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PubMed2003

Drug resistance is uncommon in pregnant women with low viral loads taking zidovudine monotherapy to prevent perinatal HIV transmission.

Zidovudine monotherapy is used to reduce perinatal HIV transmission in women with low viral loads. There are few data on the risk of drug resistance in this select cohort of women. We determined the prevalence of newly acquired mutations conferring reduced sensitivity to zidovudine after exposure during pregnancy, and found that the development of mutations was uncommon and was restricted to women treated before 1998 who had higher baseline viral loads than those currently recommended monotherapy.

Anti-HIV Agents↗

PubMed2005

CD4+ cell count, viral load, and highly active antiretroviral therapy use are independent predictors of body composition alterations in HIV-infected adults: a longitudinal study.

BACKGROUND: To understand the concurrent effects of human immunodeficiency virus (HIV) infection, the immune system, and antiretroviral therapy on body composition alterations, we examined annualized composition changes in HIV-infected adults who were receiving stable antiretroviral therapy. METHODS: With use of data from the Nutrition For Healthy Living Study, we performed multivariate analyses using longitudinal models to evaluate the relationship of CD4+ cell count, viral load, and highly active antiretroviral therapy (HAART) or antiretroviral therapy (ART) with changes in trunk and extremity composition for 110 men and 42 women who provided data relating to 194 study intervals (i.e., intervals of time between 2 assessment visits). Of these intervals, 165 involved HAART use (89.7% involved protease inhibitor-based regimens), and 29 did not involve HAART use. Patients receiving HAART or ART (who had continuous use during the interval) were compared with HAART- or ART-naive subjects. RESULTS: The median length of intervals between visits was 12.9 months (interquartile range, 12.1-17.6 months). In models adjusted for HAART or ART use, baseline CD4+ cell count was positively associated with increased trunk fat (mean increase per year, 2.3% per 100 cells/mm3; 95% confidence interval [CI], 0.7%-3.9%]) and, in men, with increased extremity fat (mean increase per year, 1.8% per 100 cells/mm3; 95% CI, 0.6%-3.0%). Increase in CD4+ cell count predicted increased extremity lean mass (mean increase per year, 0.6% per 100 cells/mm3; 95% CI, 0.05%-1.1%). Higher baseline viral load predicted fat loss (trunk fat loss per year, -5.0% per log10 copies/mL; 95% CI, -9.4% to -0.7%; extremity fat loss per year, -3.4% per log10 copies/mL; 95% CI, -6.1% to -0.6%), as did zidovudine use (trunk fat loss per year, -10.8%; 95% CI, -20.4% to -1.4%; extremity fat loss per year, -4.9%; 95% CI, -9.8% to -0.01%). HAART use independently predicted decreased bone mineral content (extremity bone mineral content loss per year, -1.6%; 95% CI, -3.1% to -0.08%) but did not predict changes in fat or lean mass. Receipt of protease inhibitor-based HAART predicted a -1.9% decrease in extremity bone mineral content per year (95% CI, -3.6% to -0.2%), and zidovudine use predicted a -2.6% decrease in trunk bone mineral content per year (95% CI, -4.4% to -0.8%). CONCLUSIONS: Baseline viral load, CD4+ cell count, and change in CD4+ cell count predicted alterations in trunk fat, extremity fat, and lean mass. HAART use and zidovudine use were associated with bone loss, and zidovudine use was associated with fat loss, but HAART use was not associated with fat mass changes.

Adult↗

PubMed2005

Evaluation of a low cost reverse transcriptase assay for plasma HIV-1 viral load monitoring.

We evaluated a low cost manual reverse transcriptase assay (ExaVir Load V.1 and V.2; Cavidi Tech AB) against commercially available HIV RNA assays that quantify viral load to assess its suitability for use in resource-constrained settings. Frozen plasma samples previously tested for RNA by RT-PCR (Roche Diagnostics) and bDNA (Bayer Diagnostics) were retested for RT activity. Text sequence obtained from HIV genotype analysis was submitted to the Stanford HIV Resistance Database V.3.9 and were examined for resistant virus. Detectable RT was present in 98% of samples (V.1; n=127) and in 95% of samples (V.2; n=69) with RNA >10,000 and >1,000 copies/ml respectively. Positive association was found between the log10 RNA copies/ml and log10 RT copies/ml equivalents variables using Pearson's correlation (V.1: r=0.89, n=189; V.2: r=0.89, n=85). The RT activity over time closely followed the trend for RNA levels in samples from 10 HIV seropositive patients with progressive disease. A strong association between RT and RNA was also found with paired samples from 19 patients taken at initiation or change of antiretroviral therapy and again within 2 months. Current (n=40) or no (n=119) exposure to efavirenz therapy had no effect on RT assay performance despite efavirenz binding tightly to the RT enzyme. Samples that demonstrated resistance to the non-nucleoside RT inhibitors (n=112) had a decrease in RT of 0.20 log10 indicating a possible decrease in RT fitness. The RT assay showed good association with current molecular assays, and V.2 is sufficiently sensitive for monitoring HIV viral load in resource-constrained settings.

Alkynes↗

PubMed2003

Viral load and disease progression as responsible for endothelial activation and/or injury in human immunodeficiency virus-1-infected patients.

The endothelium participates in haemostasis, inflammation, blood pressure regulation and other physiological systems. Consequently, endothelial dysfunction has been related to hypertension, thrombosis and atherosclerosis. Both von Willebrand factor (vWF) and tissue-type plasminogen activator (t-PA) are synthesized by the endothelium and their plasma levels increased during endothelium activation or injury. So far, they are well-known markers of endothelial cell function. Many circumstances activate or damage the endothelium, such as viruses, bacterium and inflammation. Circulating vWF and t-PA were studied in 92 unselected human immunodeficiency virus-1 (HIV-1)-infected patients [27 patients with and 65 patients without acquired immunodeficiency syndrome (AIDS)] and correlated with plasma levels of pro-inflammatory cytokines (tumour necrosis factor-alpha, interleukin-6), viral load, CD4 T-cell count and infectious status. HIV-1-infected patients had significantly higher plasma levels of vWF (152 versus 90%), tumour necrosis factor-alpha (31.3 versus 9.0 pg/ml) and interleukin-6 (3.5 versus 1.9 pg/ml) but not t-PA (5.9 versus 4.2 ng/ml) than the control group. These two endothelial markers correlated significantly with viral load and interleukin-6 levels in HIV-1-infected patients. The highest levels of vWF and t-PA were found in patients with AIDS. In conclusion, endothelial cell perturbation is present in HIV infection and may be a consequence of different mechanisms such as viral load, cytokines and advanced diseases.

Adult↗

PubMed2006

Proliferative capacity of epitope-specific CD8 T-cell responses is inversely related to viral load in chronic human immunodeficiency virus type 1 infection.

The relationship between the function of human immunodeficiency virus (HIV)-specific CD8 T-cell responses and viral load has not been defined. In this study, we used a panel of major histocompatibility complex class I tetramers to examine responses to frequently targeted CD8 T-cell epitopes in a large cohort of antiretroviral-therapy-naïve HIV type 1 clade C virus-infected persons in KwaZulu Natal, South Africa. In terms of effector functions of proliferation, cytokine production, and degranulation, only proliferation showed a significant correlation with viral load. This robust inverse relationship provides an important functional correlate of viral control relevant to both vaccine design and evaluation.

CD8-Positive T-Lymphocytes↗

PubMed2002

Association of depression with viral load, CD8 T lymphocytes, and natural killer cells in women with HIV infection.

OBJECTIVE: Clinical and epidemiology studies have implicated depression as a risk factor in the morbidity and mortality of many human diseases. This study sought to determine if depression was associated with alterations in cellular immunity variables-specifically, natural killer (NK) cells and CD8 T lymphocytes-in women with HIV infection. METHOD: Ninety-three women (63 HIV-seropositive, 30 HIV-seronegative) were studied as part of an ongoing longitudinal study conducted at two sites. Subjects underwent extensive clinical, psychiatric, and immunological evaluations. CBC counts and flow cytometry panels were conducted and NK cell activity assayed for all subjects; viral load was determined for HIV-seropositive subjects. RESULTS: The overall rate of major depression in the HIV-seropositive and HIV-seronegative women was 15.87% (N=10 of 63) and 10.00% (N=3 of 30), respectively. HIV-seropositive women had higher depressive symptom scores than did the comparison subjects (Hamilton depression scale: mean=8.62 [SD=7.26] versus mean=5.67 [SD=7.33], respectively). Both groups had similar anxiety scores. Depressive and anxiety symptoms were significantly associated with higher activated CD8 T lymphocyte counts and higher viral load levels. Major depression was associated with significantly lower natural killer cell activity, and depressive and anxiety symptom scores showed a similar correlation. CONCLUSIONS: Our findings provide the first evidence that depression may alter the function of killer lymphocytes in HIV-infected women and suggest that depression may decrease natural killer cell activity and lead to an increase in activated CD8 T lymphocytes and viral load. The rate of current major depression in these HIV-seropositive women (none of whom had current substance abuse) is approximately twice that reported for HIV-seropositive men. The rate is also consistent with studies of women with other medical illnesses and with a recent epidemiology study that associated depression with mortality in HIV-infected women with chronic depressive symptoms. Depression may have a negative impact on innate immunity. Examination of killer lymphocytes may prove useful in assessing the potential relationship between depression, immunity, and HIV disease progression in women.

Adult↗

PubMed2002

A prospective study of adherence and viral load in a large multi-center cohort of HIV-infected women.

OBJECTIVES: To examine the relationship between antiretroviral adherence and viral load, and to determine the predictors of adherence over time in HIV-infected women. DESIGN: Prospective observational study. METHODS: One-hundred sixty-one HIV-infected women who were taking antiretroviral therapy for a median of 3.0 years were recruited from the HIV Epidemiology Research Study, a multicenter cohort study of HIV infection in women. Antiretroviral adherence (percent of doses taken as prescribed) was measured over a 6-month period using MEMS caps. At baseline and follow-up, CD4 lymphocyte count and viral load were measured, and a standardized interview was administered to elicit medication history and drug use behaviors. To examine changes in adherence over time, the mean adherence to all antiretroviral agents was calculated for each monitored month. RESULTS: Adherence varied significantly over time (P < 0.001), ranging from a mean of 64% in month 1 to 45% in month 6. Nearly one-fourth of the participants had a 10% or greater decrease in adherence between consecutive months. Virologic failure occurred in 17% of women with adherence of > or = 88%, 28% of those with 45-87% adherence, 43% of those with 13-44% adherence, and 71% of those with < or = 12% adherence. In multivariate analysis, factors predicting lower adherence included active drug use, alcohol use, more frequent antiretroviral dosing, shorter duration of antiretroviral use, younger age, and lower initial CD4 lymphocyte count. CONCLUSIONS: Antiretroviral adherence is not stable over time. Interventions aimed at monitoring and improving long-term adherence in women are urgently needed.

Adult↗

PubMed2000

Increasing the number of hepatitis B vaccine injections augments anti-HBs response rate in HIV-infected patients. Effects on HIV-1 viral load.

Preventing hepatitis B by vaccination is essential in HIV-infected patients (higher progression rate of HBV infection to chronicity, lower rate of serum HBe Ag loss). However, it has been shown a decreased anti-HBs response in these individuals after a standard vaccination (3 doses of 20 micrograms). Thus, we tested the hypothesis that doubling the number of hepatitis B vaccine injections might increase anti-HBs response rate. HIV-infected patients with CD4 > 200/microliter, who were on stable antiretroviral treatment, as well as seronegative for HBV markers, and who have never been vaccinated against HBV, were given 3 intramuscular injections of Genhevac B 20 micrograms at 1 month intervals. Initial non responders were given 3 additional monthly injections. Anti-HBs titer was followed. We also evaluated the effects on HIV-1 viral load. Twenty patients with a median CD4 cell count of 470/microliter were enrolled. The response rate after three 20 micrograms injections was 55% (11/20), lower in individuals with CD4 between 200 and 500/microliter (4/12 = 33.3%), compared to patients with CD4 above 500/microliter (7/8 = 87.5%, P = 0.02). Among 9 initial non-responders, only 2 did not respond to 3 additional doses; thus, the overall response rate was 90% (18/20). Geometric mean titers of anti-HBs were 133 IU/l and 77.5 IU/l, after 3 and 6 Genhevac doses, respectively (P = 0.38). One year later, only 10/17 (58.8%) patients had protective anti-HBs. Five patients experienced a significant viral load increase, transient in 3 cases. These preliminary results suggest that doubling the number of hepatitis B vaccinations in HIV-infected patients might significantly improve anti-HBs response rate; however, close monitoring of anti-HBs is necessary because of its short-lived persistence. The effects on HIV-1 viral load are limited.

Adult↗

PubMed2004

Efficient lentiviral vector-mediated control of HIV-1 replication in CD4 lymphocytes from diverse HIV+ infected patients grouped according to CD4 count and viral load.

We present preclinical studies that demonstrate in vitro the feasibility and efficacy of lentivirus-based vector antisense gene therapy for control of HIV replication in primary T lymphocytes isolated from HIV-infected patients discordant for clinical status. VRX496 is a VSV-G-pseudotyped HIV-based vector that encodes an antisense payload against the HIV envelope gene. The antisense payload is under the control of the native LTR promoter, which is highly transactivated by tat upon HIV infection in the cell. Transfer of autologous CD4(+) T lymphocytes genetically modified with VRX496 (VRX496T) into HIV-infected patients is intended to provide a reservoir of cells capable of controlling HIV, potentially delaying AIDS onset. To determine the patient population likely to respond to VRX496 for optimal efficacy, we examined the ability of our research vector, VRX494, to modify and suppress HIV in vitro in lymphocytes isolated from 20 study subjects discordant for CD4 count and viral load. VRX494 is analogous to the clinical vector VRX496, except that it contains GFP as a marker gene instead of the 186-tag marker in the clinical vector. To transfer VRX494 to target cells we developed a novel scalable two-step transduction procedure that has been translated to the clinic in an ongoing clinical trial. This procedure achieved unprecedented transduction efficiencies of 94 +/- 5% in HIV(+) study subject cells. In addition the vector inhibited HIV replication >/=93% in culture regardless of the viral load or CD4 count of the subject or tropism of the virus strain with which they were infected. These findings demonstrate that VRX496T therapy is expected to be beneficial to patients that differ in their status in term of CD4 count and viral load. The methods described represent significant technical advances facilitating execution of lentivirus vector-mediated gene therapy for treatment of HIV and are currently being employed in the first trial evaluating lentivirus vector safety in humans.

Antigens, Surface↗

PubMed2004

Performance assessment of eight high-throughput PCR assays for viral load quantitation of oncogenic HPV types.

Infection with mucosotropic human papillomavirus (HPV) is the necessary cause of cervical intraepithelial neoplasia. Several epidemiological studies suggest that HPV viral load can be a risk factor of cervical dysplasia. The purpose of the present study was to evaluate a methodology to determine HPV viral load of eight oncogenic HPV types (16, 18, 31, 39, 45, 51, 52, and 58). The quantitation assay is based on a high-throughput real-time PCR. The E6-E7 region of HPV types 16, 18, 45, and 51 were used to amplify specific DNA sequences and cloned into a plasmid vector. The constructs for HPV types 16, 18, 45, and 51, and the whole genome for HPV types 31, 39, 52, and 58 were quantitated using a limiting dilution analysis and used to create standard curves. Type-specific HPV clones were used to determine specificity, linearity, and intra- and inter-assay variation. The sensitivity of our assay covered a dynamic range of up to seven orders of magnitude with a coefficient of intra-assay variation less than 6% and the inter-assay variation less than 20%. No cross-reactivity was observed on any of the type-specific standard curves when phylogenetically close HPV types were used as templates. Our real-time PCR methodologies are highly reproducible, sensitive, and specific over a sevenfold magnitude dynamic range.

DNA Probes, HPV↗

PubMed1995

Measuring viral load in the clinical setting.

The traditional approach of assessing new treatments in clinical end-point studies is becoming less viable for ethical and practical reasons. As more antiretroviral options become available and as treatment is commenced at earlier, asymptomatic stages of infection, alternative methods of assessing the efficacy of antiretroviral regimens are necessary. Recently developed quantitative measures of viral nucleic acids are already proving invaluable in predicting which drugs and combinations are most promising for further investigation. These approaches have been used to evaluate the effect of antiretroviral drugs in clinical trials comparing zidovudine (AZT) monotherapy with combinations of AZT/didanosine (ddI), AZT/zalcitabine (ddC), and AZT/lamivudine (3TC). In one study (Trial BW 34,225-02), levels of HIV-1 RNA in serum fell by approximately 50% in antiretroviral-naive patients on commencement of AZT monotherapy. For patients treated with AZT/ddI and AZT/ddC, reductions in serum HIV-1 RNA levels were significantly greater (80-90%). Increases in CD4 cell count corresponded with these decreases with the decrease in viral load. A second study (Trial NUCB3001) comparing the effect of AZT monotherapy with AZT/3TC therapy in antiretroviral-naive individuals showed even more marked and sustained superiority of the combination regimen over monotherapy (>1.8 log RNA copies/ml vs. 0.7 log RNA copies/ml after 4 weeks). These studies showed that the reduction in viral load achieved with combination therapies were greater and were sustained for longer periods than with monotherapy. The most pronounced effect was achieved by the AZT/3TC combination. Assessment of viral load is a practical and promising approach to monitoring antiretroviral action.

Antiviral Agents↗

PubMed2002

MHC class I alleles influence set-point viral load and survival time in simian immunodeficiency virus-infected rhesus monkeys.

In HIV-infected humans and SIV-infected rhesus macaques, host genes influence viral containment and hence the duration of the disease-free latency period. Our knowledge of the rhesus monkey immunogenetics, however, is limited. In this study, we describe partial cDNA sequences of five newly discovered rhesus macaque (Mamu) class I alleles and PCR-based typing techniques for the novel and previously published Mhc class I alleles. Using 15 primer pairs for PCR-based typing and DNA sequence analysis, we identified at least 21 Mhc class I alleles in a cohort of 91 SIV-infected macaques. The results confirm the presence of multiple class I genes in rhesus macaques. Of these alleles, Mamu-A*01 was significantly associated with lower set-point viral load and prolonged survival time. Mamu-A*1303 was associated with longer survival and a "novel" Mhc class I allele with lower set-point viral load. The alleles are frequent in rhesus macaques of Indian origin (12-22%). In addition, survival probability of individual SIV-infected rhesus monkeys increased with their number of alleles considered to be associated with longer survival. The results contribute to improve the interpretation and quality of preclinical studies in rhesus monkeys.

Alleles↗

PubMed2006

Improved endocervical sampling and HPV viral load detection by Cervex-Brush Combi.

OBJECTIVE: Liquid-based cytology (LBC) for cervical screening is becoming increasingly used. Together with SurePath LBC, various collecting devices can be utilized, among which the Cervex-Brush is the most widely used. The new Rovers Cervex-Brush Combi combines the advantages of the Cervex-Brush with the EndoCervex-Brush increasing sampling of the endocervical canal. The objective of this study was to analyse and to compare the Cervex-Brush Combi with the Cervex-Brush for the collection of squamous and endocervical cells, human papillomavirus (HPV) typing/quantification and disease detection in SurePath LBC. METHODS: Using either the Cervex-Brush or the Cervex-Brush Combi 100 consecutive SurePath LBC samples were collected using each brush type. All 200 slides were read by the FocalPoint and screened by guided screening using slide wizards. The viral load of HPV type 16 E7, 18 E7, 31 E6, 33 L1, 33 E6, 35 E4, 39 E7, 45 E7, 51 E6, 52 L1, 52 E7, 53 E6, 56 E7, 58 L1, 58 E6, 59 E7, 66 E6 and 68 E7 was determined using a TaqMan-based real-time quantitative PCR analysis. RESULTS: The mean number of sampled squamous cells did not differ between the two brush types (54 963 versus 54 595 cells). The use of the Cervex-Brush Combi, however, resulted in a two- to threefold increase in the number of sampled endocervical cells (P < 0.00001). Using the Cervex-Brush Combi slightly more lesions were detected (three versus two low-grade squamous intraepithelial lesions), and resulted in the detection of more atypical squamous cells of undetermined significance (six versus three). In the Cervex-Brush group, 60% (3/5) of abnormal smears were positive for oncogenic HPV types, whereas 66.7% (6/9) of abnormal smears in the Cervex-Brush Combi group tested positive. The median HPV viral load for samples taken with the Cervex-Brush Combi was 0.1825 copies/cell and was significantly higher than in samples taken with the Cervex-Brush (0.0042 copies/cell) (P = 0.02). CONCLUSION: Sampling with the Cervex-Brush Combi resulted in the collection of the same amount of squamous cells, but in a two to threefold harvest of endocervical cells. This led to the detection of a higher viral load for oncogenic HPV and an increase in the number of detected abnormal smears.

Cohort Studies↗

PubMed2000

Human immunodeficiency virus type 2 (HIV-2) in Portugal: clinical spectrum, circulating subtypes, virus isolation, and plasma viral load.

The human immunodeficiency virus type 2 (HIV-2) is responsible for 4. 5% of AIDS cases in Portugal. Six HIV-2 subtypes have been described so far, subtype A being proposed as more pathogenic than the rest. The relationship between the clinical status and levels of both cellular and plasma HIV-2 viraemia is not well known, nor their modifications under antiretroviral therapy. Thirty-two consecutive HIV-2 infected persons (17 men, 15 women) attending two different hospitals in Lisbon in 1997 were enrolled prospectively in the study. All but 4 individuals most likely acquired the infection through heterosexual contact. More than half of the study population was of African origin, mainly from Guinea-Bissau. Eleven (34.4%) patients had developed clinical manifestations included within the B or C groups of the CDC classification system for HIV infection, with the rest being asymptomatic. Half of the population was undergoing antiretroviral treatment at the time of the study. HIV-2 subtypes were investigated using a new Nef-based restriction fragment length polymorphism (RFLP) method that allows differentiation of the main two variants, A and B. Plasma viral load was quantified using a new quantitative competitive reverse transcriptase polymerase chain reaction (QcRT-PCR) procedure as well as the Amp-RT assay. Virus isolation was attempted from peripheral blood mononuclear cells. All but one person carried HIV-2 subtype A. Plasma viraemia examined by QcRT-PCR was measurable in 15 (50%) of 30 subjects, yielding in all instances values below 20,000 HIV-2 RNA copies per ml. Plasma RT activity could be detected in only 10 (33%) of 30 subjects, a rate much lower than that seen in HIV-1 infection. Virus was isolated from 16 (53.3%) of 30 patients. A significant correlation was found between CD4+ counts, clinical status, rate of virus isolation, and plasma viral load by both QcRT-PCR and Amp-RT. In conclusion, HIV-2 subtype A is the predominant variant circulating in Portugal among both natives and immigrants. A lower cellular and plasma viral load with respect to HIV-1 was seen in persons without immunosuppression, from whom the rate of virus recovery was extremely low.

Female↗

PubMed2002

Human papillomavirus type 16 is episomal and a high viral load may be correlated to better prognosis in tonsillar cancer.

The aim of our study was to investigate the physical state and the viral load of HPV-16 in tonsillar cancer and to correlate these findings with clinical outcome. To distinguish between integrated and episomal forms of HPV, 22 fresh-frozen tonsillar cancer samples were analysed by a method based on restriction enzyme cleavage, ligation and PCR (rliPCR). HPV-16 was detected in 11/22 and HPV-33 in 1/22 of the cancers, hence 12/22 (55%) of the tumours were HPV positive. Only extrachromosomal forms of HPV-16 were observed. Full-length episomal HPV was detected exclusively in 7/11 of the cancers, whereas both full-length and deleted forms of episomal HPV-16 were found in parallel in 2 other tumours. In 1 tumour only a deleted episomal form of HPV-16 was present. In the remaining HPV-16 positive tumour both full-length episomal as well as an 11 kbp PCR product were detected and if the 11 kbp product contained integrated HPV, or was off-size linearised episomal could not be determined. In 2 cervical cancer controls, HPV-16 was integrated and could be chromosome located. HPV-16 was quantified by real-time PCR and most tonsillar cancers contained between 10 to a few hundred copies of HPV per beta-actin. The 6 patients with tumour sections with > or =190 HPV-16 copies/beta-actin remained tumour free (p = 0.026) and had a better survival rate (p = 0.039) when compared to the 5 patients with tumours sections with < or =60 HPV-16 copies/beta-actin. In conclusion, HPV-16 is mainly episomal in tonsillar cancer. The viral load showed a wide distribution and the clinical outcome in our study was better when the HPV load was higher.

Adult↗

PubMed2005

Improved HIV-1 viral load determination based on reverse transcriptase activity recovered from human plasma.

A more sensitive version of ExaVir Load, a test that utilizes reverse transcriptase (RT) activity from virions in plasma to determine HIV-1 viral load, is described. The virions were immobilized on a gel that was washed, followed by lysis of the virions, elution of purified RT, and finally RT activity determination. The changes made to the original test were: (1) improved washing of the immobilized virions by addition of a non-lytic detergent to the wash buffer, (2) improved virion lysis procedure, including changes in salt, detergent and pH, (3) the use of larger sample volumes in the RT assay, and (4) prolonged RT reaction time. The alterations gave a tenfold increased sensitivity compared to the original version. The correlation between RT load by the current test and RNA PCR was the same as previously (r=0.90). Using colorimetric product detection, the average detection limit in a panel of 262 patient plasma from Stockholm was 0.5 fg RT/ml, corresponding to approximately 170 RNA copies/ml. None of 54 HIV-1 RNA negative samples exhibited RT. The amount of RT load positive samples were 19% for samples containing 50-400 RNA, 71% for samples with 400-1,500, and 100% among samples with >8,000 copies/ml (according to Roche Amplicor). The sensitivity could be increased further using fluorimetric detection. In conclusion, the modifications of the test described result in an important increase in sensitivity. It can now be regarded as a competitive alternative method for HIV viral load determinations.

Fluorometry↗

PubMed2001

CD4 T cell recovery is slower in patients experiencing viral load rebounds during HAART.

To determine whether viral load rebounds during HAART impact on CD4+ T cell recovery and immune reconstitution, we studied a prospective cohort of 355 antiretroviral naive patients enrolled to be randomized in a trial of three strategies of induction/maintenance HAART. The extent of immune reconstitution in blood through 72 weeks of antiretroviral treatment was evaluated. Lymphocyte subset markers (CD4, CD8, CD45RA, CD62L, CD16, CD19), activation markers (HLA-DR, CD38, CD25) were performed by cytometry analysis. Our results showed that plasma HIV-1 RNA was suppressed to below 500 copies per ml through week 72 in 240 patients (group 1) while the remaining 115 patients experienced at least one viral rebound (group 2). At baseline, CD4 cell count was higher and HIV-1 RNA was lower in group 1 than in group 2. Over 72 weeks, mean increase in CD4+ T cell count was 0.32 cell/mm3/day in group 1 and only 0.14 cell/mm3/day in group 2 (P < 0.0001). However, the patterns of changes in CD4+ and CD8+ T cell subsets during therapy were very similar across the two groups with only subtle and very limited differences. We conclude that permanent control of HIV replication could be necessary for faster immune reconstitution.

Adult↗

PubMedDate not supplied

The expression of epidermal growth factor receptor, vascular endothelial growth factor, matrix metalloproteinase-2, and cyclooxygenase-2 in relation to human papilloma viral load and persistence of human papillomavirus after conization with negative margins.

The aim of this study was to investigate the correlations between human papillomavirus (HPV) load and vascular endothelial growth factor (VEGF), epidermal growth factor receptor (EGFR), matrix metalloproteinase-2 (MMP-2), and cyclooxygenase-2 (COX-2), and to identify biomarkers that may predict high-risk HPV clearance or persistence after conization with negative margins. The following samples were analyzed: 77 paraffin-embedded specimens from patients with cervical intraepithelial neoplasia (CIN), including 27 CIN 2 conization specimens and 50 CIN 3 conization specimens. Immunohistochemical analysis was performed with antibodies to VEGF, EGFR, MMP-2, and COX-2. Hybrid capture II testing was used to detect HPV DNA. VEGF expression was significantly associated with HPV load (rho = 0.27186, P = 0.0191), while COX-2 expression was significantly and inversely associated with HPV load (rho = -0.34309, P = 0.0028). In univariate analysis, HPV load (P = 0.0112) and VEGF expression (P = 0.0274) were significantly associated with high-risk HPV clearance or persistence after conization with negative margins. In multiple regression analysis, high viral load (relative light unit/positive control > 500) and positive VEGF expression were significantly associated with high-risk HPV persistence after conization with negative margins (odds ratio [OR]: 9.915, CI: 1.891-51.994; OR: 6.661, CI: 1.208-36.722, respectively). In conclusion, VEGF expression is related to HPV load, while COX-2 expression is inversely related to HPV load, and immunohistochemical analysis of VEGF expression and HPV viral load are a significant and an independent prognostic indicator of high-risk HPV persistence after conization with negative margins.

Adult↗