Neonatal Natural Killer (NK) cells show functional impairment and expansion of a CD56 negative population of uncertain significance.
NK cells were isolated from cord blood and from adult donors. NK subpopulations were identified as positive or negative for the expression of CD56 and characterized for expression of granzyme B and surface markers by multi-parameter flow cytometry. Cell function was assessed by viral suppression and cytokine production using autologous lymphocytes infected with HIV. Activating (NKp30, NKp46) and inhibitory (Siglec-7) markers in healthy infants and adults were compared with viremic HIV-infected adults.
Cord blood contained increased frequencies of CD56 negative (CD56neg) NK cells with reduced expression of granzyme B and reduced production of IFNγ and the CC-class chemokines RANTES, MIP1α and MIP1β upon stimulation. Both CD56pos and CD56neg NK subpopulations showed impaired viral suppression in cord blood, with impairment most marked in the CD56neg subset. CD56neg NK cells from cord blood and HIV-infected adults shared decreased inhibitory and activating receptor expression when compared with CD56pos cells.
CD56neg NK cells are increased in number in normal infants and these effectors show reduced anti-viral activity. Like the expanded CD56neg population described in HIV-infected adults, these NK cells demonstrate functional impairments which may reflect inadequate development or activation.
Transmission of drug-resistant HIV (TDR) has been associated with virologic failure of “first-line,” nonnucleoside reverse transcriptase inhibitor (NNRTI)-based antiretroviral therapy (ART). A national ART program began in Peru in 2004. We evaluated the prevalence of TDR in individuals initiating ART and their virologic outcome during 2 years of ART. HIV-infected, ARV-naive subjects who met criteria to start ART in Lima, Peru were enrolled in a longitudinal observational study between July 2007 and February 2009. Blood plasma and cells obtained prior to ART initiation were assessed for antiretroviral (ARV) resistance by an oligonucleotide ligation assay (OLA) sensitive to 2% mutant at reverse transcriptase (RT) codons K103N, Y181C, G190A, and M184V and a subset by consensus sequencing. A total of 112 participants were enrolled; the mean CD4 was 134±89 cells/μl and the median plasma HIV RNA was 93,556 copies/ml (IQR 62,776–291,364). Drug resistance mutations conferring high-level resistance to ARV were rare, detected in one of 96 (1%) evaluable participants. This subject had the Y181C mutation detected in both plasma and peripheral blood mononuclear cells (PBMCs) at a concentration of 100% by OLA and consensus sequencing; nevertheless nevirapine-ART suppressed her viral replication. Consensus sequencing of 37 (19%) participants revealed multiple polymorphisms that occasionally have been associated with low-level reductions in ARV susceptibility. A low prevalence of TDR was detected among Peruvians initiating ART. Given the increasing availability of ART, continuing surveillance is needed to determine if TDR increases and the mutant codons associated with virologic failure.
Bacterial vaginosis is associated with sexual activity, but mechanisms for this association are unclear.
Cross-sectional analysis of data from women reporting sex with women who provided information on sexual behaviors as part of a study of vaginal bacteria. Vaginal bacteria were detected by semi-quantitative culture. Binomial regression with log-link evaluated associations between detection of bacteria and sexual behaviors reported to occur prior to enrollment. Linear regression evaluated associations between these behaviors and quantity of bacteria.
Of 320 women, 216 (68%) were colonized with H2O2-producing lactobacilli and 142 (44%) with G. vaginalis. Colonization with G. vaginalis was associated with >20 digital-vaginal sex acts (RR 2.01; 1.22, 3.29) or >10 toy-vaginal acts in the past 3 months (RR 1.76; 1.32, 2.36). Quantity of H2O2-producing lactobacilli was 1.3 log lower in colonized women reporting >10 acts of insertive vaginal sex toy use in the past 3 months (95%CI −2.04, −.56), 1.19 log lower with toy-vaginal sex in past 7 days (−1.75, −.62), and 0.78 log lower in women sharing toys with a partner (−1.25, −.31).
Vaginal insertive use and sharing of sex toys was associated with decreased quantities of H2O2-producing lactobacilli and higher risk of colonization with G. vaginalis.
bacterial vaginosis; lactobacilli; Gardnerella vaginalis
The presence of hydrogen peroxide (H2O2) producing Lactobacillus in the vagina may play a role in controlling genital HIV-1 shedding. Sensitive molecular methods improve our ability to characterize the vaginal microbiota; however, they cannot characterize phenotype. We assessed the concordance of H2O2-producing Lactobacillus detected by culture with quantitative PCR (qPCR) detection of Lactobacillus species commonly assumed to be H2O2-producers.
Samples were collected as part of a prospective cohort study of HIV-1 seropositive US women. Cervicovaginal lavage specimens were tested for L. crispatus and L. jensenii using 16S rRNA gene qPCR assays. Vaginal swabs were cultured for Lactobacillus and tested for H2O2-production. We calculated a kappa statistic to assess concordance between culture and qPCR.
Culture and qPCR results were available for 376 visits from 57 women. Lactobacilli were detected by culture at 308 (82%) visits, of which 233 of 308 (76%) produced H2O2. L. crispatus and/or L. jensenii were detected at 215 (57%) visits. Concordance between detection of L. crispatus and/or L. jensenii by qPCR and H2O2-producing Lactobacillus by culture was 75% (kappa = 0.45).
Among HIV-1 seropositive women, there was a moderate level of concordance between H2O2-producing Lactobacillus detected by culture and the presence of L. crispatus and/or L. jensenii by qPCR. However, one-quarter of samples with growth of H2O2-producing lactobacilli did not have L. crispatus or L. jensenii detected by qPCR. This discordance may be due to the presence of other H2O2-producing Lactobacillus species.
Lactobacillus; Lactobacillus crispatus; Lactobacillus jensenii; Bacterial vaginosis; Culture; Hydrogen peroxide; 16S rRNA gene
Objective: Evaluate predictors of vaginal colonization with lactobacilli after treatment for bacterial vaginosis (BV). Methods. Vaginal fluid specimens from women with BV underwent qPCR for Lactobacillus crispatus, L. jensenii, and L. iners pre- and posttreatment. Results. Few women with BV were colonized with L. crispatus (4/44, 9%) or L. jensenii (1/44, 2%), though all had L. iners. One month posttreatment 12/44 (27%) had L. crispatus, 12/44 (27%) L. jensenii, and 43/44 (98%) L. iners. Presence of L. jensenii posttreatment was associated with cure (Risk Ratio (RR) 1.67; 95% CI 1.09–2.56); L. crispatus showed a similar trend (RR 1.41; 95% CI 0.89–2.24, P = 0.14). Receptive oral sex was associated with 2.2-log10 lower concentration of L. crispatus (95% CI −4.38, −.02), and digital-vaginal sex with 2.6-log10 lower concentration (95% CI −4.87, −.33). Conclusion. One month after BV treatment, few women established colonization with L. crispatus or L. jensenii. Few behaviors were associated with colonization.
The National Institute for Health and clinical Excellence (NICE) depression guideline (2004) and the updated Quality and Outcomes Framework (QOF) ( 2006) in general practice have introduced the concepts of screening severity assessment, for example using the Patient Health Questionnaire 9 (PHQ-9), and ‘stepped care’ for depression.
To explore primary care practitioner perspectives on the clinical utility of the NICE guideline and the impact of the QOF on diagnosis and management of depression in routine practice.
Design and setting
Qualitative study using focus groups from four multidisciplinary practice teams with diverse populations in south Yorkshire.
Four focus groups were conducted, using a topic guide and audiotaping. There were 38 participants: GPs, nurses, doctors in training, mental health workers, and a manager. Data analysis was iterative and thematic.
The NICE guideline, with its embedded principles of holism and evidence-based practice, was viewed positively but its impact was compromised by resource and practitioner barriers to implementation. The perceived imposition of the screening questions and severity assessments (PHQ-9) with no responsive training had required practitioners to work hard to minimise negative impacts on their work, for example: constantly adapting consultations to tick boxes; avoiding triggering open displays of distress without the time to offer appropriate care; positively managing how their patients were labelled. Further confusion was experienced around the evolving content of psychological interventions for depression.
Organisational barriers to the implementation of the NICE guideline and the limited scope of the QOF highlight the need for policy makers to work more effectively with the complex realities of general practice in order to systematically improve the quality and delivery of ‘managed’ care for depression.
depression; primary health care; qualitative
We examined the relationship of proinflammatory vaginal cytokines and secretory leukocyte protease inhibitor (SLPI) with genital HIV-1 shedding after controlling for genital coinfections. Fifty-seven HIV-1-infected women in Seattle, WA (n = 38) and Rochester, NY (n = 19) were followed every 3–4 months for a total of 391 visits. At each visit, plasma and cervicovaginal lavage (CVL) were tested for HIV-1 RNA using qPCR. Vaginal samples were tested for bacterial vaginosis, yeast, hydrogen peroxide-producing Lactobacillus colonization, Trichomonas vaginalis, Neisseria gonorrhea, Chlamydia trachomatis, CMV, and HSV shedding. CVL interleukins (IL)-1β, IL-6, IL-8, and SLPI were measured using ELISA. Linear regression with generalized estimating equations examined effects of cytokine concentrations on CVL HIV-1 RNA, adjusted for plasma HIV RNA, and measured coinfections. CVL IL-1β and IL-8 were significantly associated with CVL HIV-1 RNA. This persisted after adjusting for plasma HIV-1 RNA. Higher levels of IL-1β were associated with higher concentrations of HIV-1 RNA in CVL (β = 0.25, 95% CI 0.09, 0.42), as were higher levels of IL-8 (β = 0.34, 95% CI 0.17, 0.50). Adjusting for the presence of the coinfections described, this relationship was attenuated for IL-1β (β = 0.16; 95% CI –0.01, 0.33) but still significant for IL-8 (β = 0.29; 95% CI 0.13, 0.45). The proinflammatory cytokines IL-1β and IL-8 are associated with higher cervicovaginal HIV-1 RNA concentrations, even after controlling for plasma viral load and vaginal microbial cofactors. This association suggests that there may be additional, noninfectious causes of inflammation that increase cervicovaginal HIV-1 shedding.
To assess the volume of genital fluid collected for measuring the HIV-1 RNA level in cervicovaginal fluid, phosphate-buffered saline containing 10 mM LiCl was used. Thirty-eight women provided 275 cervicovaginal specimens. The estimated median volume of cervicovaginal fluid was 0.51 ml (interquartile range, 0.33, 0.69).
Dried blood spot (DBS) samples are a convenient way to collect infant blood for HIV-1 diagnostic testing. Minimizing the risk of false positives is critical for diagnostic tests. A protocol for processing and testing DBS for infant HIV-1 diagnosis was evaluated to identify the rate and source of false positive results. DBS were created on Flinders Technology Associates (FTA) filter paper with 500 copies/punch (high) or 5000 copies/punch (very high) concentrations of HIV-1 DNA. Blank discs of filter paper punched after DBS samples were tested for carry-over of HIV-1 DNA using nested PCR for the pol region. No false positives were detected in the 40 series using high concentration DBS. In series with very high concentrations of HIV-1, 8/246 (3%) reactions were falsely positive. When tubes were spun prior to opening, contact with caps minimized, and spaces left between lanes of the gel, repeat second round PCR of five false positives resulted in only one repeat false positive PCR. This study outlines procedures that minimize false positive results for nested PCR of HIV-1 DNA from DBS.
dried blood spots; infant HIV diagnosis; PCR contamination
Bacterial vaginosis (BV) is associated with preterm delivery, but there is little evidence that treatment improves pregnancy outcomes. We examined whether oral or vaginal metronidazole treatment for BV in early pregnancy was more effective in restoring the normal vaginal environment.
This was a randomized controlled trial comparing oral and intravaginal metronidazole for treatment of BV in early pregnancy (<20 weeks). Vaginal samples collected at baseline and 4 weeks after treatment were evaluated using gram stain, culture, colorimetric detection of sialidase, and immunoassay for measurement of proinflammatory cytokines interleukins-1β, -6, -8 (IL-1β, IL-6, IL-8) and secretory leukocyte protease inhibitor (SLPI). We compared the effect of treatment between groups (using chi-square and t test) and within individuals (McNemar's test).
Of 126 subjects, 108 (86%) completed follow-up (55 oral, 53 intravaginal). Of the study population, 34% achieved therapeutic cure, and this was not different between treatment groups. BV-associated bacteria were significantly reduced in both groups, but few subjects regained colonization with protective lactobacilli. Among women who achieved therapeutic cure, the level of IL-1β dropped significantly (p < 0.001) and SLPI increased (p = 0.003). More women in the vaginal treatment group had undetectable sialidase after treatment (p = 0.013).
Treatment with oral or intravaginal metronidazole in early pregnancy reduced colonization with BV-associated bacteria but was not effective in achieving therapeutic cure or in restoring healthy vaginal lactobacilli.
We compared vaginal proinflammatory cytokine and secretory leukocyte protease inhibitor (SLPI) concentrations among pregnant and nonpregnant women according to bacterial vaginosis (BV) status. One-hundred and twenty-two women at 12–20 weeks' gestation and 133 nonpregnant controls had vaginal concentrations of interleukin (IL)-1β, IL-6, IL-8, and SLPI measured by enzyme immunoassay. Multivariable linear regression was used to evaluate factors independently associated with vaginal cytokine and SLPI response. Pregnancy and BV were both independently associated with increased vaginal concentrations of IL-1β and IL-8; pregnant women had increased concentrations of SLPI, while women with BV had decreased SLPI concentrations.
Objective. When pathologic discrepancy arises between high-grade cytology on Papanicolaou (Pap) smear and low-grade histology on cervical biopsy, Loop Electrosurgical Excisional Procedure (LEEP) is one management alternative. Our objective was to determine whether the time from initial HGSIL Pap to LEEP affects the pathologic grade of the LEEP specimen.
Study Design. We performed a retrospective case-control study identifying LEEPs performed for discrepancy over a 10-year period (1997–2007). 121 subjects were separated into two groups based on LEEP pathology (≤CIN 1 and CIN 2,3) and compared using χ2.
Results. Of the 121 LEEP specimens, 67 (55.4%) had CIN 2,3. CIN 2,3 was more often discovered when LEEP was performed within 3 months of the HGSIL Pap smear versus after 5 months (55.2% versus 16.4%, P = .096).
Conclusion. Women undergoing LEEP for discrepancy >5 months from their HGSIL Pap demonstrated a trend toward less CIN 2,3 on LEEP pathology.
Little is known about short-term bacterial fluctuations in the human vagina. This study used PCR to assess the variability in concentrations of key vaginal bacteria in healthy women and the immediate response to antibiotic treatment in women with bacterial vaginosis (BV).
Twenty-two women assessed for BV using Amsel's criteria were evaluated daily for 7 or 14 days, then at 2, 3 and 4 weeks, using a panel of 11 bacterium-specific quantitative PCR assays. Participants with BV were treated with 5 days of intravaginal metronidazole. Participants without BV had vaginal biotas dominated by lactobacilli, whose levels fluctuated with menses. With onset of menstruation, quantities of Lactobacillus jensenii and Lactobacillus crispatus decreased and were found to be inversely related to Gardnerella vaginalis concentrations (p<0.001). Women with BV had a variety of fastidious bacteria whose concentrations dropped below detection thresholds 1–5 days after starting metronidazole. Recurrent BV was characterized by initial profound decreases of BV-associated bacteria after treatment followed by subsequent increases at relapse.
The microbiota of the human vagina can be highly dynamic. Healthy women are colonized with Lactobacillus species, but levels can change dramatically over a month. Marked increases in G. vaginalis were observed during menses. Participants with BV have diverse communities of fastidious bacteria that are depleted by vaginal metronidazole therapy. Women with recurrent BV initially respond to antibiotic treatment with steep declines in bacterial concentrations, but these bacteria later reemerge, suggesting that antibiotic resistance in these bacteria is not an important factor mediating BV recurrence.
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.
Several fastidious bacteria have been associated with bacterial vaginosis (BV) using broad-range bacterial PCR methods such as consensus sequence 16S rRNA gene PCR, but their role in BV remains poorly defined. We describe changes in vaginal bacterial concentrations following metronidazole therapy for BV. Vaginal swabs were collected from women with BV diagnosed using Amsel clinical criteria, and vaginal fluid was assessed by Gram stain to generate Nugent scores. Follow-up swabs were collected 1 month after a 5-day course of vaginal 0.75% metronidazole gel and analyzed for 24 subjects with cured BV and 24 subjects with persistent BV. Changes in bacterial concentrations were measured using eight bacterium-specific 16S rRNA gene quantitative PCR assays. DNA from several fastidious BV-associated bacteria (BVAB) were present at high concentrations in the vagina prior to treatment. Successful antibiotic therapy resulted in 3- to 4-log reductions in median bacterial loads of BVAB1 (P = 0.02), BVAB2 (P = 0.0004), BVAB3 (P = 0.03), a Megasphaera-like bacterium (P < 0.0001), Atopobium species (P < 0.0001), Leptotrichia/Sneathia species (P = 0.0002), and Gardnerella vaginalis (P < 0.0001). Median posttreatment bacterial levels did not change significantly in subjects with persistent BV except for a decline in levels of BVAB3. The presence or absence of BV is reflected by vaginal concentrations of BV-associated bacteria such as BVAB1, BVAB2, Leptotrichia/Sneathia species, Atopobium species, Gardnerella vaginalis, and a Megasphaera-like bacterium, suggesting that these bacteria play an important role in BV pathogenesis and may be suitable markers of disease and treatment response.
Bacterial vaginosis (BV) is a common condition that is associated with preterm birth and acquisition of complex communities of vaginal bacteria that include several fastidious species. Treatment of BV in pregnancy has mixed effects on the risk of preterm delivery, which some hypothesize is due to variable antibiotic efficacy for the fastidious bacteria. Both oral and intravaginal metronidazole can be used to treat bacterial vaginosis in pregnancy, but little is known about the impact of different routes of antibiotic administration on concentrations of fastidious vaginal bacteria.
This was a sub-study of a larger randomized trial of oral versus vaginal metronidazole for treatment of BV in pregnancy. Fifty-three women were evaluated, including 30 women who received oral metronidazole and 23 who received intravaginal metronidazole. Bacterial taxon-specific quantitative PCR assays were used to measure concentrations of bacterial vaginosis associated bacterium (BVAB) 1, 2, and 3, Gardnerella vaginalis, Atopobium species, Leptotrichia/Sneathia species, Megasphaera species, and Lactobacillus crispatus before and after antibiotic treatment.
Concentrations of Leptotrichia and Sneathia spp. and the fastidious Clostridia-like bacterium designated BVAB1 decreased significantly with oral (p = .002, p = .02) but not vaginal therapy (p = .141, p = .126). The fastidious bacterium BVAB3 did not significantly decrease with either treatment. Concentrations of Atopobium spp., reportedly resistant to metronidazole in vitro, dropped significantly with oral (p = .002) and vaginal (p = .001) treatment. There was no significant difference in the magnitude of change in bacterial concentrations between oral and vaginal treatment arms for any of the bacterial species. Lactobacillus crispatus concentrations did not change.
Both oral and vaginal metronidazole therapy in pregnant women result in a significant decrease in concentrations of most BV-associated anaerobic bacteria, with the exception that Leptotrichia, Sneathia and BVAB1 do not significantly decrease with vaginal metronidazole therapy. These data suggest that the route of antibiotic administration has a minor impact on bacterial eradication in pregnant women with BV.
This trial is registered with ClinicalTrials.gov, number NCT00153517
Collecting whole blood on filter paper simplifies the processing, transport, and storage of specimens used for the diagnosis of human immunodeficiency virus type 1 (HIV-1) and other tests. Specimens may be collected in tropical or rural areas with minimal facilities for handling specimens. To compare simulated tropical conditions with freezer storage, we examined the stability of HIV-1 DNA in dried blood spots (DBS) stored in humid heat and at −20°C. DBS were created by spotting 50-μl aliquots of whole blood on 903 filter paper. DNA was extracted from DBS at baseline and after 2, 6, or 12 months of storage at −20°C or at 37°C with ∼85% humidity. The DNA was tested undiluted or diluted using the Amplicor HIV-1 DNA PCR (Roche), version 1.5. Each reaction was scored positive, negative, or indeterminate based on optical density. Results were compared between storage conditions and over time. A total of 1,832 reactions from 916 DBS were analyzed, including 100 DBS at baseline, 418 stored at −20°C, and 398 stored at 37°C. A chi-square test showed fewer positive reactions for DBS stored at 37°C (55%) than for those stored at −20°C (78%) (P < 0.0001). Samples stored at −20°C showed little change in the probability of detection of HIV-1 DNA over time; the odds ratio (OR) was 0.93 after storage for 1 year. Samples stored at 37°C demonstrated a significant change in detection at 1 year (OR, 0.29). We conclude that exposure of DBS to 37°C and high humidity impaired the recovery of HIV-1 DNA from DBS, whereas DNA recovery was preserved when DBS were stored frozen.
A strong and self confident primary care workforce can deliver the highest quality care and outcomes equitably and cost effectively. To meet the increasing demands being made of it, primary care needs its own thriving research culture and knowledge base.
Review of recent developments supporting primary care clinical research.
Primary care research has benefited from a small group of passionate leaders and significant investment in recent decades in some countries. Emerging from this has been innovation in research design and focus, although less is known of the effect on research output.
Primary care research is now well placed to lead a broad re-vitalisation of academic medicine, answering questions of relevance to practitioners, patients, communities and Government. Key areas for future primary care research leaders to focus on include exposing undergraduates early to primary care research, integrating this early exposure with doctoral and postdoctoral research career support, further expanding cross disciplinary approaches, and developing useful measures of output for future primary care research investment.