Acute and chronic immune biomarker changes during interferon/ribavirin treatment in HIV/HCV co-infected patients.

Chronic viral infections lead to persistent immune activation, which is alleviated by eradicating or suppressing the infection. To understand the effects of interferon treatment on immune system activation by chronic infections, we evaluated kinetic patterns of a broad spectrum of serum biomarkers during HCV treatment in HIV/HCV co-infected patients. HCV viral load and 50 biomarkers were analysed at baseline and 27 time points during pegylated interferon-alpha and ribavirin (IFN/RBV) treatment of 12 HIV/HCV co-infected patients. We evaluated biomarker changes from baseline for each time point and biomarker correlations with clinical parameters, treatment response and liver histopathology. IL-1α, IL-12p40, IL-1RA, IP-10, MIG, MIP-1α/1ß, HGF, sCD40L, TRAIL and leptin increased in the first day. IL-12p70, IL-17A, IL-10, GROα, IL-8, MCP-3, IL-4 and M-CSF peaked later during week 1. IL-1α, HGF, IP-10, MIP-1α, TRAIL, sCD40L, IL-10, IL-12p70, MCP-3, FGFb, ENA-78, TGF-ß, IL-2, IFN-γ, IL-6, IL-15, IL-7 and PDGF-BB decreased below baseline over the course of treatment. Higher BMI, baseline HCV viral load and leptin levels were associated with lack of sustained virologic response. ENA-78 was associated with sustained viral response. Positive correlations were found between liver inflammation and baseline CD4 count, sVCAM and HGF; fibrosis stage and HGF; liver steatosis, BMI and leptin. Our findings suggest IFN/RBV treatment initially increases levels of several biomarkers, but eventually leads to a decline in many immune markers. These findings shed light on the relationship between IFN treatment and immune activation by chronic viral infections, such as HCV.

Concurrent outbreaks with co-infection of norovirus and Clostridium difficile in a long-term-care facility.

We describe an outbreak of simultaneous Clostridium difficile and norovirus infections in a long-term-care facility. Thirty patients experienced acute gastroenteritis, and four had co-infection with identical C. difficile 027 and genotype II.4 New Orleans norovirus strains. Co-occurring infection requires improved understanding of risk factors, clinical impact, and testing strategies.

Predictive value of HIV-1 replication capacity and phenotypic susceptibility scores in antiretroviral treatment-experienced patients.

OBJECTIVES: The aim of the study was to determine the prognostic value of HIV replication capacity (RC) for subsequent antiretroviral (ARV) treatment response in ARV-experienced patients. METHODS: RC and phenotypic resistance testing were performed at baseline and week 12 on plasma samples from patients randomized to undergo a 12-week ARV drug-free period (ARDFP) or initiate immediate salvage therapy (no-ARDFP group) in the Options in Management with Antiretrovirals (OPTIMA) trial. Dichotomous and incremental phenotypic susceptibility scores (dPSSs and iPSSs, respectively) were calculated. The predictive value of RC and PSS for ARV therapy response and/or ARDFP was evaluated using multivariate regression analysis and Pearson correlations. RESULTS: In 146 no-ARDFP subjects, baseline RC (50.8%) did not change at week 12 and was not correlated with CD4 cell count or viral load changes at week 12 (P=0.33 and P=0.79, respectively) or at week 24 (P=0.96 and P=0.14, respectively). dPSS predicted virological but not CD4 cell count response to ARV therapy at weeks 12, 24 and 48 (P=0.002, P<0.001 and P=0.005, respectively). RC was significantly correlated with dPSS and iPSS at baseline, but did not increase their predictive value. In the 137 ARDFP patients, RC increased significantly (from 52.4 to 85.8%), but did not predict CD4 cell count and viral load changes during ARDFP (P=0.92 and P=0.26, respectively). RC after ARDFP did not predict subsequent CD4 cell count and viral load changes 12 weeks following ARV treatment reinitiation (P=0.90 and P=0.29, respectively). CONCLUSIONS: We found no additional predictive value of replication capacity for virological or immunological responses (above what PSS provides) in patients undergoing salvage ARV treatment.

HIV-HBV vaccine escape mutant infection with loss of HBV surface antibody and persistent HBV viremia on tenofovir/emtricitabine without antiviral resistance.

We report a case of acute hepatitis B virus genotype A vaccine escape mutant infection with loss of HBV vaccine-induced seropositivity in a HIV-1 infected patient. His HBV is unresponsive to tenofovir/emtricitabine treatment demonstrated by persistent viremia despite lacking known resistance mutations and while having an undetectable HIV-1 viral load.

Comparative effectiveness of dried plasma HIV-1 viral load testing in Brazil using ViveST for sample collection.

BACKGROUND: Utilization of dried plasma for HIV-1 viral load testing would significantly decrease sample shipping costs. OBJECTIVES: To describe the precision and reproducibility of ViveST(®) (ST) as a transportation method for shipping specimens for HIV-1 viral load (VL) testing. STUDY DESIGN: Thirty clinical plasma samples were used to generate replicate samples with HIV VL values of 4 log(10), 3 log(10) and 2 log(10) copies/mL for reproducibility testing and an additional 299 samples with HIV VL <50 copies/mL (99); 1.7 log(10) to 3.99 log(10) (100); and 4 log(10) to 5.99 log(10)/mL (100) were used to compare ViveST to frozen plasma samples using the VERSANT(®) HIV-1 RNA 3.0 Assay. Results were compared using Student t-test, Pearson correlation and Bland-Altman analyses. RESULTS: Mean intra-assay variance among frozen and dried plasma triplicates was 0.15 log(10) and 0.09 log(10) copies/mL respectively (n=10, P=NS). Compared to frozen plasma, there was a mean reduction of 0.3 log(10), 0.27 log(10), and 0.35 log(10) copies/mL at the 4 log(10), 3 log(10), and 2 log(10) copy/mL samples respectively (n=30, all comparisons, P<0.01). Overall correlation between 299 frozen and ViveST samples was r=0.97, where 12 of 99 undetectable frozen VL were positive with ST, and 12 of 200 frozen detectable VL were undetectable with ViveST (mean VL 2.1, 1.9 log(10) copies/mL respectively). CONCLUSIONS: HIV-1 viral load results using ViveST were reproducible, correlated well with frozen plasma, though yielding minimally lower values. Our data suggest that dried plasma for HIV-1 VL testing using ViveST has promise for use in HIV clinical practice.

Dried-plasma transport using a novel matrix and collection system for human immunodeficiency virus and hepatitis C virus virologic testing.

A novel method for the collection and transportation of dried-blood-plasma samples, SampleTanker (ST), was developed and compared to standard shipping protocols for frozen-plasma specimens containing human immunodeficiency virus type 1 (HIV-1) and/or hepatitis C virus (HCV). Matched frozen and dried 1-ml EDTA-containing plasma samples were collected and analyzed by several molecular-based virologic assays. After addition of 1.175 ml of reconstitution buffer, 1.035 ml of dried plasma was recovered. Mean intra-assay variances were 0.05, 0.05, and 0.06 log(10) copies/ml for the Versant, Amplicor, and NucliSens QT HIV-1 load assays, respectively (P, not significant). However, mean HIV-1 viral load was consistently reduced in dried samples by 0.32 to 0.51 log(10) copies/ml, depending on assay type (P < 0.05). Infectious HIV-1 was not recovered from dried ST plasma. There was no significant difference in HIV-1 viral load results obtained using ST after 8 weeks of storage at ambient temperature. Compared to frozen plasma, HIV-1 genotypic results were >99% concordant at the nucleotide and amino acid levels, as well as for resistance-associated mutations. We further demonstrated successful detection of multiple analytes, including HIV-1 viral load, HIV-1 antiretroviral resistance genotype, and HCV genotype, from a single ST unit. Dried plasma collected with ST yielded comparable results to frozen samples for multiple-analyte clinical testing. As such, ST could be a useful alternative for virologic tests and clinical trials worldwide by significantly diminishing transportation cost and the sample volume restrictions associated with dried-blood-spot technology.

Clinical utility of viral load measurements in individuals with chronic hepatitis C infection on antiviral therapy.

SUMMARY: Both absolute viral load and log decline in viral load from baseline were found clinically useful in predicting sustained virological response and lack of sustained virological response (non-sustained virological response, NSVR) to treatment. We assessed the clinical utility of hepatitis C virus (HCV) RNA quantitation and changes in viral load using the VERSANT HCV RNA 3.0 Assay (bDNA) in 351 HCV-infected individuals treated with interferon plus ribavirin. We show that viral load decision thresholds provided negative predictive values (NPVs) of >95% at week 4 using a 100 000 IU/mL cut-off and at weeks 8 and 12 using 10 000 IU/mL cut-offs. A 2-log decline from baseline provided NPVs >95% at weeks 8 and 12. Combinations of absolute viral loads and changes in viral load from baseline did not enhance the performance of the decision rules for predicting NSVR. The positive predictive values (PPVs) at weeks 8 and 12 were 59.1 and 67.3%. This study highlights the critical importance of viral quantitation in gauging therapeutic response in patients with chronic HCV infection on antiviral therapy. Early changes in viral load, measured as absolute viral loads or change in viral load from baseline, are highly predictive of NSVR at 8 and 12 weeks. PPVs are modest but these data may provide encouragement to patients who are in the early phases of treatment when side effects are frequent. Additionally, we demonstrated the need for cautious interpretation of stopping rules when the values are at or near the decision thresholds.

A vaccine to prevent herpes zoster and postherpetic neuralgia in older adults.

BACKGROUND: The incidence and severity of herpes zoster and postherpetic neuralgia increase with age in association with a progressive decline in cell-mediated immunity to varicella-zoster virus (VZV). We tested the hypothesis that vaccination against VZV would decrease the incidence, severity, or both of herpes zoster and postherpetic neuralgia among older adults. METHODS: We enrolled 38,546 adults 60 years of age or older in a randomized, double-blind, placebo-controlled trial of an investigational live attenuated Oka/Merck VZV vaccine ("zoster vaccine"). Herpes zoster was diagnosed according to clinical and laboratory criteria. The pain and discomfort associated with herpes zoster were measured repeatedly for six months. The primary end point was the burden of illness due to herpes zoster, a measure affected by the incidence, severity, and duration of the associated pain and discomfort. The secondary end point was the incidence of postherpetic neuralgia. RESULTS: More than 95 percent of the subjects continued in the study to its completion, with a median of 3.12 years of surveillance for herpes zoster. A total of 957 confirmed cases of herpes zoster (315 among vaccine recipients and 642 among placebo recipients) and 107 cases of postherpetic neuralgia (27 among vaccine recipients and 80 among placebo recipients) were included in the efficacy analysis. The use of the zoster vaccine reduced the burden of illness due to herpes zoster by 61.1 percent (P<0.001), reduced the incidence of postherpetic neuralgia by 66.5 percent (P<0.001), and reduced the incidence of herpes zoster by 51.3 percent (P<0.001). Reactions at the injection site were more frequent among vaccine recipients but were generally mild. CONCLUSIONS: The zoster vaccine markedly reduced morbidity from herpes zoster and postherpetic neuralgia among older adults.

Prevalence of antiretroviral drug resistance in the HIV-1-infected urban indigent population in San Francisco: a representative study.

We determined the prevalence of antiretroviral (ARV) resistance in HIV-1 infected indigent persons in San Francisco, California. Three hundred and twenty-seven subjects (159 (49%) ARV naïve, and 168 (51%) ARV-experienced), were recruited during 1996-97 and 1999-2000. Plasma HIV-1 viral load quantification and genotypic resistance testing were performed. Twice as many subjects received nucleoside reverse transcriptase inhibitors (NRTIs) as non-nucleoside reverse transcriptase inhibitors (NNRTIs) or protease inhibitors (PIs); resistance mutation prevalences were 30%, 14% and 16% respectively. Risk of any resistance mutations was strongly and independently associated with prior ARV exposure (OR = 1.3 per year of exposure, P < 0.0001) and with ARV exposure prior to HAART (OR = 2.5, P = 0.015). Prevalences of primary ARV resistance mutations among both treatment-naive and treatment-experienced subjects in this indigent urban population are low compared to other observational cohorts, are directly related to length and type of prior ARV exposure, and did not increase significantly between recruitment periods.

A pilot trial of indinavir, ritonavir, didanosine, and lamivudine in a once-daily four-drug regimen for HIV infection.

OBJECTIVE: To evaluate the tolerance, pharmacokinetics, and virologic and immunologic outcomes of once-daily indinavir, ritonavir, didanosine, and lamivudine in HIV-seropositive individuals. DESIGN: Open-label 24-week pilot study. PATIENTS: Ten HIV-seropositive subjects who were either antiretroviral-naive or minimally experienced with short-term single-or dual-nucleoside therapy provided informed consent and were enrolled. All subjects received didanosine (400 mg) 30 to 60 minutes before a meal followed by indinavir (1200 mg), ritonavir (400 mg), and lamivudine (300 mg) concurrent with the aforementioned meal. METHODS: Safety laboratory tests, including a complete blood cell count and amylase, lipase, liver transaminase, and nonfasting lipid monitoring as well as plasma HIV viral load and CD4+ lymphocyte count, were carried out at monthly intervals. Genotyping was performed at baseline. Pharmacokinetic studies for indinavir and ritonavir were performed at week 8. RESULTS: Nine of 10 subjects completed 24 weeks of therapy. No subject demonstrated primary protease inhibitor mutations at baseline. Toxicities experienced by subjects were typically mild and consistent with those commonly reported for each of the medications, including two cases of hematuria. By week 24, median nonfasting cholesterol and triglyceride levels increased by 49% and 108%, respectively. Median baseline plasma HIV viral load and CD4+ lymphocyte count were 29,292 (4.47 log10) copies/ml and 224 cells/mm3, respectively. Eight of 10 subjects had a plasma HIV viral load of <50 copies/ml by week 12. The 2 subjects with a detectable HIV viral load reached <50 copies/ml by week 28. Median CD4+ lymphocyte counts increased by 193 cells/mm3 at week 24. Indinavir and ritonavir plasma concentrations remained above respective inhibitory and effective concentrations (IC95 and EC50) (uncorrected for protein binding) throughout the 24-hour dosing interval for 6 of 10 and 8 of 10 subjects, respectively. CONCLUSIONS: Our pilot study demonstrates excellent virologic suppression despite low minimum protease inhibitor concentrations during a dosing interval in some patients and is supportive of further study.

Multinodular polymyositis in a patient with human immunodeficiency and hepatitis C virus coinfection.

We report a patient who developed multiple inflammatory muscle masses and generalized polymyositis in the setting of combined human immunodeficiency virus (HIV) and hepatitis C virus (HCV) infection. Magnetic resonance imaging (MRI) of muscles showed patchy edema which was particularly intense within the nodular masses. Polymerase chain reaction (PCR) showed no evidence of either virus within muscle. This report reviews earlier literature on muscle nodules associated with myositis and discusses the differential diagnosis of muscle masses in HIV infection.

Assessment of developmental toxicity of antiretroviral drugs using a rat whole embryo culture system.

BACKGROUND: Previous guidelines for HIV-infected pregnant women have recommended zidovudine (ZDV) monotherapy during the second and third trimesters of pregnancy to prevent fetal HIV infection. New guidelines suggest that women should continue or be offered combination antiretroviral therapy (including protease inhibitors) during pregnancy. Nevertheless, little animal or human toxicity data underlie these recommendations. METHODS: We used an in vitro rat whole embryo culture system to assess the embryo toxicity of various nucleoside analogues, namely, ZDV, dideoxyinosine (ddI), and 2', 3'-dideoxycytidine (ddC), and the HIV-1 protease inhibitor, indinavir, both alone and in combination. RESULTS: Although human fetal concentrations of these compounds are unknown, no gross abnormalities were detected after incubation with these agents, either alone or in combination at concentrations that would be expected to be achievable in human maternal serum (1-50 microM). ZDV in combination with ddC at >100 microM, resulted in severe growth retardation and morphologic abnormalities not seen with either agent singly. CONCLUSIONS: We conclude that the combination of ZDV/ddC results in severe concentration-dependent embryo toxicity. No growth retardation or gross morphologic abnormalities were found for any of the agents, either singly or in combination, at clinically relevant concentrations.

Adherence to protease inhibitors, HIV-1 viral load, and development of drug resistance in an indigent population.

OBJECTIVE: To examine the relationship between adherence, viral suppression and antiretroviral resistance in HIV-infected homeless and marginally housed people on protease inhibitor (PI) therapy. DESIGN AND SETTING: A cross-sectional analysis of subjects in an observational prospective cohort systematically sampled from free meal lines, homeless shelters and low-income, single-room occupancy (SRO) hotels. PARTICIPANTS: Thirty-four HIV-infected people with a median of 12 months of PI therapy. MAIN OUTCOMES: Adherence measured by periodic unannounced pill counts, electronic medication monitoring, and self-report; HIV RNA viral load; and HIV-1 genotypic changes associated with drug resistance. RESULTS: Median adherence was 89, 73, and 67% by self-report, pill count, and electronic medication monitor, respectively. Thirty-eight per cent of the population had over 90% adherence by pill count. Depending on the measure, adherence explained 36-65% of the variation in concurrent HIV RNA levels. The three adherence measures were closely related. Of 20 genotyped patients who received a new reverse transcriptase inhibitor (RTI) when starting a PI, three had primary protease gene substitutions. Of 12 genotyped patients who received a PI without a new RTI, six had primary protease gene substitutions (P < 0.03). CONCLUSION: A substantial proportion of homeless and marginally housed individuals had good adherence to PI therapy. A strong relationship was found between independent methods of measuring adherence and concurrent viral suppression. PI resistance was more closely related to the failure to change RTI when starting a PI than to the level of adherence.

Stability of plasma human immunodeficiency virus load in VACUTAINER PPT plasma preparation tubes during overnight shipment.

VACUTAINER PPT plasma preparation tubes were evaluated to determine the effects of various handling and shipping conditions on plasma human immunodeficiency virus (HIV) load determinations. Plasmas obtained from PPT tubes stored and shipped under nine different conditions were compared to conventional EDTA tube plasmas stored at -70 degrees C within 2 h after phlebotomy. Compared to viral loads in frozen EDTA plasma, those in PPT tube plasma that was frozen immediately and either separated or shipped in situ were not significantly different. Viral loads in PPT tube plasma after storage for 6 h at either room temperature or 4 degrees C, followed by shipment at ambient temperature or on wet or dry ice, were not significantly different from baseline viral loads in EDTA or PPT plasma. The results of this study indicate that the HIV load in PPT tube plasma is equivalent to that in standard EDTA plasma. Plasma viral load is not affected by storage or shipment temperature when plasma is collected in PPT tubes. Furthermore, plasmas can be shipped in spun PPT tubes, and the tubes provide a safer and more convenient method for sample collection and transport than regular EDTA tubes.

Decreased medical expenditures for care of HIV-seropositive patients. The impact of highly active antiretroviral therapy at a US Veterans Affairs Medical Center.

OBJECTIVE: To identify any changes in expenditures and in morbidity and mortality with the progression of treatment of the HIV-seropositive population from monotherapy with a nucleoside reverse transcriptase inhibitor (NRTI) [1993] through dual NRTI therapy (1995) to highly active antiretroviral therapy (HAART) [1997]. DESIGN AND SETTING: This study retrospectively compared 3 separate years of the total expenditures encountered in the management of HIV-seropositive individuals seen at a US Veterans Affairs Medical Center. INTERVENTIONS: Utilising a computerised hospital database, we identified those patients with HIV-related International Classification of Diseases, version 9 (ICD-9) codes and collected all healthcare-related expenditure data. The 3 eras selected for comparison were controlled for similar utilisation of prophylaxis against opportunistic infections, access to investigational antivirals, consistency between primary care providers and distribution of new anti-HIV therapies relative to that era. Cost data for inpatient and outpatient activities (visits and admissions) were derived from actual expenditures. Major categories were then compared, including total inpatient/outpatient expenditures and utilisation, laboratory and prescription costs, and morbidity and mortality rates. MAIN OUTCOME MEASURES AND RESULTS: The 3 periods had similar patient populations, with 86, 86 and 82% of patients in 1993, 1995 and 1997, respectively, having some degree of immunosuppression (defined as CD4+ lymphocyte counts < 500 cells/mm3). Morbidity and mortality were not changed by the addition of dual NRTI therapy. HAART therapy produced 60 and 70% declines in relative mortality when compared with the single and dual NRTI eras. Dual NRTI or HAART therapy decreased overall expenditures as compared with NRTI monotherapy. HIV-related outpatient resource utilisation other than pharmacy and laboratory costs fell by 25 and 59% in 1997 as compared with 1993 and 1995, respectively. The greatest fall in resource utilisation was for inpatient bed-days of care, where the average cost per patient fell by $US2782 between 1993 and 1997. Pharmacy and laboratory expenditures increased by $US1825 and $US231 per patient from 1993 to 1997, respectively. Overall, the impact of HAART was a decrease of $US1193 in the average total cost per patient from 1993 to 1997. CONCLUSIONS: The introduction of HAART provided a positive outcome on patient morbidity and mortality and on medical centre expenditures. The end result was a cost shift of expenditures from inpatient utilisation to outpatient pharmacy and laboratory costs. This information is important for patients and providers, who need to make clinical decisions on lifelong therapies, and for healthcare financial planners, who need to predict inpatient and outpatient healthcare utilisation during an era of limited healthcare dollars.

A double blind, randomized, placebo-controlled phase II study to assess the safety and efficacy of orally administered SP-303 for the symptomatic treatment of diarrhea in patients with AIDS.

OBJECTIVE: The aim of this study was to investigate the safety and effectiveness of orally administered SP-303 in patients with AIDS and diarrhea. METHODS: This is a multicenter, phase II, randomized, double blind, placebo-controlled study. HIV-positive subjects with a history of a CD4 count <200 or an AIDS-defining illness were admitted to an inpatient study unit and screened for diarrhea defined as at least three abnormal (i.e., soft or watery) stools and >200 g of abnormal stool weight over a 24-h period. Subjects discontinued all antidiarrheal agents >24 h before enrollment. Stool samples were studied for routine pathogens. Subjects received 500 mg p.o. of SP-303 or placebo every 6 h for 96 h (4 days). Stool frequency and weights were recorded. Subjects were monitored for symptoms and side effects and were seen 1 wk later in follow-up. RESULTS: A total of 26 subjects received SP-303, and 25 received placebo. There were no significant demographic differences between treatment arms. A total of 41 subjects (80%) were receiving antiretroviral therapy and 39 subjects (77%) were receiving at least one protease inhibitor. Stool studies revealed no pathogens in 48 of 51 patients (94%). There were no serious adverse events or laboratory abnormalities. The SP-303 treatment group demonstrated a mean reduction from baseline stool weight of 451 g/24 h versus 150 g/24 h with placebo on day 4 of treatment (p = 0.14), and a mean reduction in abnormal stool frequency of three abnormal stools in 24 h versus two in 24 h in the placebo group (p = 0.30). Daily measures analysis over 4 days of treatment demonstrated that SP-303 subjects had a significant reduction in stool weight (p = 0.008) and abnormal stool frequency (p = 0.04) when compared to placebo-treated subjects. CONCLUSIONS: SP-303 is safe and well tolerated. These results suggest that SP-303 may be effective in reducing stool weight and frequency in patients with AIDS and diarrhea.

Viral Load Monitoring in HIV Infection.

Measurement of HIV-1 viral load is now an accepted part of clinical practice for the determination of clinical prognosis and antiretroviral effectiveness in HIV infection. Consensus guidelines have been published on the appropriate use of this testing. Furthermore, recent advances in molecular technology have improved the sensitivity and reproducibility of viral load assays, and these improved assays have provided new insight into the pathogenesis of HIV disease. This article reviews new issues affecting viral load quantification, including viral subtypes, sex, compartmental differences, and other covariables.

Effects of collection, processing, and storage on RNA detection and quantification.

Historically, clinicians and researchers have relied upon the development of clinical endpoints or the use of surrogate markers in the evaluation of disease pathogenesis and in response to various therapeutic agents. In addition, microbiologic methods of detecting various pathogens have usually required the culture of an agent. The majority of bacterial pathogen culture methods have been standardized and identification has become relatively straightforward. Nonetheless, a wide variety of unculturable pathogens have been identified. Recent advances in molecular diagnostics have provided clinicians with the ability to measure directly infectious agents. Certain viral pathogens such as human immunodeficiency virus (HIV) are detectable by standard culture techniques whereas others such as hepatitis C virus (HCV) are not.

HIV quantitation in spiked vaginocervical secretions: lack of non-specific inhibitory factors. DATRI 005 Study Team. Division of AIDS Treatment Research Initiative.

The objective of this study was to assess the effect of menstrual phase on the ability to quantitate HIV-1 in vaginocervical secretions (VCS) through reconstruction experiments with HIV seronegative VCS collected throughout the menstrual cycle. Measurement of HIV-1 inoculated into both fresh and frozen VCS was undertaken by quantitative micro co-culture, p24 antigen assay and polymerase chain reaction (PCR) for both HIV-1 RNA and pro-viral DNA. Two laboratories carried out these assays over a range of viral concentrations. The study involved a randomized factorial design and the factors were: (1) diluents (phases of the menstrual cycle and controls); (2) laboratories; (3) stock concentrations; and (4) frozen versus fresh VCS samples. Each assay was assessed independently using a random effects analysis of variance (ANOVA) model. No statistical differences due to menstrual cycle were seen in the assay results of p24 antigen (P = 0.08), PBMC culture (P = 0.74), plasma culture (P = 0.13), cell-free RNA (P = 0.44), cell-associated RNA (P = 0.58) and cell-associated DNA (P = 0.43). Inter-laboratory differences were statistically significant for cell-free RNA (P < 0.001), cell-associated DNA (P < 0.001) and p24 (P < 0.001). It is concluded that VCS obtained throughout the menstrual cycle from HIV-uninfected women lacks intrinsic inhibitory factors which could limit detection and quantification by antigen, culture or nucleic acid-based technologies for HIV-1 in VCS throughout the menstrual cycle. Using a standardized collection procedure, we suggest that variation in HIV quantity over time, when reported in VCS of infected women, should be attributed to HIV-associated biologic factors, rather than non-specific or other technical factors.