Validation of the GeneXpert® CT/NG Assay for use with Male Pharyngeal and Rectal Swabs.

OBJECTIVES: The GeneXpert® CT/NG (Cepheid, Sunnyvale, CA) assay is a point-of-care (POC) molecular diagnostic assay designed to rapidly test for the presence of Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (GC). However, the test is only approved for vaginal swabs, urine, and endocervical swabs. Here, we performed an evaluation of the GeneXpert® CT/NG assay to detect the presence of CT and GC on male pharyngeal and rectal swabs. METHODS: Men who have sex with men participating in an HIV and Sexually Transmitted Infection (STI) screening program providing consent were enrolled into the study. Participants were asked to self-collect two pharyngeal and two rectal swabs. One set was tested on site using GeneXpert® and the other was sent to a reference lab for molecular testing using the APTIMA® system (Hologic, San Diego, CA). RESULTS: A total of 570 swabs were collected from 144 patients. GeneXpert® detected 13/15 rectal swabs testing CT positive by the APTIMA® assay (relative sensitivity=88.2%), 1/2 pharyngeal swabs testing CT positive (relative sensitivity=50%), and 7/9 pharyngeal swabs testing NG positive (relative sensitivity =77.8%). No discordance was observed for rectal NG swabs. CONCLUSIONS: Although less sensitive than the APTIMA® assay for the molecular detection of NG and CT, GeneXpert®'s potential as a rapid POC diagnostic still make it a viable diagnostic test for STI screening. Molecular POC diagnostics, such as this, will allow more thorough screening of at risk individuals, and enhance the ability of clinics to provide same-day diagnosis and treatment.

Community HIV-1 drug resistance is associated with transmitted drug resistance.

OBJECTIVES: As community viral load (CVL) measurements are associated with the incidence of new HIV-1 infections in a population, we hypothesized that similarly measured community drug resistance (CDR) could predict the prevalence of transmitted drug resistance (TDR). METHODS: Between 2001 and 2011, the prevalences of HIV-1 drug resistance for patients with established infection receiving HIV care (i.e. CDR) and TDR in recently infected patients were determined in San Diego. At each position in HIV-1 reverse transcriptase (RT) and protease (pro), drug resistance was evaluated both as the overall prevalence of resistance-associated mutations and by weighting each resistance position to the concurrent viral load of the patient and its proportion to the total viral load of the clinic (CVL). The weighting was the proportion of the CVL associated with patients identified with resistance at each residue. Spearman ranked correlation coefficients were used to determine associations between CDR and TDR. RESULTS: We analysed 1088 resistance tests for 971 clinic patients and baseline resistance tests for 542 recently infected patients. CDR at positions 30, 46, and 88 in pro was associated with TDR between 2001 and 2011. When CDR was weighted by the viral load of patients, CDR was associated with TDR at position 103 in RT. Each of these associations was corroborated at least once using shorter measurement intervals. CONCLUSIONS: Despite evaluation of a limited percentage of chronically infected patients in San Diego, CDR correlated with TDR at key resistance positions and therefore may be a useful tool with which to predict the prevalence of TDR.

A case cluster demonstrating the relationship between HLA concordance and virologic and disease outcomes in human immunodeficiency virus infection.

We present a detailed analysis of sexual HIV transmission from one source partner to two recipients. The HLA haplotypes between the source partner and one recipient were very similar with 7 out of 8 HLA alleles from four loci (HLA A, B, C and DRB) shared, while the other recipient shared only one allele. The immunologic outcomes between the two recipients differed dramatically, despite the absence of apparent virologic differences in their inoculums. We suggest that non-viral factors, which might be related to differences in the HLA profile, played a role in determining different CD4+ T-cells dynamics for these two recipients.

Resting cerebral blood flow: a potential biomarker of the effects of HIV in the brain.

OBJECTIVE: HIV enters the brain soon after infection causing neuronal damage and microglial/astrocyte dysfunction leading to neuropsychological impairment. We examined the impact of HIV on resting cerebral blood flow (rCBF) within the lenticular nuclei (LN) and visual cortex (VC). METHODS: This cross-sectional study used arterial spin labeling MRI (ASL-MRI) to measure rCBF within 33 HIV+ and 26 HIV- subjects. Nonparametric Wilcoxon rank sum test assessed rCBF differences due to HIV serostatus. Classification and regression tree (CART) analysis determined optimal rCBF cutoffs for differentiating HIV serostatus. The effects of neuropsychological impairment and infection duration on rCBF were evaluated. RESULTS: rCBF within the LN and VC were significantly reduced for HIV+ compared to HIV- subjects. A 2-tiered CART approach using either LN rCBF < or =50.09 mL/100 mL/min or LN rCBF >50.09 mL/100 mL/min but VC rCBF < or =37.05 mL/100 mL/min yielded an 88% (29/33) sensitivity and an 88% (23/26) specificity for differentiating by HIV serostatus. HIV+ subjects, including neuropsychologically unimpaired, had reduced rCBF within the LN (p = 0.02) and VC (p = 0.001) compared to HIV- controls. A temporal progression of brain involvement occurred with LN rCBF significantly reduced for both acute/early (<1 year of seroconversion) and chronic HIV-infected subjects, whereas rCBF in the VC was diminished for only chronic HIV-infected subjects. CONCLUSION: Resting cerebral blood flow (rCBF) using arterial spin labeling MRI has the potential to be a noninvasive neuroimaging biomarker for assessing HIV in the brain. rCBF reductions that occur soon after seroconversion possibly reflect neuronal or vascular injury among HIV+ individuals not yet expressing neuropsychological impairment.

Estimating selection pressures on HIV-1 using phylogenetic likelihood models.

Human immunodeficiency virus (HIV-1) can rapidly evolve due to selection pressures exerted by HIV-specific immune responses, antiviral agents, and to allow the virus to establish infection in different compartments in the body. Statistical models applied to HIV-1 sequence data can help to elucidate the nature of these selection pressures through comparisons of non-synonymous (or amino acid changing) and synonymous (or amino acid preserving) substitution rates. These models also need to take into account the non-independence of sequences due to their shared evolutionary history. We review how we have developed these methods and have applied them to characterize the evolution of HIV-1 in vivo. To illustrate our methods, we present an analysis of compartment-specific evolution of HIV-1 env in blood and cerebrospinal fluid and of site-to-site variation in the gag gene of subtype C HIV-1.

Docosahexaenoic acid-induced changes in phospholipids in cortex of young and aged rats: a lipidomic analysis.

The age-related decline in cognitive function has been associated with biochemical changes that can be attenuated following n-3 polyunsaturated fatty acid treatment. Dietary supplementation with docosahexaenoic acid (DHA) has been shown to reverse age-related changes in synaptic function. Here, lipidomic analyses were undertaken to examine changes in lipid classes and phospholipid species in cortical tissue of young (2-4 months) and aged (20-22 months), control- and DHA-treated (10mg daily) rats following treatment for 8 weeks, aiming to explore the mechanism of DHA action. Dietary supplementation normalised the age-related decrease in unsaturation index, reduced the levels of arachidonic acid-containing phospholipids in both young and aged animals, and gave rise to production of new phosphatidylserine and phosphatidylinositol species. These findings suggest that DHA may mediate some of its effects through alterations in the membrane lipid composition that can consequently affect the production of pro-inflammatory mediators and signalling molecular species.

Maintenance of Nef-mediated modulation of major histocompatibility complex class I and CD4 after sexual transmission of human immunodeficiency virus type 1.

Viruses encounter changing selective pressures during transmission between hosts, including host-specific immune responses and potentially varying functional demands on specific proteins. The human immunodeficiency virus type 1 Nef protein performs several functions potentially important for successful infection, including immune escape via down-regulation of class I major histocompatibility complex (MHC-I) and direct enhancement of viral infectivity and replication. Nef is also a major target of the host cytotoxic T-lymphocyte (CTL) response. To examine the impact of changing selective pressures on Nef functions following sexual transmission, we analyzed genetic and functional changes in nef clones from six transmission events. Phylogenetic analyses indicated that the diversity of nef was similar in both sources and acutely infected recipients, the patterns of selection across transmission were variable, and regions of Nef associated with distinct functions evolved similarly in sources and recipients. These results weighed against the selection of specific Nef functions by transmission or during acute infection. Measurement of Nef function provided no evidence that the down-regulation of either CD4 or MHC-I was optimized by transmission or during acute infection, although rare nef clones from sources that were impaired in these activities were not detected in recipients. Nef-specific CTL activity was detected as early as 3 weeks after infection and appeared to be an evolutionary force driving the diversification of nef. Despite the change in selective pressure between the source and recipient immune systems and concomitant genetic diversity, the majority of Nef proteins maintained robust abilities to down-regulate MHC-I and CD4. These data suggest that both functions are important for the successful establishment of infection in a new host.

Stratified response to environmental stress in a polar lichen characterized with FT-Raman microscopic analysis.

The role of Antarctic epilithic lichens in the primary colonization of rocks and in the formation of soils is receiving attention because of the production of the stress-protective biochemicals needed to combat radiation, desiccation and extremes of temperature. Raman microscopy has been used here to study the encrustations produced at the interface between the rock substratum and Buellia spp. lichen thalli; in addition to whewellite, calcium oxalate monohydrate, the presence of weddellite, the metastable dihydrate form, was confirmed in the encrustations. An unusual pigmentation of the rock surface found on detachment of the lichen growths is identified as beta-carotene from its characteristic Raman bands at 1525, 1191, 1157 and 1003 cm(-1); normally, beta-carotene, which has been identified as a UV-radiation protectant, is found at the exposed upper surface of the biological organism. The interface between the detached lichen thalli and the rock also contains whewellite as the sole biomineralization product--which suggests a possible strategy for the formulation of weddelite in the growing Buellia spp. colony as an anti-desiccant.

Evidence for increased T cell turnover and decreased thymic output in HIV infection.

The effects of HIV infection upon the thymus and peripheral T cell turnover have been implicated in the pathogenesis of AIDS. In this study, we investigated whether decreased thymic output, increased T cell proliferation, or both can occur in HIV infection. We measured peripheral blood levels of TCR rearrangement excision circles (TREC) and parameters of cell proliferation, including Ki67 expression and ex vivo bromodeoxyuridine incorporation in 22 individuals with early untreated HIV disease and in 15 HIV-infected individuals undergoing temporary interruption of therapy. We found an inverse association between increased T cell proliferation with rapid viral recrudescence and a decrease in TREC levels. However, during early HIV infection, we found that CD45RO-CD27high (naive) CD4+ T cell proliferation did not increase, despite a loss of TREC within naive CD4+ T cells. A possible explanation for this is that decreased thymic output occurs in HIV-infected humans. This suggests that the loss of TREC during HIV infection can arise from a combination of increased T cell proliferation and decreased thymic output, and that both mechanisms can contribute to the perturbations in T cell homeostasis that underlie the pathogenesis of AIDS.

Differential involvement of NMDA, AMPA/kainate, and dopamine receptors in the nucleus accumbens core in the acquisition and performance of pavlovian approach behavior.

Stimuli paired with primary rewards can acquire emotional valence and the ability to elicit automatic, Pavlovian approach responses that have been shown to be mediated by the nucleus accumbens. The present experiment investigated the effects of infusions of glutamatergic or dopaminergic receptor antagonists into the core of the nucleus accumbens on the acquisition and performance of Pavlovian discriminated approach to an appetitive conditioned stimulus. Rats were trained on an autoshaping task in which a conditioned stimulus (CS+; a lever) was inserted into the operant chamber for 10 sec, after which a food pellet was delivered. Presentation of another lever (CS-) was never followed by food. Subjects developed a conditioned response of approaching and contacting the CS+ selectively, although food delivery was not in any way contingent on the animals' response. A triple dissociation in the effects of AP-5, LY293558 [(3SR, 4aRS, 6RS, 8aRS)-6-[2-(iH-tetrazol-5-yl)ethyl]-1,2,3,4,4a,5,6,7,8,8a-decahydroiso-quinoline-3-carboxylic acid], and alpha-flupenthixol infused into the nucleus accumbens core on the acquisition and performance of this conditioned response was observed. The AMPA/kainate receptor antagonist LY293558 disrupted discriminated approach performance but not acquisition, as evidenced by increased approaches to the CS-. In contrast, the NMDA receptor antagonist AP-5 impaired only the acquisition, but not performance, of autoshaping whereas the dopamine D1/D2 receptor antagonist alpha-flupenthixol decreased approaches to the CS+ during both acquisition and performance. The data are discussed with reference to dissociable interactions of these receptor types with limbic cortical and dopaminergic afferents to the nucleus accumbens core during the acquisition and expression of Pavlovian conditioned approach.

Reduced susceptibility of human immunodeficiency virus type 1 (HIV-1) from patients with primary HIV infection to nonnucleoside reverse transcriptase inhibitors is associated with variation at novel amino acid sites.

Recently, significant numbers of individuals with primary human immunodeficiency virus (HIV) infection have been found to harbor viral strains with reduced susceptibility to antiretroviral drugs. In one study, HIV from 16% of such antiretroviral-naive individuals was shown to have a susceptibility to nonnucleoside reverse transcriptase (RT) inhibitors (NNRTIs) between 2.5- and 10-fold lower than that of a wild-type control. Mutations in the RT domain that had previously been associated with antiretroviral resistance were not shared by these strains. We have analyzed by logistic regression 46 variable amino acid sites in RT for their effect on susceptibility and have identified two novel sites influencing susceptibility to NNRTIs: amino acids 135 and 283 in RT. Eight different combinations of amino acids at these sites were observed among these patients. These combinations showed a 14-fold range in mean susceptibility to both nevirapine and delavirdine. In vitro mutagenesis of the control strain combined with a phenotypic assay confirmed the significance of amino acid variation at these sites for susceptibility to NNRTIs.

Transmission and prevalence of HIV resistance among treatment-naive subjects.

Recent reports demonstrate the transmission of drug-resistant HIV-1 variants to newly infected individuals, although estimates of the prevalence of drug resistance among populations from different geographic regions are highly varied. The interpretation and comparison of available study results are confounded by the lack of consensus regarding the nomenclature and reporting of antiviral resistance. This report re-evaluates previously presented and published data using uniform criteria for genotypic and phenotypic drug resistance. Treatment-inexperienced and recently or chronically infected populations are reviewed. The prevalence of transmitted drug resistance ranges from 1% to 11% among recently infected persons using these criteria. Programmes to monitor and characterize drug resistance among newly infected persons and their source partners are essential to evaluate the selection processes that influence the transmission of certain genetic variants of HIV. Temporal trends in the prevalence of transmitted drug resistance among diverse populations are necessary to evaluate the potential need for selective and generalized drug resistance screening programmes among newly infected, treatment-naive patients.

Reduced antiretroviral drug susceptibility among patients with primary HIV infection.

CONTEXT: The transmission of drug-resistant human immunodeficiency virus (HIV) has been documented, but the prevalence of such transmission is unknown. OBJECTIVE: To assess the spectrum and frequency of antiretroviral susceptibility among subjects with primary HIV infection. DESIGN, SETTING, AND PATIENTS: Retrospective analysis of 141 subjects identified from clinical research centers in 5 major metropolitan areas, enrolled from 1989 to 1998, with HIV seroconversion within the preceding 12 months and no more than 7 days' prior antiretroviral (ARV) therapy. MAIN OUTCOME MEASURES: Phenotypic and genotypic ARV susceptibility of HIV from plasma samples. RESULTS: The transmission of drug-resistant HIV as assessed by a greater than 10-fold reduction in ARV susceptibility to 1 or more drugs was observed in 3 (2%) of 141 subjects, including to a nonnucleoside reverse transcriptase inhibitor in 1 patient and to a nucleoside reverse transcriptase inhibitor and a protease inhibitor in 2 patients. Population-based sequence analysis of these 3 samples identified multidrug-resistance mutations in reverse transcriptase (M184V, T215Y, K219K/R) and protease (L101/V, K20R, M361, M46I, G48V, L63P, A71T, V771, V82T, 184V, L90M) in the 2 latter patient samples, along with numerous polymorphisms. A reduction in susceptibility of greater than 2.5- to 10-fold to 1 or more drugs was observed in viral isolates from 36 patients (26%). Sequence analysis of these 36 samples identified well-characterized drug resistance mutation in reverse transcriptase and protease in only 1 of these patients. CONCLUSIONS: Reductions in drug susceptibility of more than 10-fold were rare among this cohort of recently HIV-infected subjects and were distributed among each of the 3 major classes of ARV drugs tested. Reductions in susceptibility of more than 2.5- to 10-fold to certain ARV drugs of unknown clinical significance were highly prevalent among newly infected patients. Resistance testing may be warranted to monitor the frequency of drug resistance over time and to assess the potential for geographic variability.

Viral dynamics of acute HIV-1 infection.

Viral dynamics were intensively investigated in eight patients with acute HIV infection to define the earliest rates of change in plasma HIV RNA before and after the start of antiretroviral therapy. We report the first estimates of the basic reproductive number (R(0)), the number of cells infected by the progeny of an infected cell during its lifetime when target cells are not depleted. The mean initial viral doubling time was 10 h, and the peak of viremia occurred 21 d after reported HIV exposure. The spontaneous rate of decline (alpha) was highly variable among individuals. The phase 1 viral decay rate (delta(I) = 0.3/day) in subjects initiating potent antiretroviral therapy during acute HIV infection was similar to estimates from treated subjects with chronic HIV infection. The doubling time in two subjects who discontinued antiretroviral therapy was almost five times slower than during acute infection. The mean basic reproductive number (R(0)) of 19.3 during the logarithmic growth phase of primary HIV infection suggested that a vaccine or postexposure prophylaxis of at least 95% efficacy would be needed to extinguish productive viral infection in the absence of drug resistance or viral latency. These measurements provide a basis for comparison of vaccine and other strategies and support the validity of the simian immunodeficiency virus macaque model of acute HIV infection.

The value of patient-reported adherence to antiretroviral therapy in predicting virologic and immunologic response. California Collaborative Treatment Group.

OBJECTIVE: To correlate self-reported antiretroviral adherence with virologic suppression. DESIGN: Prospective observational study of adherence to therapy nested in a randomized comparative trial of frequent versus infrequent monitoring of plasma HIV RNA. SETTING: Five university-affiliated HIV clinics. PATIENTS: A group of 173 HIV-infected patients with a mean baseline CD4 count of 142 x 10(6) cells/l (range 3-515) of whom 164 and 119 completed adherence questionnaires at 2 and 6 months, respectively. INTERVENTION: Individualized, unrestricted antiretroviral therapy. MEASUREMENTS: Patients were classified into four groups by adherence to therapy in the previous 4 weeks (< 80%, 80-95%, 95-99%, 100%). Plasma HIV RNA levels and CD4 lymphocyte counts were measured bimonthly. RESULTS: Recreational drug or alcohol use was associated with decreased adherence, whereas frequency of HIV RNA monitoring, demographic variables, (age, gender, education, and risk group) and stage of disease had no effect. Greater HIV suppression at 6 months was seen across four categories of increasing adherence (P = 0.009 for linear trend). Patients reporting < 80% adherence at 6 months had a 0.2 log10 copies/ml increase in HIV RNA and a loss of 19 x 10(6) CD4 cells/l compared with a 1.1 log10 copies/ml decrease in HIV RNA and an increase of 72 x 10(6) CD4 cells/l in those reporting 100% adherence (P = 0.02). CONCLUSION: Self-reported poor adherence (< 80%) and drug or alcohol use predicted non-response of HIV RNA at 6 months of antiretroviral therapy.

Cell-associated viral RNA expression during acute infection with HIV type 1.

The mechanism of decline in viremia following acute infection with HIV is unknown. To characterize this process virologically, the expression of viral RNAs was analyzed in samples of peripheral blood mononuclear cells (PBMCs) from a patient who experienced a 100-fold decline in plasma viremia over a 13-day period prior to the initiation of antiretroviral therapy. Cell-associated viral RNA declined in association with the decline in plasma virus. During the initial 7 days of observation, plasma viremia declined more than 10-fold with no change in the ratio of unspliced to multiply spliced mRNAs. The efficiency of viral gene expression did not decline during the study period and varied from 380 to 2800 unspliced RNA copies per productively infected cell. Together, these data indicate no change in the relative proportion of cells in late- and early-stage gene expression during the initial decline and provide evidence against shortening of the viral replication cycle by immune surveillance. However, the prevalence of productively infected cells declined markedly during the 13 days of observation, from 1 in 250 to 1 in 25,000 PBMCs. These data are compatible with depletion of available target cells during the initial decline in viremia. As the level of plasma virus stabilized after 8 days of observation, the ratio of unspliced to multiply spliced mRNAs rose; this rise was due to a relatively greater decline in multiply spliced mRNA. These data suggest the possible onset of a blockade to new infection events (for example, by neutralizing antibody or chemokines), causing an increase in the relative proportion of cells in late-stage gene expression. They may also be explained, however, by the persistence of cell-associated virions together with the near disappearance of productively infected cells from the circulation.

Effective group behavioral intervention for older periodontal patients.

A randomized clinical trial assessed the effect of a group-based behavior modification intervention on oral hygiene skills, adherence and clinical outcomes for older periodontal patients. Subjects (n = 107) were aged 50-70 yr with moderate periodontal disease. They were randomly assigned to usual care or intervention. Intervention consisted of 5 weekly, 90-min sessions that included skill training, self-monitoring, weekly feedback about bleeding points and group support focused on long-term habit change. Four-month follow-up indicated significant improvements in the intervention versus the usual periodontal maintenance group for oral hygiene skills and self-reported flossing (p < 0.001), plaque, gingival bleeding, bleeding upon probing throughout the mouth, and pocket depth for sulcus depths that measured between 3 and 6 mm at baseline (p < 0.009). Group oral health intervention provides an effective and relatively inexpensive means of helping patients improve their self-care skills and achieve high levels of adherence to an effective self-care regimen.

Making the most of a teachable moment: a smokeless-tobacco cessation intervention in the dental office.

OBJECTIVES: Primary care medical clinics are good settings for smoking interventions. This study extends this strategy with a smokeless tobacco intervention delivered by dentists and dental hygienists in the course of routine dental care. METHODS: Male users of moist snuff and chewing tobacco (n = 518) were identified by questionnaire in clinic waiting rooms and then randomly assigned to either usual care or intervention. The intervention included a routine oral examination with special attention to the part of the mouth in which tobacco was kept and an explanation of the health risks of using smokeless tobacco. After receiving unequivocal advice to stop using tobacco, each patient viewed a 9-minute videotape, received a self-help manual, and was briefly counseled by the dental hygienist. RESULTS: Long-term success was defined as no smokeless tobacco use at both 3- and 12-month follow-ups, with those lost to follow-up counted as smokeless tobacco users. The intervention increased the proportion of patients who quit by about one half (12.5% vs 18.4%, P < .05). CONCLUSIONS: These results demonstrate the efficacy of a brief dental office intervention for the general population of smokeless tobacco users.

Cell surface CD4 downregulation and resistance to superinfection induced by a defective provirus of HIV-1.

Proviral sequences encoding defective HIV-1 regulatory genes have been detected previously in infected individuals; however, the role of these defective genomes in pathogenesis is unclear. The hypothesis that such replication-defective genomes might induce downregulation of the cellular receptor for HIV-1 (CD4) was tested. CEM cells were stably transfected with a provirus that contains a mutation in the splice site immediately 5' of the rev coding sequence. This mutant expresses early HIV-1 mRNAs but is defective for replication. Cells expressing this defective provirus displayed markedly reduced surface CD4. This downregulation of CD4 was dependent on an intact nef gene and was sufficient to cause resistance to superinfection by wild-type virus. These findings indicate that Nef as expressed by replication-defective HIV-1 can downregulate CD4. This perturbation of the T lymphocyte cell membrane is a potential basis for pathogenicity of defective HIV-1.