A Spectroscopic Approach to Unravel the Local Conformations of a G-Quadruplex Using CD-Active Fluorescent Base Analogues.

The formation of a stable G-quadruplex (GQ) can inhibit the increased telomerase activity that is common in most cancers. The global structure and the thermal stability of the GQs are usually evaluated by spectroscopic methods and thermal denaturation properties. However, most biochemical processes involving GQs might require local conformational changes at the guanine tetrad (G4) level. These local conformational changes of individual G4 layers during protein and drug interactions have not yet been explored in detail. In this study, we monitored the local conformations of individual G4 layers in GQs using 6-methylisoxanthopterine (6MI) chromophores, which are circular dichroism (CD)-active fluorescent base analogues of guanine, as local conformational probes. A synthetic, tetramolecular, parallel GQ with site-specifically positioned 6MI monomers or dimers was used as the experimental construct. Analytical ultracentrifugation studies and gel electrophoretic studies showed that properly positioned 6MI monomers and dimers could form stable GQs with CD-active fluorescent G4 layers. The local conformation of individual fluorescent G4 layers in the GQ structure was then tracked by monitoring the absorbance, fluorescence intensity, thermal melting, fluorescence quenching, and CD changes of the incorporated probes. Overall, these studies showed that site-specifically incorporated fluorescent base analogues could be used as probes to monitor the local conformational changes of individual G4 layers of a GQ structure. This method can be applied to explore the details of small molecule-GQ interaction at the level of the individual G4 layers, which may prove to be useful in designing drugs to treat GQ-related genetic disorders, cancer, and aging.

Continued Transmission of HIV Among Young Adults Who Inject Drugs in San Francisco: Still Room for Improvement.

We measured HIV incidence rate, trend and risk factors in 564 HIV-negative young people (< 30 years) who inject drugs (PWID) in San Francisco between 2000 and 2014. HIV incidence was 0.93/100 person-years (PY; 95% CI 0.50, 1.73). Incidence varied between 0.62/100 PY in 2000-2002 and 1.06/100 PY in 2012-2014 (P for trend = 1.0). HIV incidence varied significantly (P < 0.01) by race/ethnicity: among Hispanics it was 8.19/100 PY (95% CI 3.41, 19.68), African-Americans 4.59/100 PY (95% CI 1.15, 18.37), and Whites 0.26/100 PY (95% CI 0.06, 1.03). Male participants who reported sex with men (MSM) had higher HIV incidence (2.63/100 PY; 95% CI 1.31, 5.25) compared to males who did not report MSM (0.50/100 PY; 95% CI 0.12, 1.99) (P = 0.01). Despite an overall stable HIV incidence trend, incidence was elevated among African-American and Hispanic PWID, and men who have sex with men. Addressing prevention needs in these key populations is critical for the goal of eliminating HIV transmission.

Challenges in detecting HIV persistence during potentially curative interventions: a study of the Berlin patient.

There is intense interest in developing curative interventions for HIV. How such a cure will be quantified and defined is not known. We applied a series of measurements of HIV persistence to the study of an HIV-infected adult who has exhibited evidence of cure after allogeneic hematopoietic stem cell transplant from a homozygous CCR5Δ32 donor. Samples from blood, spinal fluid, lymph node, and gut were analyzed in multiple laboratories using different approaches. No HIV DNA or RNA was detected in peripheral blood mononuclear cells (PBMC), spinal fluid, lymph node, or terminal ileum, and no replication-competent virus could be cultured from PBMCs. However, HIV RNA was detected in plasma (2 laboratories) and HIV DNA was detected in the rectum (1 laboratory) at levels considerably lower than those expected in ART-suppressed patients. It was not possible to obtain sequence data from plasma or gut, while an X4 sequence from PBMC did not match the pre-transplant sequence. HIV antibody levels were readily detectable but declined over time; T cell responses were largely absent. The occasional, low-level PCR signals raise the possibility that some HIV nucleic acid might persist, although they could also be false positives. Since HIV levels in well-treated individuals are near the limits of detection of current assays, more sensitive assays need to be developed and validated. The absence of recrudescent HIV replication and waning HIV-specific immune responses five years after withdrawal of treatment provide proof of a clinical cure.

Aspergillus galactomannan antigen in the bronchoalveolar lavage fluid for the diagnosis of invasive aspergillosis in lung transplant recipients.

BACKGROUND: The clinical utility of Platelia Aspergillus enzyme immunoassay (EIA) for galactomannan (GM) antigen detection in bronchoalveolar lavage (BAL) for the diagnosis of invasive aspergillosis (IA) in lung transplant recipients is not known. METHODS: BAL fluid samples from consecutive lung transplant recipients who underwent bronchoscopy were prospectively analyzed for GM. RESULTS: A total of 333 BAL samples from 116 patients were tested. Invasive aspergillosis was documented in 5.2% (6/116) of the patients. Samples analyzed included 9 BALs from two patients with proven IA, 19 BALs from four patients with probable IA, and 305 BALs from 110 patients without IA. At the index cutoff value of > or =0.5, the sensitivity was 60%; specificity was 95%, with positive and negative likelihood ratios of 14 and 0.41, respectively. Increasing the index cutoff value to > or =1.0 yielded a sensitivity of 60%, a specificity of 98%, and the positive and negative likelihood ratios of 28 and 0.40, respectively. Two of six patients with IA receiving antifungal prophylaxis had false-negative results. CONCLUSIONS: A Platelia EIA index cut-off > or =1.0 in the BAL fluid in a lung transplant recipient with a compatible clinical illness may be considered as suggestive of IA.

Prospective Aspergillus galactomannan antigen testing in pediatric hematopoietic stem cell transplant recipients.

BACKGROUND: The galactomannan (GM) assay is an approved noninvasive test for detection of invasive aspergillosis (IA) that has been validated in adult patients with hematologic malignancies who are undergoing bone marrow transplantation. There have been few studies with this assay in pediatric patients, but early reports suggest that there may be differences in the performance such that false-positive GM tests in pediatric patients are more common than in adult patients. METHODS: We performed a prospective study in pediatric hematopoietic stem cell transplant recipients with twice-weekly sampling for GM detection during the highest risk periods of neutropenia and graft-versus-host disease. We analyzed 826 serum samples from 64 patients, including 15 serum samples from one patient diagnosed with probable IA according to defined criteria. RESULTS: Twenty of 811 samples tested positive on repeat testing (specificity, 97.5%; 95% CI: 96.2-98.4%) including samples from 8 of 63 patients without clinical evidence of IA according to study criteria (specificity, 87.3%; 95% CI: 76.9-93.4%). Eleven patients received piperacillin/tazobactam therapy, and 4 of the 11 patients had a positive assay result coinciding with the dates of piperacillin/tazobactam administration. When samples from these patients were excluded, specificity increased to 98.4% (95% CI: 97.2-99.1%) by sample and to 91.5% (95% CI: 81.6-96.3%) by patient. CONCLUSIONS: The GM assay holds promise for early, noninvasive diagnosis of IA in high-risk children and false-positive results were not common or unexplainable. This study supports further validation of this assay in a large-scale, pediatric-dedicated format.

Seroreversion in subjects receiving antiretroviral therapy during acute/early HIV infection.

BACKGROUND: We assessed human immunodeficiency virus (HIV) antibody seroreversion among individuals initiating antiretroviral therapy (ART) during acute/early HIV infection and determined whether seroreversion was associated with loss of cytotoxic T lymphocyte responses. METHODS: Subjects in a cohort with acute/early HIV infection (<12 months into infection) who initiated ART within 28 days after study entry and maintained HIV type 1 ribonucleic acid levels of < or =500 copies/mL for >24 weeks were selected. Two clinically available second-generation enzyme immunoassays (EIAs) and a confirmatory Western blot were used to screen subjects for antibody reversion. Those with negative screening test results underwent additional antibody testing, including a third-generation EIA, and were assessed for cytotoxic T lymphocyte responses. RESULTS: Of 87 subjects identified, 12 (14%) had negative antibody test results at the start of ART; all 12 had seroconversion, although 1 had seroconversion only on a third-generation EIA. Of the 87 subjects, 6 (7%) had seroreversion on at least 1 EIA antibody assay while receiving ART during a median follow-up of 90 weeks. The only clinical predictor of seroreversion was a low baseline "detuned" (less sensitive) antibody. Cytotoxic T lymphocyte responses to HIV Gag peptides were detected in 4 of 5 subjects with seroreversion who could be tested. All 5 who had seroreversion who stopped ART experienced virologic rebound and antibody evolution. CONCLUSIONS: HIV antibody seroconversion on second-generation EIA antibody tests may fail to occur when ART is initiated early. Seroreversion was not uncommon among subjects treated early, although cytotoxic T lymphocyte responses to HIV antigens remained detectable in most subjects. Antibody seroreversion did not indicate viral eradication. A third-generation EIA was the most sensitive test for HIV antibodies.

Performance comparison of the 4th generation Bio-Rad Laboratories GS HIV Combo Ag/Ab EIA on the EVOLIS™ automated system versus Abbott ARCHITECT HIV Ag/Ab Combo, Ortho Anti-HIV 1+2 EIA on Vitros ECi and Siemens HIV-1/O/2 enhanced on Advia Centaur.

BACKGROUND: A multisite study was conducted to evaluate the performance of the Bio-Rad 4th generation GS HIV Combo Ag/Ab EIA versus Abbott 4th generation ARCHITECT HIV Ag/Ab Combo. The performance of two 3rd generation EIAs, Ortho Diagnostics Anti-HIV 1+2 EIA and Siemens HIV 1/O/2 was also evaluated. OBJECTIVE: Study objective was comparison of analytical HIV-1 p24 antigen detection, sensitivity in HIV-1 seroconversion panels, specificity in blood donors and two HIV false reactive panels. STUDY DESIGN: Analytical sensitivity was evaluated with International HIV-1 p24 antigen standards, the AFFSAPS (pg/mL) and WHO 90/636 (IU/mL) standards; sensitivity in acute infection was compared on 55 seroconversion samples, and specificity was evaluated on 1000 negative blood donors and two false reactive panels. RESULTS: GS HIV Combo Ag/Ab demonstrated better analytical HIV antigen sensitivity compared to ARCHITECT HIV Ag/Ab Combo: 0.41 IU/mL versus 1.2 IU/mL (WHO) and 12.7 pg/mL versus 20.1 pg/mL (AFSSAPS); GS HIV Combo Ag/Ab EIA also demonstrated slightly better specificity compared to ARCHITECT HIV Ag/Ab Combo (100% versus 99.7%). The 4th generation HIV Combo tests detected seroconversion 7-11 days earlier than the 3rd generation HIV antibody only EIAs. CONCLUSION: Both 4th generation immunoassays demonstrated excellent performance in sensitivity, with the reduction of the serological window period (7-11 days earlier detection than the 3rd generation HIV tests). However, GS HIV Combo Ag/Ab demonstrated improved HIV antigen analytical sensitivity and slightly better specificity when compared to ARCHITECT HIV Ag/Ab Combo assay, with higher positive predictive values (PPV) for low prevalence populations.

Performance evaluation of the Bio-Rad Laboratories GS HIV Combo Ag/Ab EIA, a 4th generation HIV assay for the simultaneous detection of HIV p24 antigen and antibodies to HIV-1 (groups M and O) and HIV-2 in human serum or plasma.

BACKGROUND: A multi-center study was conducted to evaluate the Bio-Rad GS HIV Combo Ag/Ab EIA, a 4th generation HIV-1/HIV-2 assay for the simultaneous detection of HIV p24 antigen and antibodies to HIV-1 (groups M and O) and HIV-2 in human serum or plasma in adult and pediatric populations. OBJECTIVES: The objectives of the study were to assess assay performance for the detection of acute HIV infections; sensitivity in known HIV positive samples; percent agreement with HIV status; specificity in low and high risk individuals of unknown HIV status; and to compare assay performance to a 3rd generation HIV assay. STUDY DESIGN: The evaluation included testing 9150 samples at four U.S. clinical trial sites, using three kit lots. Unlinked samples were from routine testing, repositories or purchased from vendors. RESULTS: GS HIV Combo Ag/Ab EIA detection in samples from individuals in two separate populations with acute HIV infection was 95.2% (20/21) and 86.4% (38/44). Sensitivity was 100% (1603/1603) in known antibody positive [HIV-1 Groups M and O, and HIV-2] samples. HIV p24 antigen detection was 100% (53/53) in HIV-1 culture supernatants. HIV-1 seroconversion panel detection improved by a range of 0-20 days compared to a 3rd generation HIV test. Specificity was 99.9% (5989/5996) in low risk, 99.9% (959/960) in high risk and 100% (100/100) in pediatric populations. CONCLUSION: The GS HIV Combo Ag/Ab EIA significantly reduced the diagnostic window when compared to the 3rd generation screening assay, enabling earlier diagnosis of HIV infection. The performance parameters of the Bio-Rad GS HIV Combo Ag/Ab EIA are well suited for use in HIV diagnostic settings.

Acute HIV-1 infection is highly prevalent in Ugandan adults with suspected malaria.

OBJECTIVES: Acute febrile illnesses consistent with malaria are the most common presentation at health clinics in sub-Saharan Africa, accounting for 30-50% of outpatient visits. The symptoms of acute HIV infection can mimic acute malaria. We investigated whether acute HIV infections could be identified among adults with suspected malaria at rural health centers in Uganda. DESIGN: A cross-sectional study of 1000 consecutive patients referred for malaria blood smears at each of seven government health centers, of which 2893 (41%) were 13 years or older and tested for HIV. METHODS: HIV enzyme immunoassay antibody testing was performed on dried blood spots and confirmed by western blot. Enzyme immunoassay-nonreactive and enzyme immunoassay-reactive, western blot-unconfirmed samples were pooled (10/pool) and tested for HIV RNA by nucleic acid amplification testing. We defined acute HIV infection as HIV-1 RNA positive with a negative or indeterminate HIV-1 western blot pattern and early HIV infection as HIV-1 RNA positive with a positive western blot pattern, but with a BED-corrected optical density of below 0.8. RESULTS: Of 2893 patients evaluated, 324 (11%) had test results indicating HIV infection. Overall, 30 patients (1.0%) had acute HIV infection, 56 (1.8%) had early HIV infection, and 238 (8%) had established HIV infection. Acute HIV infections were more prevalent at sites with higher HIV prevalence and lower malaria endemicity. CONCLUSION: At multiple sites in Uganda, 1-3% of adults with suspected malaria had acute or early HIV infection. These findings highlight a major opportunity for expanding recognition of acute and early HIV infection in Africa.

Detection of galactomannan antigenemia by enzyme immunoassay for the diagnosis of invasive aspergillosis: variables that affect performance.

Invasive aspergillosis (IA) is a frequent complication of blood or marrow transplantation. Previous studies have reported that the Aspergillus galactomannan enzyme immunoassay (GM EIA) may be a useful diagnostic tool for IA, but its sensitivity is variable. We examined the performance of the GM EIA in 986 serum samples from 67 patients. Results demonstrated that decreasing the index cutoff for positivity to 0.5 increased its sensitivity, with minimal loss of specificity. The low cutoff increased the duration of test positivity before diagnosis by clinical means. Sensitivity was highest in patients who did not receive preventative mold-active antifungals (87.5%). A rabbit model demonstrated that the level of circulating antigen correlated with the tissue fungus burden. A quantifiable response to antifungal therapy in clinical samples and the rabbit model supports the development of this assay for early diagnosis and therapeutic monitoring. The 0.5 cutoff may allow for better performance as an early diagnostic test.