Alanine aminotransferase levels are not significantly elevated in patients with HIV/HBV co-infection and lamivudine resistance.

In hepatitis B virus (HBV) monoinfection, alanine aminotransferase (ALT) levels are linearly correlated with HBV DNA levels and lamivudine resistance. In human immunodeficiency virus (HIV)/HBV co-infection, little is known about the association between ALT, HBV DNA, and lamivudine resistance. We assessed HBV DNA, lamivudine resistance and ALT levels in 45 time points in 11 patients with HIV/HBV co-infection during lamivudine-containing antiretroviral therapy. High HBV DNA levels (>10(6) copies/mL) and lamivudine resistance developed in 45% and 91% of patients, respectively. However, ALT levels were not elevated in the setting of high HBV DNA levels (mean ALT, 48 IU/mL) or lamivudine resistance (mean ALT, 44 IU/mL). HBV viraemia and lamivudine resistance during extended lamivudine-containing antiretroviral therapy are common in HIV/HBV co-infection, occurring in the absence of significant ALT elevations. In HIV/HBV co-infection, measurement of HBV DNA and HBV resistance mutations may identify HBV virological failure before biochemical changes and should be routinely used in the management of HIV/HBV co-infection.

A study of HIV RNA viral load in AIDS patients with bacterial pneumonia.

We examined the effect of bacterial pneumonia on the magnitude of circulating plasma HIV RNA in HIV-infected patients. Serum samples from 13 adult HIV-infected patients (median CD4 count = 83 cells/microl) were assayed for HIV RNA using the reverse transcriptase polymerase chain reaction assay (a) before bacterial pneumonia, (b) during the acute phase, and (c) after the recovery from the disease. Patients remained on constant antiretroviral therapy: HIV RNA was detected in all samples tested. The medians before, during, and after bacterial pneumonia were 60,000 copies per ml, 245,000 copies per ml, and 84,000 copies per ml, respectively. All 13 patients had increased HIV RNA levels on developing pneumonia. There was a decline in the level of HIV RNA with recovery from pneumonia in 12 of 13 patients. The difference between the HIV RNA levels before and after pneumonia was not significant, nor was there significant difference in the CD4 counts before and after pneumonia. In conclusion, bacterial pneumonia is associated with a consistent, transient increase in HIV RNA of variable magnitude in AIDS patients. Interpretation of HIV RNA changes for clinical management of AIDS patients must take into account this reversible elevation during infections.

Changes in virus load markers during AIDS-associated opportunistic diseases in human immunodeficiency virus-infected persons.

Human immunodeficiency virus (HIV) load markers are being used increasingly to monitor disease progression and evaluate antiretroviral therapy. This study examined plasma HIV RNA and p24 antigen levels before, during, and after 15 AIDS-associated opportunistic disease events in patients with AIDS (median CD4 cell count = 65/microL). Plasma HIV RNA was detected during 13 of the 15 events (median level before an event = 21,000 copies/mL). There was an increase in the level of plasma HIV RNA with the onset of an AIDS-associated opportunistic disease during 11 of 13 events for which HIV RNA was detectable (median level during an event = 145,000 copies/mL). There was a decline in the level of HIV RNA with the recovery from disease (median level after an event = 29,700 copies/mL). In contrast, there was no consistent or significant change in p24 antigen levels or CD4 cell counts with either the onset of or recovery from an event. Clinical interpretation of plasma HIV RNA changes must take into account this reversible elevation during AIDS-associated opportunistic disease.

Gender is not a factor in serum human immunodeficiency virus type 1 RNA levels in patients with viremia.

We investigated gender as a factor in viral load measurements for human immunodeficiency virus-infected patients. Forty antiretroviral-therapy-naive, age- and CD4-matched women and men were tested for serum RNA and p24 antigen levels prior to antiretroviral therapy and at approximately 12 weeks after therapy. No gender differences were observed for these two markers of viral load.

Comparison of HIV type 1 RNA plasma viremia, p24 antigenemia, and unintegrated DNA as viral load markers in pediatric patients.

Better surrogate markers need to be developed to evaluate therapy in HIV-infected children. This study evaluated plasma RNA, immune complex-dissociated p24 antigenemia, and unintegrated DNA (uDNA) in HIV-infected pediatric patients. Ten children were followed from initiation of nucleoside antiretroviral therapy at intervals up to 24 months. Prior to initiation of therapy, HIV RNA was detected in 10 of 10 patients (median, 76,000 Eq/ml), p24 antigen was detected in 8 of 10 patients (median, 193 pg/ml), and uDNA was detected in 6 of 7 patients (median, 10% uDNA). After 12 months the RNA decreased in all patients and became undetectable in six. In contrast, p24 antigenemia decreased in 6 of 10 patients, remained undetectable in 1, and increased in 3. HIV uDNA decreased in six of six patients and became undetectable in three. There was no overall change in CD4 cell count. Plasma RNA and uDNA levels are both sensitive markers of nucleoside therapy in children; however, they do not covary strongly.

Antiretroviral therapy is associated with a decrease in unintegrated HIV-1 DNA in pediatric patients.

Good markers for monitoring the efficacy of antiretroviral therapy in children do not currently exist. This study examined the effect of antiretroviral therapy on human immunodeficiency virus (HIV-1) unintegrated DNA (uDNA), integrated DNA (iDNA), percent uDNA, immune complex dissociated (ICD) p24 antigenemia, and plasma viral titer. Seven children were followed at therapy initiation and at approximately 3- and 10-month intervals. HIV-1 uDNA was detected in all children prior to start of therapy (average percent uDNA, 43%). At 3 months, the percent HIV uDNA decreased in all patients to an average of 18% (p = 0.01) and at 10 months decreased to an average of 1%. In contrast, the amount of HIV iDNA was relatively constant after initiation of therapy. ICD HIV p24 antigen was detected in all patients prior to therapy (average, 538 pg/ml). Over the study period, the ICD p24 antigen level decreased in three patients and remained relatively unchanged in four patients. Plasma cultures of HIV-1 were positive in only one of the seven patients prior to therapy. Among the methods evaluated, measurement of uDNA was the only parameter which reliable decreased after initiation of nucleoside therapy.

Rapid decrease in unintegrated human immunodeficiency virus DNA after the initiation of nucleoside therapy.

Better markers for determining therapeutic efficacy of antiretroviral drugs are needed for human immunodeficiency virus (HIV) infection. The amounts of unintegrated HIV DNA (uDNA) were sequentially determined in peripheral blood mononuclear cells (PBMC) from 20 HIV-infected patients starting nucleoside therapy. HIV copy number was determined using a quantitative polymerase chain reaction assay. Before therapy, 19 of 20 patients had detectable HIV uDNA. The average percentage of uDNA was 42%. After 1, 4, and 8 weeks of nucleoside therapy the average decreased to 23% (P < .001), 7%, and 3%, respectively. The amount of HIV uDNA decreased in all 19 patients during the first week and was undetectable in 14 by 8 weeks. Thus, measurement of HIV uDNA has many characteristics needed for a good marker of therapeutic efficacy of antiretroviral drugs, including detectability in a high proportion of patients, large and rapid response to initiation of therapy, and a biologically plausible mechanism.

Parathyroid hormone transport effects and hormonal processing in primary cultured rat proximal tubular cells.

The development of satisfactory cell culture models for the study of parathyroid hormone (PTH)-induced inhibition of Pi transport has proven difficult. Using subcellular fractionation techniques we investigated the response of primary cultures of rat proximal tubular cells to PTH-(1-34). Specific binding of 125I-bPTH-(1-34) occurred at 2 degrees C. After 5 min of rewarming, trypsin-releasable radioactivity decreased from 90 to 50%, indicating internalization of the ligand. Cell disruption, followed by density centrifugation with 17% Percoll either directly after binding at 2 degrees C or post-rewarming for 20 min, showed a shift of 125I label from the plasma membrane (5'-nucleotidase) to lysosomal fractions (beta-D-glucosaminidase), confirming the sequential occurrence of cell surface binding, internalization and transport to lysosomes of 125I-bPTH-(1-34). Reculture at 37 degrees C revealed steady accumulation of trichloroacetic acid-soluble radioactivity in the medium, indicating degradation of 125I-bPTH-(1-34). Phosphate transport in the absence of sodium was minimal. Incubation of the cells with bPTH-(1-34) resulted in up to 50% inhibition of sodium-dependent phosphate transport. Prior phosphate depletion abrogated the response to PTH.

Increased detection of human immunodeficiency virus antigenemia after dissociation of immune complexes at low pH.

This study compared the number of patients with detectable human immunodeficiency virus (HIV) antigenemia after immune complex (IC) dissociation by established methods using either 0.5 NCl or 1.5 M glycine buffer. Without IC dissociation, HIV antigen was detected in 43% of patients. After dissociation, the HCl method detected only an additional 7% of patients (P = 0.09), while the glycine method detected an additional 34% (P < 0.001). However, care must be taken in setting the threshold of the standards, and confirmatory neutralization assays should be performed to ensure specificity of HIV antigen enzyme immunoassay after IC dissociation.

Quantitation of unintegrated HIV-1 DNA in asymptomatic patients in the presence or absence of antiretroviral therapy.

The objective of this work was to determine the amount of unintegrated human immunodeficiency virus (HIV) DNA (HIV uDNA) in asymptomatic individuals in the presence or absence of antiretroviral therapy. Twenty-one healthy seropositive individuals with no history of any opportunistic infection or previous use of nucleoside antiretrovirals, and 9 similarly asymptomatic individuals who had initiated nucleoside antiretroviral therapy within the last 24 months were studied. All patients had CD4 lymphocyte counts above 400/microliters. All subjects administered antiretrovirals received 400-600 mg of zidovudine daily for 2-24 months. Two individuals additionally received 400 mg of dideoxyinosine (ddI) daily for 4 and 5 months. Patient peripheral blood mononuclear cells (PBMCs) were examined for integrated and unintegrated HIV DNA by a quantitative PCR assay. In addition, CD4 counts were measured, and free and immune complex dissociated p24 antigen was detected in plasma by ELISA. The mean percentage of HIV uDNA in asymptomatic individuals not on therapy was 59%, with 95% confidence limits from 50 to 69%. In contrast, patients on therapy had a mean of only 13% HIV uDNA, with confidence limits from 2 to 25% (p < 0.001). These findings indicate that a significant amount of HIV DNA in infected, healthy patients not on therapy is in the unintegrated form, and that the amount of HIV uDNA in asymptomatic patients on nucleoside therapy is much less. The amount of HIV uDNA in PBMCs deserves further study as a new marker of the efficacy of antiretroviral therapy.

Renal transplantation in patients 65 years old or older.

Between January 1982 and August 1989, cadaveric renal transplantation was performed in 22 patients 65 years old or older. Mean recipient age was 68 years (range 65 to 73 years). There were 17 men and 5 women. Additional risk factors included retransplantation (3 patients), high (greater than 30%) panel reactive antibody (4) and diabetes (1). All patients received cyclosporine as part of the immunosuppressive regimen. The 3-year actuarial patient and allograft survival rates were 89% and 71%, respectively. There were 6 graft losses due to chronic rejection (2 patients), renal vein thrombosis (1), myocardial infarction (1), withdrawal of immunosuppression because of sepsis (1) and primary nonfunction (1). Of the 16 patients with a functioning graft 12 currently have a serum creatinine of less than 2.0 mg./dl. These results suggest that cadaveric renal transplantation is an acceptable form of treatment for patients older than 65 years with end stage renal disease.

Detection of human immunodeficiency virus type 1 RNA in plasma samples from high-risk pediatric patients by using the self-sustained sequence replication reaction.

There is an urgent need for rapid and sensitive methods to assess human immunodeficiency virus (HIV) infection in infants and children. We evaluated an approach by using the self-sustained sequence replication reaction (3SR) to amplify HIV type 1 (HIV-1) RNA directly. The amplified RNA product was then detected by bead-based sandwich oligonucleotide capture hybridization and rare earth metal chelate time-resolved fluorescence. The sensitivity of this technology was determined to be less than 12 HIV-1 RNA copies with an amplification level of 10(10)-fold with purified HIV-1 RNA. Plasma samples from 19 high-risk pediatric patients younger than 5 years of age were examined, and results were compared with viral culture of patient plasma. Results from plasma culture and 3SR amplification agreed for 14 of these patients and disagreed for 5. Of the five samples which did not agree, four were positive by 3SR and negative by culture and one was positive by culture and negative by 3SR but became positive by 3SR at a subsequent testing. We conclude that 3SR amplification coupled with time-resolved fluorescence is a promising technology for investigating the relationship between the presence of HIV-1 RNA in plasma and progression of disease in HIV-infected pediatric patients. This technology should be important in the assessment of HIV-1 infection, in evaluating drug therapies, and in understanding the pathogenesis and transmission of the virus.

Preservation of renal function by percutaneous renal angioplasty in high-risk elderly patients: short-term outcome.

In 17 elderly patients, 19 angioplasties (17 nonostial, 2 ostial) were performed to treat acute decreases in renal function caused by high-grade renal artery stenosis in patients considered to be high-risk surgical candidates. Seventeen angioplasties (percutaneous transluminal renal angioplasty, PTRA) were technically successful and 7 patients showed improved renal function, as reflected by a fall in mean serum creatinine from 566 to 180 mumol/l (6.4 to 2.1 mg/dl). Four others had stabilization of function and 3 out of 4 with acute oliguria improved. Complications included femoral hematoma (4), minor peripheral embolism (3), renal artery thrombosis (1) renal artery dissection (1). One fatal complication was thrombosis of the aortic bifurcation due to catheterization. Four other patients died of cardiovascular causes unrelated to PTRA. Eleven patients experienced stabilization or improvement in renal function, but five out of six PTRA failures required maintenance hemodialysis and died in the hospital. Percutaneous transluminal angioplasty may offer the best change of favorable outcome in selected severely ill elderly patients with uremia, hypertension and renal artery stenosis.

Decreases in unintegrated HIV DNA are associated with antiretroviral therapy in AIDS patients.

Better markers are needed to monitor the efficacy of antiretroviral drugs in persons infected with human immunodeficiency virus (HIV). We investigated the effects of zidovudine (ZDV) and dideoxycytidine (ddC) on the presence of unintegrated HIV-1 DNA in peripheral blood mononuclear cells (PBMCs) from AIDS patients. DNA was extracted from PBMCs and separated into low molecular weight (unintegrated) and high molecular weight (integrated) chromosomal fractions. These DNA fractions were then amplified by a quantitative polymerase chain reaction (PCR) and the amount and percentage of unintegrated HIV DNA were determined. Very high levels of unintegrated HIV DNA were found in AIDS patients not receiving treatment with ZDV or ddC (median = 95% unintegrated HIV DNA). In contrast, most patients who had received 4 or more weeks of antiretroviral therapy had lower levels of unintegrated HIV DNA (median = 30% unintegrated HIV DNA for patients receiving ZDV). Paired samples taken from five patients before and after therapy showed a striking reduction in the percentage of unintegrated HIV DNA. The decrease in the proportion of unintegrated HIV DNA in AIDS patients was due to both a reduction in the copy number of unintegrated HIV DNA and an increase in the copy number of integrated HIV DNA. Thus, measurements of unintegrated and integrated HIV DNA may be useful in providing objective assessments of the effectiveness of antiretroviral therapies.

Human immunodeficiency virus infection of enterocytes and mononuclear cells in human jejunal mucosa.

Intestinal malabsorption is a recognized cause of malnutrition in patients infected with human immunodeficiency virus. However, the relationships among human immunodeficiency virus infection, morphological changes in the intestine, and development of intestinal malabsorption are not well established. Nine patients infected with human immunodeficiency virus underwent tests of intestinal absorption and jejunal biopsies for morphometric measurements, enzyme assays, and virus detection by in situ hybridization. Steatorrhea and low lactase activities were found in more than 85% of the patients. All biopsy specimens were abnormal with reversal of the ratio of villus length to crypt depth in seven and enlarged enterocyte nuclear size in nine. Human immunodeficiency virus was detected in five jejunal biopsy specimens, within villus enterocytes of one patient who had the most severe malabsorption of the group and in four other biopsy specimens in mononuclear infiltrating cells of the lamina propria. These results suggest that human immunodeficiency virus infection of the small intestinal mucosa is an early event that is associated with altered enterocyte differentiation and function.

HIV-1 proviral copy number in blood mononuclear cells from AIDS patients on zidovudine therapy.

The effect of treatment with 400-1,200 mg/day of zidovudine (ZDV) on HIV DNA concentrations in patient peripheral blood mononuclear cells (PBMCs) was studied in six patients during a 5- to 14-month period of therapy. HIV DNA was measured in PBMCs at intervals using a recently developed quantitative polymerase chain reaction assay. The amount of HIV DNA ranged from 2,000 to 40,000 copies of provirus per microgram of cellular DNA. The HIV provirus copy number showed little change with time in five patients, and increased and then remained constant in one patient. Thus, prolonged treatment with ZDV does not decrease the levels of HIV DNA in PBMCs.

Laboratory tests used in diagnosis and treatment of AIDS.

Detailed knowledge of the structure of the human immunodeficiency virus (HIV) triggered a rapid development of methods for diagnosing the infection. The enzyme-linked immunoadsorbent assay (ELISA) determining the presence of antibodies to the protein components of the virus in toto is highly sensitive and provides thus the basic screening approach. It is however somewhat less specific and therefore all positive results are verified by the Western blot method which detects individual HIV proteins and possesses a 99.9% specificity. In sporadic cases the latent period between HIV infection and the possibility to establish antibody response may extend to six months and even longer. The polymerase chain reaction (PCR) revealing the presence of DNA HIV in infected cells is suitable for detecting these seronegative patients. It is also the method of choice in diagnosing HIV infection in children of infected mothers since HIV antibodies are in newborns mostly of maternal origin. HIV antibodies, particularly the antigen p 24, can be quantified in serum by using ELISA. As their amounts correlate with the severity of the disease determination of antigen p 24 concentration is an indicator of the therapeutic efficacy of preparations such as AZT. The recently developed quantitative PCR is a further potentially valuable method for assessing the therapeutic effect since treatment should reduce the amount of cellular HIV DNA. With the exception of testing the sensitivity of HIV strains to antiretrovirus preparations, HIV cultures are rarely set up since they are technically demanding and involve a considerable risk.

Quantitation of human immunodeficiency virus DNA by using the polymerase chain reaction.

The polymerase chain reaction was used to measure the DNA copy number of human immunodeficiency virus (HIV). Differences in polymerase chain reaction amplification efficiency were controlled by amplifying known amounts of HIV DNA in parallel with samples. This technique is a sensitive, accurate, and reproducible method for the quantitation of HIV DNA.

Detection of human immunodeficiency virus in infected CEM cells using fluorescent DNA probes and a laser-based computerized image cytofluorometry system.

Quantitative nonradioactive methods to measure the human immunodeficiency virus (HIV) in individually infected cells are needed for the direct assessment of HIV infection, for the evaluation of antiviral chemotherapy and for testing the efficacy of vaccines. As a first step in accomplishing this goal, we built an argon ion laser-based computerized image cytofluorometry (ALCIC) system and determined this instrument's ability to quantitatively measure HIV nucleotides in infected lymphocytes. ALCIC consisted of a 43-mW argon ion laser connected to a Zeiss Universal microscope via a fiberoptic cable, a charged coupled-device video camera, a video frame grabber and array processor and a Micro Vax II computer using computer programs written in FORTRAN. HIV RNA and DNA were detected in infected CEM lymphocytes in culture by in situ hybridization using acetylaminofluorene (AAF)-labeled HIV-DNA probes, a rabbit anti-AAF antibody and a fluorescein-labeled goat anti-rabbit antibody. ALCIC measurements showed that 61% of the CEM cells were infected and that quantitative differences were distinguishable within this group. The levels of fluorescein isothiocyanate (FITC) fluorescence were sixfold or more greater than that observed with the same system using a 100-W mercury lamp for illumination; the improved intensity of the laser-based system is due to greater excitation intensity of the laser and the matching of the excitation spectrum to the peak wavelength of FITC. ALCIC has potential clinical value for determining the effect of antiviral agents on HIV infection and for assessing the susceptibility of different cell types to infection.