Evaluation of Performance Characteristics of the Aptima CMV Quant Assay for the Detection and Quantitation of CMV DNA in Plasma Samples.

Quantification of Cytomegalovirus (CMV) DNA has become the standard of care in the diagnosis and management of CMV infection in transplant recipients. The objective of the study was to evaluate performance characteristics of the Aptima CMV Quant assay in comparison to Abbott RealTime CMV assay, Qiagen Artus CMV RGQ MDx assay, and Roche cobas CMV test using plasma samples. The performance of the Aptima assay was evaluated by comparing the Exact Diagnostics CMV verification panel and positive controls, Hologic CMV internal reproducibility panel, and SeraCare CMV DNA qualification panel to the RealTime assay. Clinical agreement was evaluated using 389 clinical plasma samples comparing the Aptima assay to three comparator assays. The Aptima assay demonstrated good linearity and strong linear correlation between the assays (R2 = 0.99); the intra- and interassay reproducibility was excellent overall (SD = 0.09 to 0.14 and SD = 0.04 to 0.14, respectively); 95% limit of detection (LOD) is 32 IU/mL and LOQ is 45 IU/mL. The SeraCare qualification panel yielded a strong linear correlation (R2 = 0.99). A total of 262 positive samples were analyzed to compare Aptima and Realtime assays using Deming regression and Bland-Altman analysis and demonstrated a mean bias of 0.092 Log10 IU/mL. Artus (85) and cobas (159) positive samples were compared to the Aptima assay using Deming regression and Bland-Altman analyses and showed mean bias of 0.184 and -0.208 Log10 IU/mL, respectively. The findings demonstrate that the Aptima assay is sensitive and accurate in quantifying CMV in plasma specimens on the fully automated Panther system and that the results were comparable to the other FDA-approved CMV assays.

Concordance of SARS-CoV-2 Results in Self-collected Nasal Swabs vs Swabs Collected by Health Care Workers in Children and Adolescents.

Importance: Despite the expansion of SARS-CoV-2 testing, available tests have not received Emergency Use Authorization for performance with self-collected anterior nares (nasal) swabs from children younger than 14 years because the effect of pediatric self-swabbing on SARS-CoV-2 test sensitivity is unknown. Objective: To characterize the ability of school-aged children to self-collect nasal swabs for SARS-CoV-2 testing compared with collection by health care workers. Design, Setting, and Participants: Cross-sectional study of 197 symptomatic children and adolescents aged 4 to 14 years old. Individuals were recruited based on results of testing in the Children's Healthcare of Atlanta system from July to August 2021. Exposures: Children and adolescents were given instructional material consisting of a short instructional video and a handout with written and visual steps for self-swab collection. Participants first provided a self-collected nasal swab. Health care workers then collected a second specimen. Main Outcomes and Measures: The primary outcome was SARS-CoV-2 detection and relative quantitation by cycle threshold (Ct) in self- vs health care worker-collected nasal swabs when tested with a real-time reverse transcriptase-polymerase chain reaction test with Emergency Use Authorization. Results: Among the study participants, 108 of 194 (55.7%) were male and the median age was 9 years (IQR, 6-11). Of the 196 participants, 87 (44.4%) tested positive for SARS-CoV-2 and 105 (53.6%) tested negative by both self- and health care worker-collected swabs. Two children tested positive by self- or health care worker-collected swab alone; 1 child had an invalid health care worker swab. Compared with health care worker-collected swabs, self-collected swabs had 97.8% (95% CI, 94.7%-100.0%) and 98.1% (95% CI, 95.6%-100.0%) positive and negative percent agreement, respectively, and SARS-CoV-2 Ct values did not differ significantly between groups (mean [SD] Ct, self-swab: 26.7 [5.4] vs health care worker swab: 26.3 [6.0]; P = .65). Conclusions and Relevance: After hearing and seeing simple instructional materials, children and adolescents aged 4 to 14 years self-collected nasal swabs that closely agreed on SARS-CoV-2 detection with swabs collected by health care workers.

Bioaerosol Sampling for Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) in a Referral Center with Critically Ill Coronavirus Disease 2019 (COVID-19) Patients March-May 2020.

BACKGROUND: Previous research has shown that rooms of patients with coronavirus disease 2019 (COVID-19) present the potential for healthcare-associated transmission through aerosols containing severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). However, data on the presence of these aerosols outside of patient rooms are limited. We investigated whether virus-containing aerosols were present in nursing stations and patient room hallways in a referral center with critically ill COVID-19 patients. METHODS: Eight National Institute for Occupational Safety and Health BC 251 2-stage cyclone samplers were set up throughout 6 units, including nursing stations and visitor corridors in intensive care units and general medical units, for 6 h each sampling period. Samplers were placed on tripods which held 2 samplers positioned 102 cm and 152 cm above the floor. Units were sampled for 3 days. Extracted samples underwent reverse transcription polymerase chain reaction for selected gene regions of the SARS-CoV-2 virus nucleocapsid and the housekeeping gene human RNase P as an internal control. RESULTS: The units sampled varied in the number of laboratory-confirmed COVID-19 patients present on the days of sampling. Some of the units included patient rooms under negative pressure, while most were maintained at a neutral pressure. Of 528 aerosol samples collected, none were positive for SARS-CoV-2 RNA by the estimated limit of detection of 8 viral copies/m3 of air. CONCLUSIONS: Aerosolized SARS-CoV-2 outside of patient rooms was undetectable. While healthcare personnel should avoid unmasked close contact with each other, these findings may provide reassurance for the use of alternatives to tight-fitting respirators in areas outside of patient rooms during the current pandemic.

Single-Amplicon Multiplex Real-Time Reverse Transcription-PCR with Tiled Probes To Detect SARS-CoV-2 spike Mutations Associated with Variants of Concern.

To provide an accessible and inexpensive method to surveil for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mutations, we developed a multiplex real-time reverse transcription-PCR (rRT-PCR) assay, the Spike single-nucleotide polymorphism (SNP) assay, to detect specific mutations in the spike receptor binding domain. A single primer pair was designed to amplify a 348-bp region of spike, and probes were initially designed to detect K417, E484K, and N501Y. The assay was evaluated using characterized variant sample pools and residual nasopharyngeal samples. Variant calls were confirmed by SARS-CoV-2 genome sequencing in a subset of samples. Subsequently, a fourth probe was designed to detect L452R. The lower limit of 95% detection was 2.46 to 2.48 log10 genome equivalents (GE)/ml for the three initial targets (∼1 to 2 GE/reaction). Among 253 residual nasopharyngeal swabs with detectable SARS-CoV-2 RNA, the Spike SNP assay was positive in 238 (94.1%) samples. All 220 samples with threshold cycle (CT) values of <30 for the SARS-CoV-2 N2 target were detected, whereas 18/33 samples with N2 CT values of ≥30 were detected. Spike SNP results were confirmed by sequencing in 50/50 samples (100%). Addition of the 452R probe did not affect performance for the original targets. The Spike SNP assay accurately identifies SARS-CoV-2 mutations in the receptor binding domain, and it can be quickly modified to detect new mutations that emerge.

Metagenomic Sequencing To Detect Respiratory Viruses in Persons under Investigation for COVID-19.

Broad testing for respiratory viruses among persons under investigation (PUIs) for SARS-CoV-2 has been performed inconsistently, limiting our understanding of alternative viral infections and coinfections in these patients. RNA metagenomic next-generation sequencing (mNGS) offers an agnostic tool for the detection of both SARS-CoV-2 and other RNA respiratory viruses in PUIs. Here, we used RNA mNGS to assess the frequencies of alternative viral infections in SARS-CoV-2 RT-PCR-negative PUIs (n = 30) and viral coinfections in SARS-CoV-2 RT-PCR-positive PUIs (n = 45). mNGS identified all viruses detected by routine clinical testing (influenza A [n = 3], human metapneumovirus [n = 2], and human coronavirus OC43 [n = 2], and human coronavirus HKU1 [n = 1]). mNGS also identified both coinfections (1, 2.2%) and alternative viral infections (4, 13.3%) that were not detected by routine clinical workup (respiratory syncytial virus [n = 3], human metapneumovirus [n = 1], and human coronavirus NL63 [n = 1]). Among SARS-CoV-2 RT-PCR-positive PUIs, lower cycle threshold (CT ) values correlated with greater SARS-CoV-2 read recovery by mNGS (R2, 0.65; P < 0.001). Our results suggest that current broad-spectrum molecular testing algorithms identify most respiratory viral infections among SARS-CoV-2 PUIs, when available and implemented consistently.

Evaluation of Performance Characteristics of Panther Fusion Assays for Detection of Respiratory Viruses from Nasopharyngeal and Lower Respiratory Tract Specimens.

Accurate and rapid diagnosis is needed for timely intervention and clinical management of acute respiratory infections. This study evaluated performance characteristics of the Panther Fusion assay for the detection of influenza A virus (Flu A), influenza B virus (Flu B), respiratory syncytial virus (RSV), parainfluenza viruses 1 to 3 (Para 1 to 3), human metapneumovirus (hMPV), rhinovirus (RV), and adenovirus (Adeno) targets in comparison to those of the eSensor and Lyra assays using 395 nasopharyngeal (NP) and 104 lower respiratory tract (LRT) specimens. Based on the consensus positive result established (positive result in 2 of the 3 assays), the NP specimens for the Fusion and eSensor assays had 100% positive percent agreement (PPA) for all the analytes and the Lyra assays had 100% PPA for Flu A and Adeno analytes. A 100% negative percent agreement (NPA) was observed for all the Lyra analytes, whereas those for the Fusion targets ranged from 98.4 to 100% and those for the eSensor ranged from 99.4 to 100% for all the analytes except RV. For the LRT specimens, Fusion had 100% PPA and 100% NPA for all the targets except hMPV. There was a 100% PPA for eSensor analytes; the NPA ranged from 98 to 100%, except for RV. For the Lyra assays, the PPA ranged between 50 and 100%, while the NPA was 100% for all the targets except Adeno. The Fusion assay performed similarly to the eSensor assay for majority of the targets tested and provides laboratories with a fully automated random-access system to test for a broad array of viral respiratory pathogens.

Fecal microbiota transplantation promotes reduction of antimicrobial resistance by strain replacement.

Multidrug-resistant organism (MDRO) colonization is a fundamental challenge in antimicrobial resistance. Limited studies have shown that fecal microbiota transplantation (FMT) can reduce MDRO colonization, but its mechanisms are poorly understood. We conducted a randomized, controlled trial of FMT for MDRO decolonization in renal transplant recipients called PREMIX (NCT02922816). Eleven participants were enrolled and randomized 1:1 to FMT or an observation period followed by delayed FMT if stool cultures were MDRO positive at day 36. Participants who were MDRO positive after one FMT were treated with a second FMT. At last visit, eight of nine patients who completed all treatments were MDRO culture negative. FMT-treated participants had longer time to recurrent MDRO infection versus PREMIX-eligible controls who were not treated with FMT. Key taxa (Akkermansia muciniphila, Alistipes putredinis, Phocaeicola dorei, Phascolarctobacterium faecium, Alistipes species, Mesosutterella massiliensis, Barnesiella intestinihominis, and Faecalibacterium prausnitzii) from the single feces donor used in the study that engrafted in recipients and metabolites such as short-chain fatty acids and bile acids in FMT-responding participants uncovered leads for rational microbiome therapeutic and diagnostic development. Metagenomic analyses revealed a previously unobserved mechanism of MDRO eradication by conspecific strain competition in an FMT-treated subset. Susceptible Enterobacterales strains that replaced baseline extended-spectrum ß-lactamase-producing strains were not detectable in donor microbiota manufactured as FMT doses but in one case were detectable in the recipient before FMT. These data suggest that FMT may provide a path to exploit strain competition to reduce MDRO colonization.

Effect of trichomoniasis therapy on genital HIV viral burden among African women.

BACKGROUND: Our objective was to test the hypothesis that treatment for trichomoniasis among HIV-infected women not taking antiretrovirals in South Africa would be associated with decreased HIV genital shedding. METHODS: HIV-infected women presenting for routine HIV care were screened for trichomoniasis using self-collected vaginal swabs with a rapid point-of-care immunochromatographic antigen test. Women testing positive were offered enrollment into a prospective cohort study, if they had documented HIV infection, were aged 18 to 50 years, and were not receiving antiretroviral therapy. Recent use of postexposure prophylaxis or antibiotic therapy, active genital ulcers, or systemic illness were exclusion criteria. Cervical swabs were collected for gonococcal and chlamydial testing, and those testing positive were excluded. Women were treated with directly observed oral therapy with 2 g of oral metronidazole. A follow-up visit was scheduled 1 month after therapy, and partner letters were provided. Paired cervical wicks and plasma were collected for viral load measurement. RESULTS: In all, 557 women were screened. Sixty tested positive for trichomoniasis, 10 subsequently met exclusion criteria, and 4 were lost to follow-up. Of 46 women evaluated at follow-up, 37 (80.4%) were cured. Plasma viral load was not significantly different after therapy (P = 0.93). Genital tract viral load decreased by 0.5 log10 (P < 0.01). The mean genital tract viral load (log10) decreased from 4.66 (<3.52-6.46) to 4.18 (<3.52-6.48) (P < 0.01) after therapy. CONCLUSIONS: Screening and treatment of vaginal trichomoniasis decrease genital shedding of HIV among South African women not receiving antiretrovirals at 1 month after therapy.

Unrecognized introductions of SARS-CoV-2 into the US state of Georgia shaped the early epidemic.

In early 2020, as diagnostic and surveillance responses for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) ramped up, attention focused primarily on returning international travelers. Here, we build on existing studies characterizing early patterns of SARS-CoV-2 spread within the USA by analyzing detailed clinical, molecular, and viral genomic data from the state of Georgia through March 2020. We find evidence for multiple early introductions into Georgia, despite relatively sparse sampling. Most sampled sequences likely stemmed from a single or small number of introductions from Asia three weeks prior to the state's first detected infection. Our analysis of sequences from domestic travelers demonstrates widespread circulation of closely related viruses in multiple US states by the end of March 2020. Our findings indicate that the exclusive focus on identifying SARS-CoV-2 in returning international travelers early in the pandemic may have led to a failure to recognize locally circulating infections for several weeks and point toward a critical need for implementing rapid, broadly targeted surveillance efforts for future pandemics.

Association of Secretor Status and Recent Norovirus Infection With Gut Microbiome Diversity Metrics in a Veterans Affairs Population.

Norovirus infection causing acute gastroenteritis could lead to adverse effects on the gut microbiome. We assessed the association of microbiome diversity with norovirus infection and secretor status in patients from Veterans Affairs medical centers. Alpha diversity metrics were lower among patients with acute gastroenteritis but were similar for other comparisons.

Characterization of 2009 pandemic influenza A (H1N1) virus specimens with a positive hemagglutinin 1 signal in the Luminex xTAG respiratory viral panel assay.

This retrospective review analyzed Luminex xTAG respiratory viral panel (RVP) results for 2009 pandemic influenza A (H1N1) virus specimens. Comparing median fluorescence intensity (MFI) signals for the influenza A virus and hemagglutinin 1 (H1) reactions for specimens with very low positive (MFI < 1,000) or "no-call" H1 results reliably distinguished 2009 H1N1 from seasonal virus.

Validation of High-Sensitivity Severe Acute Respiratory Syndrome Coronavirus 2 Testing for Stool-Toward the New Normal for Fecal Microbiota Transplantation.

INTRODUCTION: Mounting evidence demonstrates potential for fecal-oral transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The US Food and Drug Administration now requires SARS-CoV-2 testing of potential feces donors before the use of stool manufactured for fecal microbiota transplantation. We sought to develop and validate a high-sensitivity SARS-CoV-2 reverse transcriptase polymerase chain reaction (RT-PCR) procedure for testing stool specimens. METHODS: A modified extraction method was used with an RT-PCR assay adapted from the Centers for Disease Control and Prevention PCR protocol for respiratory specimens. Contrived specimens were created using pre-COVID-19 banked stool specimens and spiking in known concentrations of SARS-CoV-2-specific nucleic acid. The highest transcript concentration at which 2/2 or 1/2 SARS-CoV-2 targets were detected in 9/10 replicates was defined as the dual-target limit and single-target limit of detection, respectively. The clinical performance of the assay was evaluated with stool samples collected from 17 nasopharyngeal swab RT-PCR-positive patients and 14 nasopharyngeal RT-PCR-negative patients. RESULTS: The dual-target and single-target limit of detection were 56 copies/µL and 3 copies/µL, respectively. SARS-CoV-2 was detected at concentrations as low as 0.6 copies/µL. Clinical stool samples from known COVID-19-positive patients demonstrated the detection of SARS-CoV-2 in stool up to 29 days from symptom onset with a high agreement with nasopharyngeal swab tests (kappa statistic of 0.95, P value < 0.001). DISCUSSION: The described RT-PCR test is a sensitive and flexible approach for the detection of SARS-CoV-2 in stool specimens. We propose an integrated screening approach that incorporates this stool test to support continuation of fecal microbiota transplantation programs.

Sampling for SARS-CoV-2 Aerosols in Hospital Patient Rooms.

Evidence varies as to how far aerosols spread from individuals infected with SARS-CoV-2 in hospital rooms. We investigated the presence of aerosols containing SARS-CoV-2 inside of dedicated COVID-19 patient rooms. Three National Institute for Occupational Safety and Health BC 251 two-stage cyclone samplers were set up in each patient room for a six-hour sampling period. Samplers were place on tripods, which each held two samplers at various heights above the floor. Extracted samples underwent reverse transcription polymerase chain reaction for selected gene regions of the SARS-CoV-2 virus nucleocapsid. Patient medical data were compared between participants in rooms where virus-containing aerosols were detected and those where they were not. Of 576 aerosols samples collected from 19 different rooms across 32 participants, 3% (19) were positive for SARS-CoV-2, the majority from near the head and foot of the bed. Seven of the positive samples were collected inside a single patient room. No significant differences in participant clinical characteristics were found between patients in rooms with positive and negative aerosol samples. SARS-CoV-2 viral aerosols were detected from the patient rooms of nine participants (28%). These findings provide reassurance that personal protective equipment that was recommended for this virus is appropriate given its spread in hospital rooms.

Performance characteristics of the MultiCode-RTx hepatitis C virus load test targeting the 3' untranslated region.

Viral load testing for hepatitis C virus (HCV) RNA has become a key parameter in the diagnosis of infection and treatment monitoring. This study evaluated the performance characteristics of the MultiCode-RTx HCV assay (MultiCode; EraGen Biosciences, Inc., Madison, WI), a real-time PCR test targeting the 3' untranslated region (UTR) of the HCV genome, compared to the TaqMan HCV load ASR assay (TaqMan; Roche Diagnostics, Indianapolis, IN) that targets the 5' UTR. For plasma specimens, the MultiCode assay had a limit of detection of 2.3 log10 IU/ml and a linear range of at least 6.7 log10 IU/ml. Comparison of plasma viral loads obtained by the MultiCode and TaqMan tests showed that they were in very close agreement (mean difference, -0.1 log10 IU/ml). No genotype bias was observed for genotypes 1, 2, and 3. When the MultiCode assay was evaluated with the MagNA Pure and easyMAG extraction methods, the viral loads for the easyMAG extraction were consistently higher for all specimens tested (mean difference, 0.45 log10 IU/ml). Aside from the limit of detection, the performance characteristics of the MultiCode assay were similar to the TaqMan assay for the clinical application of HCV load testing.

Performance evaluation of the Aptima HIV-1 RNA Quant assay on the Panther system using the standard and dilution protocols.

BACKGROUND: Currently, FDA-approved HIV-1 viral load (VL) assays use venipuncture-derived plasma. The Hologic Panther system uses 0.7 mL total volume for the Aptima HIV-1 Quant Assay standard (APT-Quant-std) and dilution (APT-Quant-dil) protocols. However, smaller plasma volumes from fingerstick whole blood (FSB) collected in EDTA-microtainer tubes (MCT) could provide an easier sample collection method for HIV-1 VL testing. OBJECTIVES: To evaluate the performance of the APT-Quant-std compared to the Roche CAP/CTM and Abbott m2000RT VL assays and an alternative APTQuant 1:7 dilution protocol, the latter using 100 µL of MCT-derived plasma from FSB. STUDY DESIGN: Linearity was determined using commercial HIV-1 RNA plasma controls. Dilutions ranging 1.56-2.95 log10 copies/mL were prepared to determine the APT-Quant-dil Limit of Quantitation (LOQ) using Probit analysis. Specificity of APT-Quant-std was calculated using 326 HIVnegative samples. To evaluate agreement, 329 plasma specimens were tested with APT-Quant-std, CAP/CTM, and m2000RT. Forty-seven matched venipuncture and MCT-derived plasma specimens were tested with APT-Quant-std and APT-Quant-dil. RESULTS: Among the RNA controls, specificity was 99.69 % for APT-Quant-std. The R2 values were 0.988 (APT-Quant-std/CAP/CTM), 0.980 (APT-Quant-std/ m2000RT), and 0.997 (APT-Quant-std/APT-Quant-dil). The APT-Quant-dil LOQ was estimated at 2.7 log10 copies/mL (500 copies/mL) (95 %CI 2.62-2.87). At 2.3 log10 copies/mL (200 copies/mL), the overall agreement was 91.0 % for APT-Quant-std/CAP/CTM, 85.7 % for APT-Quant-std/m2000RT, and 82.9 % for APT-Quant-std/APT-Quant-dil. Quantified APT-Quant-std results were on average 0.2 log10 copies/mL higher than CAP/CTM and m2000RT and 0.14 log10 copies/mL higher than APT-Quant-dil. CONCLUSION: APT-Quant showed similar performance compared to the CAP/CTM and m2000RT assays and remains sensitive and accurate using the dilution protocol.

A commutable cytomegalovirus calibrator is required to improve the agreement of viral load values between laboratories.

BACKGROUND: Viral load testing for cytomegalovirus (CMV) is an important diagnostic tool for the management of transplant recipients and immunocompromised individuals; however, inconsistency among laboratories in quantitative measurements of viral load limits interinstitutional comparisons. These inconsistencies stem from the lack of assays cleared by the US Food and Drug Administration, the absence of international standards, the wide variety of CMV-extraction and -detection methods, and differences in materials used for calibration. A critical component of standardization is the use of calibrators that are traceable and commutable. METHODS: Bland-Altman plots and prediction ellipses were used to test the commutability of 2 CMV calibrators for 2 different quantification methods. RESULTS: Tests with 2 methods showed 1 calibrator to be commutable and the other to be noncommutable. The results for the commutable calibrator were within the 95% prediction interval of the clinical samples in the Bland-Altman plot and within the 95% prediction ellipse for a simulated commutable calibrator, whereas the results for the noncommutable calibrator were not within these prediction intervals. When used to calibrate patient results, only the commutable calibrator, the OptiQuant CMV(tc) Calibration Panel, significantly improved the comparability of viral loads for the 2 different measurement methods. CONCLUSIONS: This study demonstrates that an important goal in the effort to improve healthcare for patients with CMV-related disease is the establishment of traceable and commutable reference materials, including both calibrators and controls. .

Detection of fastidious vaginal bacteria in women with HIV infection and bacterial vaginosis.

BACKGROUND: Fastidious bacteria have been associated with bacterial vaginosis (BV) using PCR methods. We assessed the prevalence of these bacteria in HIV-1 infected women and their relationship with vaginal pH and shedding of HIV-1 RNA. METHODS: 64 cervicovaginal lavage (CVL) samples were collected from 51 women. Vaginal microbiota were characterized using 8 bacterium-specific quantitative PCR assays. RESULTS: Women with the fastidious bacteria Bacterial Vaginosis Associated Bacterium (BVAB) 1, 2, and 3 showed a trend to increased HIV-1 shedding (OR 2.59-3.07, P = .14-.17). Absence of Lactobacillus crispatus (P < .005) and presence of BVAB2 (P < .001) were associated with elevated vaginal pH. BVAB1, 2, and 3 were highly specific indicators of BV in HIV-infected women, with specificities of 89%-93%. CONCLUSIONS: Fastidious bacteria (BVAB 1, 2, and 3) remain specific indicators of BV in HIV-infected women, and BVAB2 may contribute to the elevated vaginal pH that is a hallmark of this syndrome.

Antiretroviral drug concentrations and HIV RNA in the genital tract of HIV-infected women receiving long-term highly active antiretroviral therapy.

OBJECTIVE: Our objective was to determine antiretroviral drug concentrations and human immunodeficiency virus (HIV) RNA rebound in cervicovaginal fluid (CVF) in relation to blood plasma (BP) in women receiving suppressive highly active antiretroviral therapy (HAART). METHODS: Thirty-four HIV-infected women who had plasma HIV RNA levels < or =80 copies/mL for at least 6 months were enrolled. Sixty-eight paired CVF and BP drug concentrations and HIV RNA levels were determined before and 3-4 h after drug administration. For each woman and antiretroviral drug, the CVF:BP drug concentration ratios before and after drug administration were calculated. The nonparametric Wilcoxon rank sum test was used to determine if these ratios were different from 1.0. RESULTS: Lamivudine (administered to 20 patients) and tenofovir (administered to 16) had significantly higher concentrations in CVF than in BP before drug administration, with mean CVF:BP concentration ratios of 3.19 (95% confidence interval, 1.2-8.5) and 5.2 (95% confidence interval, 1.2-22.6), respectively. Efavirenz (administered to 13 patients) and lopinavir (administered to 6) had significantly lower concentrations in CVF, with mean CVF:BP concentration ratios of 0.01 (95% confidence interval, 0.00-0.03) and 0.03 (0.01-0.11), respectively. During the study visit (median time after enrollment, 6 months), BP and CVF detectable HIV RNA levels were observed 7 patients (20.6%) and 1 patient (2.9%), respectively. CONCLUSION: Despite lower CVF concentrations of key HAART components, such as efavirenz and lopinavir, virologic rebound was rare. The high concentrations of tenofovir and lamivudine in CVF may have implications for the prevention of sexual transmission during HAART and for pre-exposure or postexposure prophylaxis.

Genital tract leukocytes and shedding of genital HIV type 1 RNA.

BACKGROUND: The mechanism of human immunodeficiency virus (HIV) transmission via heterosexual intercourse is unknown. We sought to determine whether the presence of inflammatory cells in the vagina is associated with the presence of genital tract HIV type 1 (HIV-1) RNA. METHODS: Analysis of a longitudinal prospective cohort was performed. Women with HIV-1 infection were assessed with use of paired plasma and cervicovaginal lavage specimens. Viral load measurements were performed using nucleic acid sequence-based amplification. White blood cells found in the genital tract (GT WBCs) were quantified using a hemacytometer. Common lower genital tract infections assessed for association with viral shedding (i.e., genital tract viral load [GTVL]) included bacterial vaginosis, candidiasis, and trichomoniasis. Generalized estimating equations were used to estimate the prevalence and odds of detectable GTVL by GT WBC. The association was examined both in the presence and in the absence of lower genital tract infections. RESULTS: A total of 97 women and 642 visits were included in the analysis. Median duration of follow-up was 30.4 months. Thirty women (31%) had detectable GTVL at any visit. The median CD4 cell count at baseline was 525 cells/muL. Most women were antiretroviral therapy naive at baseline. After adjustment for plasma viral load, the odds of detectable GTVL increased as GT WBC increased, with an odds ratio of 1.36 (95% confidence interval, 1.1-1.7) per 1000-cell increase in GT WBC among women without lower genital tract infections. After adjustment for plasma viral load and lower genital tract infections by incorporating them in a regression model, GT WBC remained significantly associated with GTVL, with an adjusted odds ratio of 1.22 (95% confidence interval, 1.08-1.37). CONCLUSIONS: The presence of GT WBC is associated with an increased risk of detectable GTVL.

Stability of Trichomonas vaginalis DNA in urine specimens.

Trichomonas vaginalis is an important pathogen in both men and women. Culture is considered the diagnostic gold standard, although studies have shown that PCR is more sensitive than either culture or wet mount for the diagnosis of T. vaginalis infections. We sought to identify a simple method for stabilizing T. vaginalis DNA in urine samples that could be easily applied to molecular testing. The stability of T. vaginalis DNA in 40 urine samples was assessed by storage for various times at either 4 degrees C or room temperature with or without the Becton Dickinson urine preservative transport (UPT) kit. Overall, there was better stability of T. vaginalis DNA when specimens were stored at 4 degrees C than when they were stored at 20 to 22 degrees C and when the UPT system was used. T. vaginalis DNA was stable in specimens stored without using the UPT at 4 degrees C for about 3 days and at room temperature for only 1 day. For specimens placed in the UPT within 24 h (times of 1, 6, and 24 h) of collection, the DNA was stable for up to 30 days when stored at 4 degrees C. For specimens stored at room temperature, the urine should be added to the UPT ideally within 1 hour of collection, and in this case the DNA remained stable for up to 30 days. When storing specimens at room temperature, a delay of 24 h prior to adding to UPT led to an unacceptably high loss of assay sensitivity.