.

Clinical samples from immunocompromised patients were screened for polyomaviral sequences by nested polymerase chain reaction (PCR) to evaluate the association of these viral infections with progressive multifocal leukoencephalopathy (PML). JC virus (JCV) DNA was detected in 19 of 23 CSF samples and all four brain samples from patients with PML. Neither BK virus (BKV) nor simian virus 40 (SV40) DNA were detected in these samples. No evidence was found to support the hypothesis that polyomaviral DNA is present in the central nervous system of immunosuppressed patients without PML (CSF n = 67, brain n = 19). JCV DNA was not detected in any peripheral blood sample included in this study. JCV DNA was detected in urine from three of eight patients with PML, but was also amplified from three of 29 urine samples from patients without PML, JCV, and not SV40 or BKV, was associated with PML in this study.

Acute hepatitis C infection in patients undergoing therapy for haematological malignancies: a clinical and virological study.

Patients receiving multiple transfusions are at risk of acquiring hepatitis C (HCV) infection from a donor population which is unscreened for hepatitis C antibodies (anti-HCV). Prior to the introduction of blood donor screening for anti-HCV in the U.K., a group of patients undergoing therapy for haematological malignancies, with repeatedly abnormal liver function tests, were investigated for acute HCV infection. Thirty-two patients had repeatedly raised serum transaminases, and eight of these (25%) had evidence of an acute HCV infection. The diagnosis was made by the detection of HCV-RNA in the patients' serum using a complementary DNA/polymerase chain reaction (cDNA/PCR) procedure. All eight patients had received myeloablative chemotherapy and three had undergone bone marrow transplantation. HCV infection contributed significantly to the morbidity of this group of patients in the short term whilst they were undergoing treatment for their underlying haematological condition. The long-term effects have yet to be evaluated. In an attempt to decrease hepatic damage due to HCV, three patients were placed on interferon therapy. None showed a sustained reduction in serum transaminases or HCV viraemia. It is hoped that the introduction of anti-HCV screening of blood donors, will reduce the frequency of transfusion-acquired HCV infections. Early observations suggest that this is the case, as we have seen no new cases of HCV infection in our unit since the introduction of donor screening in September 1991.

Effect of nutrients on defined bacterial plaques and Streptococcus mutans C67-1 implantation in a model mouth.

Actinomyces viscosus WVU 627, Streptococcus oralis LPA-1 and Veillonella dispar OMZ 193 were cocultured on teeth in a model mouth for 66 h. Synthetic saliva containing bovine salivary glycoprotein supported bacterial growth, although the delivery of an intermittent nutrient supplement, containing 1% (w/v) glucose or sucrose, gave greater bacterial cell and viable counts. When Streptococcus mutans C67-1 was super-inoculated onto 24-hour mixed plaques, it became established under all regimens, but there was pronounced colonization resistance. With saliva only, the proportion of S. mutans at 66 h was less than 0.5% of the total cultivable microflora. When a glucose supplement was delivered for 1 h every 6 h, S. mutans attained a final proportion of 2.4%. With sucrose, both S. mutans C67-1 and its non-cariogenic glucan-deficient mutant, C67-25, attained similar proportions of 15-20%. These experiments indicate how this model can be used to study the factors influencing colonizing ability and microbial interactions in biofilms under controlled conditions.

Colonization resistance of defined bacterial plaques to Streptococcus mutans implantation on teeth in a model mouth.

We investigated the ability of Streptococcus mutans C67-1 to colonize simple bacterial plaques and the effects of age and stability of the pre-formed plaque on colonization resistance. Mixed-plaques of Actinomyces viscosus WVU627, 'Streptococcus mitior' LPA-1, and Veillonella dispar OMZ193 were grown on tooth segments, mounted back to back for simulation of approximal sites in a model mouth for 66 h. S. mutans C67-1 was either included in the original inoculum or super-inoculated onto the developing plaque. Inclusion of S. mutans C67-1 did not alter the total viable counts, but the proportional composition changed due to inter-species interactions. Colonization resistance of the mixed-plaque samples developed within 24 h, although S. mutans C67-1 was always able to colonize these stagnation sites. Colonization resistance of 24-hour plaque against a fresh isolate, S. mutans CP3, was also studied. There was greater colonization resistance by the basic plaque to this organism, compared with S. mutans C67-1, although the reasons for this were not clear. These initial experiments demonstrate the way in which the factors involved in bacterial colonization resistance in microbial films on teeth can be studied under controlled conditions.

A laboratory microcosm (artificial mouth) for the culture and continuous pH measurement of oral bacteria on surfaces.

A laboratory microcosm has been designed for the cultivation of bacteria on surfaces subjected to an adjustable supply of fluids. Bacteria are grown as a microbial film on halved premolar teeth, mounted back to back. Synthetic saliva is dropped slowly over the teeth throughout experiments. A nutrient supplement is provided at regular intervals. The drops of fluid retained by the teeth can be sampled for metabolic end-products. Alternatively, a miniature glass electrode may be set into one half of a tooth assembly to monitor the pH continuously at the stagnation site between tooth segments. Up to six replicate culture flasks and six electrodes can be accommodated in a single experiment. Satisfactory electrode performance was maintained during 66 h experiments. In initial 48 h experiments, teeth were inoculated with Streptococcus rattus BHT or 'Streptococcus mitior' LPA-1 in pure culture and provided with 1% (w/v) glucose for 1 h every 6 h. Bacteria produced typical responses to glucose feeds leading to the formation of 'Stephan'-like curves of pH-fall. Under these conditions, 'Strep. mitior' was more acidogenic than Strep. rattus and the pattern of acid production was distinct for each organism.

The role of H2O2 and the lactoperoxidase-SCN(-)-H2O2 system on the interaction between two bacteria originating from human dental plaque, Streptococcus rattus (mutans) BHT and Streptococcus mitior LPA-1, grown on human teeth in an artificial mouth.

Teeth were inoculated with either the organisms separately or with a freshly-prepared mixture of both. The apparatus was swept with 5 per cent (v/v) CO2 in either air or N2, and incubated for 90 h. A nutrient supplement containing 1 per cent (w/v) glucose was supplied for 1 h in every 6 h. Both organisms achieved similar numbers when grown aerobically in pure culture, yet in mixed culture there was pronounced inhibition of BHT (p less than 0.001). When the synthetic saliva was supplemented with catalase the strain BHT count in mixed culture was much higher (p less than 0.001). It was concluded, therefore, that the strain LPA-1 produced inhibitory levels of hydrogen peroxide (H2O2) on the tooth surface under aerobic conditions. This was supported by finding that a lower viable count of LPA-1 in pure culture was attained when lactoperoxidase (LPO) was included in the saliva (p less than 0.005), as all components of the LPO-SCN-H2O2 system were presumably present. With the N2-CO2 mixture, conditions were not strictly anaerobic and both catalase and LPO increased all viable counts. Under these conditions, therefore, when H2O2 was limiting, LPO protected bacteria against its bactericidal effect.

Effect of inoculation sequence and nutrients upon Streptococcus mutans BHT and Streptococcus mitior LPA-1 growing on human teeth in an artificial mouth.

Human teeth in an artificial mouth were inoculated with Streptococcus mutans BHT, Streptococcus mitior LPA-1, or sequentially with both organisms. Incubation was continued for 90 h. Mixed populations were largest when a nutrient supplement containing 5.0% (w/v) sucrose was supplied. Fewer organisms were recovered from experiments with synthetic saliva only, or when a supplement containing 0.05% (w/v) glucose was available. The inoculation sequence determined the total viable count and a larger population resulted when Strep. mutans was the initial colonizer (P less than 0.01). Strep. mutans was always able to become established even when super-infected on to a 24 h plaque of Strep. mitior. The final proportion of Strep mutans was lower when it was the superinfecting organism and the sucrose (P less than 0.01) or glucose (P less than 0.05) nutrient supplement was provided. This work confirms the importance of inoculation sequence and presence of sugars in plaque accumulation and demonstrates the fundamental role of microbial interactions in this process.