James E. McLaughlin

Quantification of human herpesvirus 6 in immunocompetent persons and post-mortem tissues from AIDS patients by PCR.

A quantitative competitive PCR assay for human herpesvirus 6 (HHV-6) was developed. Firstly, viral burden was determined in the blood of 25 healthy persons. Using 1 microgram of DNA, the prevalence of HHV-6 was 36% (9/25). Eight persons had viral loads of < or = 32 HHV-6 genomes/microgram DNA. The viral burden in the ninth individual was 1.2 x 10(6) HHV-6 genome copies/microgram DNA, which remained constant over a period of 10 months. This demonstrates the persistence of a high HHV-6 load in the absence of apparent disease. Secondly, HHV-6 burden was determined in 100 post-mortem tissues from seven AIDS patients and three controls. For all tissues combined, there was a statistically significant higher median viral load in AIDS patients (56 copies/microgram DNA, range 0-43321) compared to controls (10 copies/microgram DNA, range 0-423) (P = 0.04). The precision and reproducibility of this assay will allow hypotheses concerning the pathogenic potential of HHV-6 to be tested quantitatively.

Distinct HIV-1 long terminal repeat quasispecies present in nervous tissues compared to that in lung, blood and lymphoid tissues of an AIDS patient

ObjectiveTo investigate the phylogenetic relationship of HIV-1 proviral long terminal repeat (LTR) variants present in postmortem samples of lymph node, spleen, lung, dorsal root ganglion and spinal cord as well as in the peripheral blood of an HIV-1-infected patient dying with AIDS. Design and methodsPostmortem tissues were studied by a combination of histology, cell culture and molecular analyses. The patient had a stable CD4 count of 10×106/l during the 12 months preceding death. A 540 base-pair fragment of the LTR including U3/R/U5 was amplified using polymerase chain reaction on proviral DNA from the five postmortem tissues and peripheral blood mononuclear cells obtained 2 months prior to death. The population of viral variants was determined by sequencing at least five plasmid clones of the amplicons. The relationship between the variants present in different body sites was investigated using molecular phylogeny methods. ResultsHIV-1 was present in all organs analysed and correlated with the presence of abnormal histology. Genetic variation leading to divergence from the consensus sequence was more frequently present in characterized transcription factor binding sites within the LTR (P<0.0001) although the HIV-1 LTR quasispecies in the different body sites showed similar, relatively low levels of divergence (intra-organ median heterogeneity ranging from 0.0094 to 0.017). Phylogenetic analysis showed that the spinal cord and dorsal root ganglion harboured an LTR population genetically distinct from that present in other organs and more closely related to a previously characterized neurotropic strain of HIV (strain JRcsf). ConclusionThe independent clustering of HIV-1 LTR variants present in spinal cord and dorsal root ganglion shows that HIV-1 LTR evolution can occur in a compartmentalized fashion. The data show that the LTR is an important region to analyse in sequence variation studies of HIV since it may play a role in nervous tissue adaptation of HIV-1 and neuropathogenicity. Outgrowth of HIV-1 LTR variants that are most fit for the utilization of tissue-specific transcription factors can occur in the nervous tissue.

HIV-associated brain pathology in the United Kingdom: an epidemiological study

Objectives:To examine the epidemiology of HIV-associated neuropathology in the United Kingdom and to investigate whether the prevalence of different forms of HIV-associated brain pathology varies with exposure category. Design:The study was a cross-sectional survey; data was analysed from the Medical Research Council National AIDS Neuropathology database. Setting:Information was gathered from throughout England, Scotland and Wales. Subjects:Individuals who died from AIDS in the United Kingdom and had a postmortem examination. The database comprised 7% of all AIDS deaths in the United Kingdom between 1982 and 1993. Main outcome:Neuropathological diagnoses based on internationally accepted neuropathological terminology of AIDS-related brain lesions. Results:HIV encephalitis was the most prevalent pathological diagnosis, occurring in 25.3% [95% confidence interval (CI), 21.0–29.6] of the study sample. Statistically significant independent associations for the occurrence of HIV encephalitis were found for injecting drug use (odds ratio, 6.86; 95% CI, 2.91–16.17), and age less than 30 years at death (odds ratio, 3.58; 95% CI, 1.99-6.44). Vascular lesions were significantly higher among blood product recipients, 95% of whom were haemophiliacs. Conclusions:This was the first epidemiological investigation of HIV-associated brain pathology in the United Kingdom. HIV encephalitis appeared to occur more frequently in injecting drug users and those who died younger. Whereas the findings must be interpreted cautiously, one hypothesis was that differences in the route of transmission may have affected the manifestation of HIV-associated brain damage.

A clinico-pathological audit of opportunistic viral infections in HIV-infected patients.

OBJECTIVEnTo determine the prevalence of opportunistic viral infections in multiple tissues at postmortems of HIV-infected patients, and to relate these findings to their antemortem clinical course.nnnDESIGNnA study of viral infections in 16 tissues of HIV-positive postmortem cases, by a combination of histology and cell culture (virus isolation). Clinical details were abstracted retrospectively from patient records.nnnPATIENTSnForty-seven consecutive autopsies, performed between 1985 and 1992.nnnSETTINGnAutopsies were conducted by a single pathologist in a single London teaching hospital.nnnRESULTSnOpportunistic viral infections were detected in 72% of all cases, comprising cytomegalovirus (CMV, 66%), herpes simplex virus (11%), JC virus (6%) and adenovirus (2%). The most commonly infected tissues were lung, adrenal, gastrointestinal tract and central nervous system, although all tissue sites sampled could potentially support viral replication. Of 464 tissues tested by both histology and cell culture, histology alone detected CMV in 45 tissues and cell culture alone detected CMV in 31 tissues. We determined that CMV detection in postmortem gastrointestinal tissues and central nervous tissue was significantly associated with antemortem undiagnosed diarrhoea and encephalitis, respectively.nnnCONCLUSIONnThere is a high prevalence of opportunistic viral infections in late-stage HIV disease, which is best detected postmortem by the use of both histology and cell culture. Many of these infections correlate with undiagnosed symptoms antemortem. The ability of sensitive methods for virus detection to alert the clinician to such cases antemortem should be critically evaluated, as should attempts to influence the natural history of these infections by antiviral drugs. Continuing clinico-pathological audit is important for AIDS patients in order to monitor the impact of known opportunistic viral infections and to identify others which may emerge as immunosuppression becomes more profound.

Interactions between β-herpesviruses and human immunodeficiency virus in vivo : Evidence for increased human immunodeficiency viral load in the presence of human herpesvirus 6

In vitro, β‐herpesviruses can stimulate or inhibit HIV replication under particular circumstances. In order to investigate the effects of β‐herpesvirus infection on HIV replication and vice versa at an organ level, we determined the quantitative relationships between cytomegalovirus (CMV), human herpesviruses (HHV) 6 and 7, and HIV‐1 proviral DNA using quantitative competitive PCR methods in 141 organs collected at autopsy from 11 AIDS patients. The presence of HHV‐6 DNA in an organ was significantly associated with elevated HIV‐1 proviral DNA (difference in HIV median loads, 1.3 log10 genomes; P = 0.004). Consistent with this, there was a trend for the presence of HIV‐1 proviral DNA to be associated with an elevated HHV‐6 load (0.44 log10 difference; P = 0.07). In contrast, there were no significant differences between viral loads in the combinations of either CMV or HHV‐7 with HIV‐1 proviral DNA load. Pairwise combinations of the β‐herpesviruses revealed that the quantity of HHV‐7 was increased in the presence of HHV‐6 (difference in median loads, 1.3 log10; P = 0.001) and the quantity of HHV‐6 was increased in the presence of HHV‐7 (difference in median loads, 0.7 log10; P = 0.002). These results demonstrate that the presence of HHV‐6 in an organ is significantly associated with an elevated HIV‐1 proviral load and have implications for understanding HIV pathogenesis in the human host and the role that β‐herpesviruses, especially HHV‐6, might play as cofactors in the HIV disease process. J. Med. Virol. 57:278–282, 1999.

Cure of Acanthamoeba Cerebral Abscess in a Liver Transplant Patient

Acanthamoeba‐related cerebral abscess and encephalitis are rare but usually fatal, being caused by free‐living amoebic infections usually occurring in immunocompromised patients. In patients receiving transplants, a literature review showed that the infection is universally fatal. The diagnosis is often missed despite appropriate investigations including lumbar puncture, computerized tomography, and brain biopsy. We present the first reported liver transplant patient with Acanthamoeba cerebral abscess. The diagnosis was made in brain tissue removed at decompressive frontal lobectomy. He was successfully treated with a 3‐month course of co‐trimoxazole and rifampicin. There was no recurrence of the disease after 11 years of follow‐up. Liver Transpl 14:308–312, 2008.

Histopathological detection of owl's eye inclusions is still specific for cytomegalovirus in the era of human herpesviruses 6 and 7

Background—Cytomegalovirus (CMV) is the prototype member of the β-herpesvirinae, which can cause multiple organ dysfunction in the immunocompromised host. Human herpesvirus 6 (HHV-6) and HHV-7 are newer members of the β-herpesvirinae that can cause febrile illness in young children and are also possible pathogens in the immunocompromised patient. Aim—CMV is detected in histopathological sections by visualisation of owls eye inclusion bodies. The aim of this study was to quantify the relation between CMV, HHV-6, and HHV-7 viral loads and the presence of owls eye inclusions in histological sections. Methods—Histopathological examination of postmortem material and recording of owls eye inclusion bodies were performed. CMV, HHV-6, and HHV-7 were detected by qualitative and quantitative polymerase chain reaction (PCR) from the same postmortem samples. Statistical analysis of the histopathological and PCR results was performed. Results—There was a significant association between the detection of owls eye inclusion bodies and positive CMV PCR (p < 0.001); the median CMV viral load was significantly higher in samples that were positive for owls eye inclusions (p < 0.001). No association was found between the presence of owls eye inclusions and HHV-6 or HHV-7 positivity. Conclusion—Histological detection of owls eye inclusion bodies is an insensitive but highly specific method for detecting CMV organ involvement. Owls eye inclusion bodies are not associated with HHV-6 or HHV-7 infection.

Shorter survival in HIV-positive patients with diarrhoea who excrete adenovirus from the GI tract

Adenoviruses have been described as a cause of diarrhoea in patients infected with the human immunodeficiency virus (HIV). The prevalence of adenoviruses was studied in all HIV‐positive patients presenting with diarrhoea at the Royal Free Hospital in London between 1991 and 1995. In addition, all postmortems carried out in HIV‐positive individuals registered at the same centre between 1990 and 1997 were reviewed for evidence of adenovirus infection. Adenovirus was detected in 16.1% of patients presenting with diarrhoea. These individuals had a significantly lower CD4 count and were more likely to have had a diagnosis of acquired immunodeficiency syndrome (AIDS) than patients with diarrhoea in whom adenovirus was not detected. The median survival was 1 year compared with 2.4 years for those without adenoviruses; this difference remained significant (P = .008) after controlling for differences in CD4 counts between the groups. Gastrointestinal adenovirus excretion occurs at an advanced stage of HIV disease, and is associated with a poor prognosis. We suggest that adenoviruses may contribute to mortality in this population. J. Med. Virol. 58:280–285, 1999.

Quantitative differences in the distribution of zidovudine resistance mutations in multiple post-mortem tissues from AIDS patients

Replication of HIV introduces errors into the genome which are responsible for conferring a growth advantage over wildtype virus when drugs such as zidovudine (ZDV) exert a selective pressure. The molecular basis for HIV‐1 resistance to ZDV has been mapped to codons 41, 67, 70, 215 and 219 of the reverse transcriptase gene both in vitro and in clinical samples of blood. This study has investigated the relationship between the quantitative prevalence of ZDV resistance in multiple organs of the same individual. Proviral HIV‐1 load was measured by quantitative‐competitive PCR in 90 samples from organs of 11 patients dying with AIDS. Nine of these patients had been prescribed zidovudine. The distribution of wildtype and mutant sequences at the positions 41, 67, 70, 215 and 219 of the reverse transcriptase was assessed using a point mutation assay. The results showed that the highest proviral loads were predominately found in lymph node, spleen and lung and there was a significant association between viral load and resistance to ZDV (P = 0.008). Inter‐organ distribution of wildtype and mutant sequences at codons 41, 67, 70, 215 and 219 was frequently not uniform and in some patients differed markedly between the lymphoreticular system and other organs. These results demonstrate that treatment of HIV‐1 infection with zidovudine does not exert uniform selective pressures in multiple organs. These findings have implications for the interpretation of resistance data and design of treatment strategies for HIV, arguing in particular that alterations in therapeutic regimens should consider the likelihood of different resistance patterns being present in multiple sites within the same individual. J. Med. Virol. 55:138–146, 1998.