Ca Sabin

Laboratory control values for CD4 and CD8 T lymphocytes. Implications for HIV‐1 diagnosis

With the advent of standard flow cytometric methods using two‐colour fluorescence on samples of whole blood, it is possible to establish the ranges of CD3. CD 4 and CDS T lymphocyte subsets in the routine laboratory, and also to assist the definition of HIV‐1‐related deviations from these normal values. In 676 HIV‐1‐seronegative individuals the lymphocyte subset percentages and absolute counts were determined. The samples taken mostly in the morning. The groups included heterosexual controls, people with various clotting disorders but without lymphocyte abnormalities as well as seronegative homosexual men as the appropriate controls for the HIV‐1‐infected groups. The stability of CD4% and CD8% values was demonstrated throughout life, and in children CD4 values < 25% could be regarded as abnormal. The absolute counts of all T cell subsets decreased from birth until the age of 10 years. In adolescents and adults the absolute numbers (mean±s.d.) of lymphocytes, CD3, CD4 and CD8 cells were 1·90±0·55, 1·45±0·46, 0·83±0·29 and 0·56± 0·23 ± 109/l, respectively. In patients with haemophilia A and B the mean values did not differ significantly. In homosexual men higher CD8 levels were seen compared with heterosexual men and 27% had an inverted CD4/CD8 ratio but mostly without CD4 lymphopenia (CD4<0·4 ± 109/l). However, some healthy uninfected people were‘physiologically’ lymphopenic without having inverted CD4/CD8 ratios. When the variations‘within persons’ were studied longitudinally over a 5‐year period, the absolute CD4 counts tended to be fixed at different levels. As a marked contrast, over 60% of asymptomatic HIV‐1+ patients exhibited low CD4 counts <0·4 ± 109/l together with inverted CD4/CD8 ratios. Such combined changes among the heterosexual and HIV‐1‐seronegative homosexual groups were as rare as 1·4% and 3%, respectively. For this reason, when the lymphocyte tests show <0·4 ± 109/l CD4 count and a CD4/CD8 ratio of less than unity, the individuals need to be investigated further for chronicity of this disorder, the signs of viral infections such as HIV‐1 and other causes of immunodeficiency.

Interrelationships among Quantity of Human Cytomegalovirus (HCMV) DNA in Blood, Donor-Recipient Serostatus, and Administration of Methylprednisolone as Risk Factors for HCMV Disease following Liver Transplantation

Longitudinal analysis of 162 liver transplant recipients identified 51 patients who were viremic. Virus load was determined in 47 of these patients using quantitative-competitive polymerase chain reaction. Peak virus load was significantly higher in 20 symptomatic patients than 27 asymptomatic patients (P < .0001). Elevated virus load, donor seropositivity, and total methylprednisolone dosage were risk factors for human cytomegalovirus (HCMV) disease (odds ratio [OR], 2.22/0.25 log10 increase in virus load, P = .001; OR, 4.11, P = .05; OR, 1.30/1-g increment in methylprednisolone, P = .01). Methylprednisolone and virus load were independent risk factors in a multivariate analysis (OR, 2.70/1-g increase, P = .003; OR, 1.61/0.25 log10 increase, P = .03, respectively). Virus loads of 10(4.75)-10(5.25) genomes/mL of blood were associated with an increased disease probability; the latter was shifted to lower virus loads with increasing quantities of methylprednisolone. These data illustrate the central role of virus load in HCMV pathogenesis.

Assessing health-related quality-of-life in individuals with haemophilia.

The objectives of this study were to analyse current levels of health‐related quality‐of‐life (HR‐QoL) in individuals with severe haemophilia and to assess the scope for these levels to improve. To do this, 249 individuals with severe, moderate and mild haemophilia were asked to complete Medical Outcomes Study (MOS) Short‐Form 36 (SF‐36) and EuroQol (EQ‐5D) questionnaires. Access was also gained to two appropriate normative data sets. The results from these questionnaires showed that HIV status, history of orthopaedic surgery and bleeding frequency in the previous calendar year were not strong predictors of HR‐QoL for individuals with severe haemophilia. However, for the majority of scales, age was found to be a strong predictor of HR‐QoL for this patient group. The results from the analysis also showed that compared to individuals with moderate/mild haemophilia and the UK male normative population, individuals with severe haemophilia generally recorded poorer levels of HR‐QoL. These results suggest, therefore, that individuals with severe haemophilia have reduced levels of HR‐QoL compared to individuals with moderate/mild haemophilia and the general population, irrespective of differences in age. The results also suggest that the scope for primary prophylaxis to increase HR‐QoL in individuals with severe haemophilia is significant.

Longitudinal fluctuations in cytomegalovirus load in bone marrow transplant patients: relationship between peak virus load, donor/recipient serostatus, acute GVHD and CMV disease

Quantitative competitive PCR was used to monitor the quantity of cytomegalovirus (HCMV) in 1647 blood samples from 110 BMT recipients. DNAemia was detected in 49/110 (45%) of the patients, of whom 15/49 experienced HCMV disease. Peak virus load during surveillance was elevated in symptomatic (median 4.5 log10 genomes/ml) vs asymptomatic patients (median 3.6 log10 genomes/ml, P = 0.002) and was also significantly elevated in HCMV seropositive recipients of seronegative marrow, (R+D−, median 5.0 log10), compared to those in the R−D− and R+D+ groups (P < 0.01 and <0.005). odds ratios for disease per 0.25 log10 increase in viral load, recipient seropositivity and aGVHD were 1.43 (P = 0.004), 6.60 (P = 0.05) and 3.17 (P = 0.08), respectively. In multivariate logistic regression analysis only elevated viral load remained a significant risk factor for HCMV disease. The computed disease probability viral load curve showed a rapid increase in disease risk at viral loads between 3.8 and 5.5 log10 genomes/ml in blood, and odds ratios for disease were determined for different threshold viral loads. These data demonstrate the central role of viral load in the pathogenesis of HCMV in BMT recipients and provide an additional marker for targeting and monitoring therapy.

British HIV Association guidelines for the routine investigation and monitoring of adult HIV-1-infected individuals 2011.

1. Levels of evidence 1.1 Reference 2. Introduction 3. Auditable targets 4. Table summaries 4.1 Initial diagnosis 4.2 Assessment of ART‐naïve individuals 4.3 ART initiation 4.4 Initial assessment following commencement of ART 4.5 Routine monitoring on ART 4.6 References 5. Newly diagnosed and transferring HIV‐positive individuals 5.1 Initial HIV‐1 diagnosis 5.2 Tests to determine whether acquisition of HIV infection is recent 5.3 Individuals transferring care from a different HIV healthcare setting 5.4 Communication with general practitioners and shared care 5.5 Recommendations 5.6 References 6. Patient history 6.1 Initial HIV‐1 diagnosis 6.2 Monitoring of ART‐naïve patients 6.3 Pre‐ART initiation assessment 6.4 Monitoring individuals established on ART 6.5 Assessment of adherence 6.6 Recommendations 6.7 References 7. Examination 7.1 Recommendations 8. Identifying the need for psychological support 8.1 References 9. Assessment of immune status 9.1 CD4 T cell counts 9.2 CD4 T cell percentage 9.3 References 10. HIV viral load 10.1 Initial diagnosis/ART naïve 10.2 Post ART initiation 10.3 Individuals established on ART 10.4 Recommendations 10.5 References 11. Technical aspects of viral load testing 11.1 References 12. Viral load kinetics during ART and viral load ‘blips’ 12.1 References 13. Proviral DNA load 13.1 References 14. Resistance testing 14.1 Initial HIV‐1 diagnosis 14.2 ART‐naïve 14.3 Post treatment initiation 14.4 ART‐experienced 14.5 References 15. Subtype determination 15.1 Disease progression 15.2 Transmission 15.3 Performance of molecular diagnostic assays 15.4 Response to therapy 15.5 Development of drug resistance 15.6 References 16. Other tests to guide use of specific antiretroviral agents 16.1 Tropism testing 16.2 HLA B*5701 testing 16.3 References 17. Therapeutic drug monitoring 17.1 Recommendations 17.2 References 18. Biochemistry testing 18.1 Introduction 18.2 Liver function 18.3 Renal function 18.4 Dyslipidaemia in HIV‐infected individuals 18.5 Other biomarkers 18.6 Bone disease in HIV‐infected patients 18.7 References 19. Haematology 19.1 Haematological assessment and monitoring 19.2 Recommendations 19.3 References 20. Serology 20.1 Overview 20.2 Hepatitis viruses 20.3 Herpes viruses 20.4 Measles and rubella 20.5 Cytomegalovirus (CMV) 20.6 References 21. Other microbiological screening 21.1 Tuberculosis screening 21.2 Toxoplasma serology 21.3 Tropical screening 21.4 References 22. Sexual health screening including anal and cervical cytology 22.1 Sexual history taking, counselling and sexually transmitted infection (STI) screening 22.2 Cervical and anal cytology 22.3 Recommendations 22.4 References 23. Routine monitoring recommended for specific patient groups 23.1 Women 23.2 Older age 23.3 Injecting drug users 23.4 Individuals coinfected with HBV and HCV 23.5 Late presenters 23.6 References Appendix

Heat-mediated immune complex dissociation and enzyme-linked immunosorbent assay signal amplification render p24 antigen detection in plasma as sensitive as HIV-1 RNA detection by polymerase chain reaction

OBJECTIVE: To study the prevalence, incidence and predictive value for progression to AIDS of the HIV-1 syncytium-inducing (SI) phenotype in HIV-infected injecting drug users (IDU) compared with HIV-infected homosexual men. DESIGN: Two prospective cohort studies on HIV-1 infection among IDU and homosexual men. METHODS: HIV-infected IDU (n = 225) and homosexual men (n = 366) without AIDS were studied from March 1989 through December 1993. Data on laboratory markers, including the presence of SI variants, demographics, behavioural characteristics and clinical events were collected at every visit. RESULTS: At baseline, SI variants were detected in 4% of IDU and 17% of homosexual men. During the study period 18 IDU and 68 homosexual men switched from non-SI to SI phenotype (4-year cumulative incidence, 14.6 and 28.4%, respectively) before AIDS diagnosis. Among participants with a documented date of HIV infection the cumulative incidence of SI was lower among IDU than homosexual men (4-year cumulative incidence, 6.2 and 20.7%, respectively). At AIDS diagnosis, 21% of all AIDS cases among IDU had the SI phenotype compared with 54% among homosexual men. In both risk groups an accelerated CD4 decline was found after the non-SI-to-SI switch. The SI phenotype appeared to be a predictor of AIDS (multivariate relative hazard, 5.33), independent of CD4 cell count and p24 antigen at baseline. In the multivariate time-dependent analysis, the relative hazard of SI phenotype decreased considerably, which is consistent with the hypothesis that the effect of SI phenotype on progression to AIDS is mediated by CD4 cell count. CONCLUSION: The SI phenotype is associated with accelerated CD4 decline and progression to AIDS in both risk groups. The remarkable lower prevalence and incidence of the SI phenotype among IDU may implicate a difference in pathogenesis and natural history of HIV infection linked to transmission group.

Cytomegalovirus (CMV) viraemia detected by polymerase chain reaction identifies a group of HIV-positive patients at high risk of CMV disease

Background: Cytomegalovirus (CMV) disease is a major cause of morbidity in patients with HIV infection. Despite treatment, CMV retinitis causes substantial visual loss, especially in patients with CD4 cell counts below 50 × 106/l. Although routine ophthalmological screening of these patients has been recommended, no controlled trials have evaluated how frequently it should be performed. The aim of this study was to assess whether CMV polymerase chain reaction (PCR) results could direct ophthalmological screening to patients at high risk of CMV retinitis. Methods: In a prospective study of HIV‐positive patients with CD4 cell counts below 50 × 106/l, CMV viraemia was detected by qualitative PCR of whole blood. Patients who were CMV PCR‐viraemic were allocated to monthly virological and ophthalmological follow‐up; patients who were PCR‐negative received 3‐monthly virological and ophthalmological follow‐up. CMV viral load was determined in all CMV‐positive samples using a quantitative competitive PCR. Results: Nineteen out of 97 patients developed CMV disease over the first 12 months of the study. Sixteen (59%) out of 27 patients who were CMV‐positive developed disease compared with three (4%) out of 70 of patients who were PCR‐negative (P = 0.0001). A positive CMV PCR result was significantly associated with the development of disease (P= 0.0001), with a relative hazard of 20.15 [95% confidence interval (CI), 5.80–69.98]. Median CMV viral load was significantly higher in those individuals who went on to develop CMV disease (P = 0.02). In PCR‐positive patients, each 0.25 log10 increase in viral load increased the risk of disease (relative hazard, 1.37; 95% CI, 1.15–1.63; P = 0.0004). Conclusions: CMV PCR predicts the development of CMV disease and can be used to target ophthalmological resources to those patients at highest risk of retinitis. Asymptomatic patients who are PCR‐positive represent a high‐risk group in whom controlled trials of pre‐emptive therapy could be conducted.

Prospective study of human betaherpesviruses after renal transplantation - Association of human herpesvirus 7 and cytomegalovirus co-infection with cytomegalovirus disease and increased rejection

BACKGROUND Human herpesvirus 6 (HHV-6) and HHV-7 are two lymphotropic herpesviruses, which, like cytomegalovirus (CMV), have the potential to be pathogenic in immunocompromised individuals. We have conducted a prospective investigation to compare the natural history of HHV-6 and HHV-7 infection with that of CMV after renal transplantation. METHODS Polymerase chain reaction was used to identify infections and quantify the viral load of CMV, HHV-6, and HHV-7 in peripheral blood samples from 52 renal transplant recipients. Betaherpesvirus infections were related to defined clinical criteria obtained by detailed examination of the clinical records of each patient for the immediate 120-day posttransplant period. RESULTS CMV was the most commonly detected virus after transplant (58% of patients), followed by HHV-7 (46%) and HHV-6 (23%). Examining the time to first polymerase chain reaction positivity, HHV-7 infection was detected earlier than CMV (P=0.05). The median maximum CMV viral load was significantly higher than those for HHV-6 (P=0.01) and HHV-7 (P<0.0001) and a trend for HHV-7 viral load to be greater than HHV-6 (P=0.08). Clinicopathological analyses revealed that, in those patients with rejection, HHV-7 was associated with more episodes of rejection (P=0.02). In addition, there was a significant increase in CMV disease occurring in patients with CMV and HHV-7 co-infection compared to those with CMV infection only (P=0.04). CONCLUSIONS HHV-7 should be further investigated as a possible co-factor in the development of CMV disease in renal transplant patients and may potentially exacerbate graft rejection. No clear pathological role was observed for HHV-6.

CD8+,CD38+ lymphocyte percent: a useful immunological marker for monitoring HIV-1-infected patients.

We investigated the relationship between three prognostic markers, CD4 lymphocyte count, serum beta2-microglobulin (beta2M) levels, and CD8+,CD38+ lymphocyte percent, and the association with the rate of development of AIDS. The markers were measured regularly throughout follow-up in 224 patients. The risk of developing AIDS during follow-up was investigated using Cox proportional hazards models. Time-updated values of the prognostic markers were used, which modelled the risk of AIDS according to the latest measurement of the marker rather than using a single value of the marker at baseline. During a median follow-up period of 13.6 months (range 0.5-31.9 months), 34 cases of AIDS occurred. In a univariate analysis, all three markers predicted the development of AIDS; a 10% increase in the percentage of CD8+ T cells expressing CD38+ resulted in an 88% increase in the risk of AIDS (95% confidence interval: 53-130%; p < 0.0001). After adjustment for the current CD4 count and beta2M, a 10% increase in the CD8+,CD38+ population was associated with a 37% increase in the risk of AIDS (95% confidence interval: 4-81%; p = 0.02). Thus, the percentage CD8+,CD38+ level predicts the development of AIDS independently of the latest CD4 count and beta2M. This assay is therefore potentially useful in conjunction with blood CD4 counts and serum beta2M levels in patient management and clinical trial design.

Cytomegalovirus retinitis in AIDS patients : influence of cytomegaloviral load on response to ganciclovir, time to recurrence and survival

Objectives: Despite life‐long maintenance therapy, cytomegalovirus (CMV) retinitis frequently progresses in patients with AIDS. Virological markers that could clarify pathogenesis and identify risk factors for progression are required. Design and methods: We prospectively recruited 45 patients with CMV retinitis. Blood and urine samples were collected before and after induction therapy, and on a monthly basis thereafter during routine medical and ophthalmological assessment, and at any time retinitis progressed. CMV load was measured by quantitative‐ competitive polymerase chain reaction (PCR). Results: The median time to first progression of retinitis was 78 days and to death was 8.7 months. Eighty‐five per cent of patients who were PCR‐positive at diagnosis of retinitis became PCR‐negative after 21 days of ganciclovir induction therapy. Six patients who remained PCR‐positive after 21 days of treatment had a significantly higher CMV load at presentation (P = 0.005), and a shorter time to first progression of retinitis of 40 days. High CMV loads in blood at presentation were associated with a shorter time to progression (P = 0.16; relative hazard, 1.57) and a significantly shorter time to death (P = 0.004; relative hazard, 1.76). This significant relationship with survival remained after adjustment for potential confounding variables (CD4 count, age, method of drug administration). Conclusions: We conclude that CMV load in the blood of AIDS patients is an important factor in the pathogenesis of retinitis, and quantification of CMV could be used to both select patients for controlled clinical trials and to optimize individual anti‐CMV induction therapy.