Expanding U.S. Laboratory Capacity for Neisseria gonorrhoeae Antimicrobial Susceptibility Testing and Whole-Genome Sequencing through the CDC's Antibiotic Resistance Laboratory Network.

U.S. gonorrhea rates are rising, and antibiotic-resistant Neisseria gonorrhoeae (AR-Ng) is an urgent public health threat. Since implementation of nucleic acid amplification tests for N. gonorrhoeae identification, the capacity for culturing N. gonorrhoeae in the United States has declined, along with the ability to perform culture-based antimicrobial susceptibility testing (AST). Yet AST is critical for detecting and monitoring AR-Ng. In 2016, the CDC established the Antibiotic Resistance Laboratory Network (AR Lab Network) to shore up the national capacity for detecting several resistance threats including N. gonorrhoeae AR-Ng testing, a subactivity of the CDC's AR Lab Network, is performed in a tiered network of approximately 35 local laboratories, four regional laboratories (state public health laboratories in Maryland, Tennessee, Texas, and Washington), and the CDC's national reference laboratory. Local laboratories receive specimens from approximately 60 clinics associated with the Gonococcal Isolate Surveillance Project (GISP), enhanced GISP (eGISP), and the program Strengthening the U.S. Response to Resistant Gonorrhea (SURRG). They isolate and ship up to 20,000 isolates to regional laboratories for culture-based agar dilution AST with seven antibiotics and for whole-genome sequencing of up to 5,000 isolates. The CDC further examines concerning isolates and monitors genetic AR markers. During 2017 and 2018, the network tested 8,214 and 8,628 N. gonorrhoeae isolates, respectively, and the CDC received 531 and 646 concerning isolates and 605 and 3,159 sequences, respectively. In summary, the AR Lab Network supported the laboratory capacity for N. gonorrhoeae AST and associated genetic marker detection, expanding preexisting notification and analysis systems for resistance detection. Continued, robust AST and genomic capacity can help inform national public health monitoring and intervention.

Increases in Endogenous or Exogenous Progestins Promote Virus-Target Cell Interactions within the Non-human Primate Female Reproductive Tract.

Currently, there are mounting data suggesting that HIV-1 acquisition in women can be affected by the use of certain hormonal contraceptives. However, in non-human primate models, endogenous or exogenous progestin-dominant states are shown to increase acquisition. To gain mechanistic insights into this increased acquisition, we studied how mucosal barrier function and CD4+ T-cell and CD68+ macrophage density and localization changed in the presence of natural progestins or after injection with high-dose DMPA. The presence of natural or injected progestins increased virus penetration of the columnar epithelium and the infiltration of susceptible cells into a thinned squamous epithelium of the vaginal vault, increasing the likelihood of potential virus interactions with target cells. These data suggest that increasing either endogenous or exogenous progestin can alter female reproductive tract barrier properties and provide plausible mechanisms for increased HIV-1 acquisition risk in the presence of increased progestin levels.

Population-attributable fraction of tubal factor infertility associated with chlamydia.

BACKGROUND: Chlamydia trachomatis infection is highly prevalent among young women in the United States. Prevention of long-term sequelae of infection, including tubal factor infertility, is a primary goal of chlamydia screening and treatment activities. However, the population-attributable fraction of tubal factor infertility associated with chlamydia is unclear, and optimal measures for assessing tubal factor infertility and prior chlamydia in epidemiological studies have not been established. Black women have increased rates of chlamydia and tubal factor infertility compared with White women but have been underrepresented in prior studies of the association of chlamydia and tubal factor infertility. OBJECTIVES: The objectives of the study were to estimate the population-attributable fraction of tubal factor infertility associated with Chlamydia trachomatis infection by race (Black, non-Black) and assess how different definitions of Chlamydia trachomatis seropositivity and tubal factor infertility affect population-attributable fraction estimates. STUDY DESIGN: We conducted a case-control study, enrolling infertile women attending infertility practices in Birmingham, AL, and Pittsburgh, PA, during October 2012 through June 2015. Tubal factor infertility case status was primarily defined by unilateral or bilateral fallopian tube occlusion (cases) or bilateral fallopian tube patency (controls) on hysterosalpingogram. Alternate tubal factor infertility definitions incorporated history suggestive of tubal damage or were based on laparoscopic evidence of tubal damage. We aimed to enroll all eligible women, with an expected ratio of 1 and 3 controls per case for Black and non-Black women, respectively. We assessed Chlamydia trachomatis seropositivity with a commercial assay and a more sensitive research assay; our primary measure of seropositivity was defined as positivity on either assay. We estimated Chlamydia trachomatis seropositivity and calculated Chlamydia trachomatis-tubal factor infertility odds ratios and population-attributable fraction, stratified by race. RESULTS: We enrolled 107 Black women (47 cases, 60 controls) and 620 non-Black women (140 cases, 480 controls). Chlamydia trachomatis seropositivity by either assay was 81% (95% confidence interval, 73-89%) among Black and 31% (95% confidence interval, 28-35%) among non-Black participants (P < .001). Using the primary Chlamydia trachomatis seropositivity and tubal factor infertility definitions, no significant association was detected between chlamydia and tubal factor infertility among Blacks (odds ratio, 1.22, 95% confidence interval, 0.45-3.28) or non-Blacks (odds ratio, 1.41, 95% confidence interval, 0.95-2.09), and the estimated population-attributable fraction was 15% (95% confidence interval, -97% to 68%) among Blacks and 11% (95% confidence interval, -3% to 23%) among non-Blacks. Use of alternate serological measures and tubal factor infertility definitions had an impact on the magnitude of the chlamydia-tubal factor infertility association and resulted in a significant association among non-Blacks. CONCLUSION: Low population-attributable fraction estimates suggest factors in addition to chlamydia contribute to tubal factor infertility in the study population. However, high background Chlamydia trachomatis seropositivity among controls, most striking among Black participants, could have obscured an association with tubal factor infertility and resulted in a population-attributable fraction that underestimates the true etiological role of chlamydia. Choice of chlamydia and tubal factor infertility definitions also has an impact on the odds ratio and population-attributable fraction estimates.

Development of a rectal sexually transmitted infection (STI) Model in Rhesus macaques using Chlamydia trachomatis serovars E and L2.

BACKGROUND: Rectal STI coinfection models enhance the understanding of rectal HIV transmission risk factors. MATERIALS AND METHODS: Rhesus macaques (n=9) were exposed to one of three rectal Chlamydia trachomatis (CT) challenges: C. trachomatis L2 (CT-L2 ); C. trachomatis serovar E (CT-E), followed by CT-L2 ; or CT-E, treatment/clearance, then CT-L2 . Infections were monitored by PCR. Weekly blood and rectal secretion/lavage samples were collected for cytokine analyzes and/or epithelial sloughing, occult, and overt blood determinations. RESULTS: Chlamydial infections were successfully established in each animal, with varying degrees of persistence. Mucosal IL-1beta was upregulated in animals consecutively infected with CT-E then CT-L2 (P=.05). Epithelial sloughing was also significantly increased post-infection in this group (P=.0003). CONCLUSIONS: This study demonstrates successful rectal infection of rhesus macaques with CT-E and CT-L2 and describes measures of assessing rectal inflammation and pathology. Different infection strategies yield varying inflammatory and pathologic outcomes, providing well-described models for future SIV/SHIV susceptibility studies.

Combination Emtricitabine and Tenofovir Disoproxil Fumarate Prevents Vaginal Simian/Human Immunodeficiency Virus Infection in Macaques Harboring Chlamydia trachomatis and Trichomonas vaginalis.

Genital inflammation associated with sexually transmitted infections increases susceptibility to human immunodeficiency virus (HIV), but it is unclear whether the increased risk can reduce the efficacy of pre-exposure prophylaxis (PrEP). We investigated whether coinfection of macaques with Chlamydia trachomatis and Trichomonas vaginalis decreases the prophylactic efficacy of oral emtricitabine (FTC)/tenofovir disoproxil fumarate (TDF). Macaques were exposed to simian/human immunodeficiency virus (SHIV) vaginally each week for up to 16 weeks and received placebo or FTC/TDF pericoitally. All animals in the placebo group were infected with SHIV, while 4 of 6 PrEP recipients remained uninfected (P= .03). Oral FTC/TDF maintains efficacy in a macaque model of sexually transmitted coinfection, although the infection of 2 macaques signals a modest loss of PrEP activity.

Macaque models of enhanced susceptibility to HIV.

There are few nonhuman primate models of enhanced HIV susceptibility. Such models can improve comprehension of HIV acquisition risk factors and provide rigorous testing platforms for preclinical prevention strategies. This paper reviews past, current, and proposed research on macaque HIV acquisition risk models and identifies areas where modeling is significantly lacking. We compare different experimental approaches and provide practical considerations for designing macaque susceptibility studies. Modifiable (mucosal and systemic coinfections, hormonal contraception, and rectal lubricants) and non-modifiable (hormonal fluctuations) risk factors are highlighted. Risk acquisition models via vaginal, rectal, and penile challenge routes are discussed. There is no consensus on the best statistical model for evaluating increased susceptibility, and additional research is required. The use of enhanced susceptibility macaque models would benefit multiple facets of the HIV research field, including basic acquisition and pathogenesis studies as well as the vaccine and other biomedical preventions pipeline.

Analysis of putative mucosal SHIV susceptibility factors during repeated DMPA treatments in pigtail macaques.

BACKGROUND: Depot-medroxyprogesterone acetate (DMPA) has been associated in some studies with increased HIV susceptibility in women. We used a pigtail macaque model to document the effects of repeated DMPA treatments and their potential contribution to increased SHIV susceptibility. METHODS: Nine pigtails were administered 2.5, 1.5, or 0.5 mg/kg DMPA in study weeks one and four. Menstrual cycling, vaginal epithelial thickness, and other SHIV susceptibility factors were monitored for a mean of 24 study weeks. RESULTS: All DMPA treatments suppressed menstrual cycling and increased vaginal pH. The vaginal epithelium thinned naturally during baseline menstrual cycles (from mean of 351 to 161 µm in late-luteal phase). Following DMPA, the non-nucleated layer was temporarily absent. Two weeks post-second DMPA injection, mean epithelial thickness was 53, 45, and 167 µm for the descending doses, respectively. CONCLUSIONS: All animals showed temporal vaginal epithelial thinning with loss of the non-nucleated layer, and vaginal pH changes post-DMPA injections.

Relationship of menstrual cycle and vaginal infection in female rhesus macaques challenged with repeated, low doses of SIVmac251.

Varying susceptibility during menstrual cycling could be a factor for S(H)IV infection risk in female rhesus macaques. We retrospectively determined vaginal SIV infection time points relative to the menstrual cycle in a group of rhesus macaques (n=11) enrolled in an HIV transmission trial. Eight of nine rhesus macaques became infected around menstruation time.

Increased susceptibility to vaginal simian/human immunodeficiency virus transmission in pig-tailed macaques coinfected with Chlamydia trachomatis and Trichomonas vaginalis.

BACKGROUND: Sexually transmitted infections (STIs) are associated with an increased risk of human immunodeficiency virus (HIV) infection, but their biological effect on HIV susceptibility is not fully understood. METHODS: Female pig-tailed macaques inoculated with Chlamydia trachomatis and Trichomonas vaginalis (n = 9) or medium (controls; n = 7) were repeatedly challenged intravaginally with SHIVSF162p3. Virus levels were evaluated by real-time polymerase chain reaction, plasma and genital cytokine levels by Luminex assays, and STI clinical signs by colposcopy. RESULTS: Simian/HIV (SHIV) susceptibility was enhanced in STI-positive macaques (P = .04, by the log-rank test; relative risk, 2.5 [95% confidence interval, 1.1-5.6]). All STI-positive macaques were SHIV infected, whereas 3 controls (43%) remained uninfected. Moreover, relative to STI-negative animals, SHIV infections occurred earlier in the menstrual cycle in STI-positive macaques (P = .01, by the Wilcoxon test). Levels of inflammatory cytokines (interferon γ, interleukin 6, and granulocyte colony-stimulating factor [G-CSF]) were higher in STI-positive macaques during STI inoculation and SHIV exposure periods (P ≤ .05, by the Wilcoxon test). CONCLUSIONS: C. trachomatis and T. vaginalis infection increase the susceptibility to SHIV, likely because of prolonged genital tract inflammation. These novel data demonstrate a biological link between these nonulcerative STIs and the risk of SHIV infection, supporting epidemiological associations of HIV and STIs. This study establishes a macaque model for studies of high-risk HIV transmission and prevention.

Preclinical evaluation of the immunomodulatory lymphocyte trafficking drug FTY720 for HIV prevention in the female genital mucosa of macaques.

FTY720 has been shown to reduce inflammatory cytokines and immune cells in the genital mucosa of macaques. This pilot study examined the ability of FTY720 to inhibit HIV acquisition. Systemic treatment with FTY720 failed to prevent or delay vaginal SHIV transmission.

Evaluation of pigtail macaques as a model for the effects of copper intrauterine devices on HIV infection.

BACKGROUND: Long-acting, hormonal contraception may increase HIV risk. Copper intrauterine devices (IUDs) could serve as non-hormonal alternatives. We pilot a pigtail macaque model for evaluating HIV susceptibility factors during copper IUD use. METHODS: Frameless and flexible GyneFix(®) copper IUDs were surgically implanted into three SHIVSF 162p3 -positive macaques via hysterotomy and monitored for up to 4 months. Four macaques served as non-IUD controls. RESULTS: All animals retained the devices without complications. No consistent change in vaginal viral RNA or inflammatory cytokines was seen. Two animals had altered menstrual cycles and experienced marked thinning of vaginal epithelium after IUD insertion. Histological examination of uterine tissue at necropsy revealed endometrial ulceration and lymphocytic inflammation with glandular loss at sites of direct IUD contact. CONCLUSIONS: Although the need for insertion surgery could limit its usefulness, this model will allow studies on copper IUDs and SHIV shedding, disease progression, and HIV susceptibility factors.

SHIV susceptibility changes during the menstrual cycle of pigtail macaques.

BACKGROUND: Hormonal changes during menstrual cycling may affect susceptibility to HIV. METHODS: We determined the simian human immunodeficiency virus (SHIV) acquisition time point in 43 cycling pigtail macaques infected by repeated vaginal virus exposures initiated randomly in the cycle. RESULTS: SHIV infection was first detected in the follicular phase in 38 macaques (88%), and in the luteal phase in five macaques (12%), indicating a statistically significant timing difference. Assuming a 7-day eclipse phase, most infections occurred during or following a high-progesterone period associated with menstruation, vaginal epithelium thinning, and suppressed mucosal immunity. CONCLUSIONS: This raises questions whether other high-progesterone conditions (pregnancy, hormonal contraception) similarly affect HIV risk.

Development of a rectal sexually transmitted infection--HIV coinfection model utilizing Chlamydia trachomatis and SHIVSF162p3.

BACKGROUND: Rectal sexually transmitted infections (STIs) may increase HIV susceptibility in men who have sex with men (MSM), and Chlamydia trachomatis is prevalent among HIV-positive MSM. To study STIs and HIV infection in MSM, we first evaluated whether cynomolgus macaques can sustain both C. trachomatis and SHIVSF162p3 infections. METHODS: Four SHIVSF162p3 -positive male cynomolgus macaques were used (n = 3 rectally inoculated with 10(6) IFU; n = 1 control). Systemic and rectal SHIV RNA levels and cytokines were measured by real-time PCR and Luminex assays, respectively. RESULTS: Macaques were successfully Chlamydia infected. Rectal SHIV shedding (P = 0.02 χ(2) ) and levels of G-CSF, IL-1ra, IL-6, IL-8, IFN-γ, and TNF-α (P ≤ 0.01, Mann-Whitney) in rectal secretions increased following infection. CONCLUSIONS: These pilot data successfully demonstrate rectal C. trachomatis-SHIV coinfection in cynomolgus macaques and suggest the feasibility of a rectal C. trachomatis model for SHIV susceptibility and biomedical prevention studies in the context of rectal STIs.

Evaluation of the lymphocyte trafficking drug FTY720 in vaginal tissues.

BACKGROUND: FTY720 is an immunomodulatory agent that reduces lymphocytes in peripheral tissues and circulation. Such agents may be effective as vaginal microbicides for HIV prevention. Systemic or vaginal application of FTY720 may reduce lymphocyte concentrations in genital tissues, reducing HIV target cell numbers. METHODS: Five female pigtail macaques received topical vaginal gel FTY720 (n = 2), intravenous (i.v.) FTY720 (n = 2), or placebo gel (n = 1) in this pilot study. Circulating and mucosal lymphocytes and genital mucosa, cytokines, and tissue histology were analyzed to document topical and i.v. FTY720 effects. RESULTS: Topical and i.v. FTY720 appeared to decrease the levels of cervicovaginal IL-8, IL-1ra, and genital inflammatory cells. Small sample size precluded statistical analysis. Topical administration had no overt adverse effects. CONCLUSIONS: This study introduces FTY720 as an immunomodulatory agent for the vaginal mucosa, compares topical effects to those of i.v. administration, and provides the basis for future studies involving FTY720 for HIV prevention.

Lessons learned for public health workforce development: An evaluation of the centers for disease control and prevention's laboratory leadership service fellowship.

The Centers for Disease Control and Prevention launched the Laboratory Leadership Service (LLS) Fellowship Program in July 2015 to develop public health laboratory (PHL) leaders who will improve PHL quality and safety. This article describes a retrospective, summative evaluation to determine the extent to which LLS has met its short-term goals for PHL workforce development. The evaluation relied on existing data from routine LLS data collection and reporting, supplemented with a new alumni survey. The purpose of the design was threefold: 1) to reduce data collection burden on program staff and participants, 2) to assess the value and limits of routine fellowship data for comprehensive public health workforce development program evaluation, and 3) to identify ways to improve LLS's routine data collections for program evaluation. We used descriptive statistics, qualitative analysis, and participatory methods (i.e., a data party) to analyze and interpret data. Results show LLS short-term outcome achievement and highlight opportunities for program improvement, particularly related to the design of certain training requirements and for future evaluations. Overall, the evaluation contributes to lessons learned for PHL workforce development efforts, including how routine data collections can contribute to comprehensive public health workforce development evaluations.

Development of a pigtail macaque model of sexually transmitted infection/HIV coinfection using Chlamydia trachomatis, Trichomonas vaginalis, and SHIV(SF162P3).

BACKGROUND: Sexually transmitted infections (STIs) are associated with an increased risk of HIV infection. To model the interaction between STIs and HIV infection, we evaluated the capacity of the pigtail macaque model to sustain triple infection with Trichomonas vaginalis, Chlamydia trachomatis, and SHIV(SF162P3). METHODS: Seven SHIV(SF162P3) -infected pigtail macaques were inoculated with T. vaginalis only (n = 2), C. trachomatis only (n = 1), both T. vaginalis and C. trachomatis (n = 2), or control media (no STI; n = 2). Infections were confirmed by culture and/or nucleic acid testing. Genital mucosa was visualized by colposcopy. RESULTS: Characteristic gynecologic signs were observed for both STIs, but not in control animals. Manifestations were most prominent at days 7-10 post-infection. STIs persisted between 4 and 6 weeks and were cleared with antibiotics. CONCLUSIONS: These pilot studies demonstrate the first successful STI-SHIV triple infection of pigtail macaques, with clinical presentation of genital STI symptoms similar to those observed in humans.

Elevated cervical white blood cell infiltrate is associated with genital HIV detection in a longitudinal cohort of antiretroviral therapy-adherent women.

BACKGROUND: Identification of factors associated with the presence of human immunodeficiency virus (HIV) in female genital secretions is critical for intervention strategies targeting transmission and eliminating replication of genital virus. We sought to monitor the prevalence of genital HIV shedding in antiretroviral therapy-adherent women over time and to assess changes in the genital microenvironment. METHODS: Levels of cell-free HIV (HIV RNA) and HIV-infected cells (HIV DNA) were monitored in peripheral blood samples and cervical and vaginal fluid samples at monthly intervals in 11 women for 1 year. Genital tract infections and fluctuations in cervical and vaginal white blood cell counts were also evaluated at each study visit. RESULTS: Plasma HIV was undetectable at the majority of study visits; when detected, it was only at low levels. Throughout the study, genital HIV RNA and DNA were detected in each person. Combined genital HIV (RNA and DNA) was detected at 49.2% of study visits and was associated with an elevated concentration of cervical white blood cell infiltrate (odds ratio, 2.52 [95% confidence interval, 1.01-6.22]; P = .04). Infiltrate was not associated with a clinical disorder or patient-reported symptoms. CONCLUSIONS: Despite antiretroviral therapy adherence and clinically suppressed plasma viremia, HIV was intermittently detected in genital secretions and was associated with subclinical inflammation and cells trafficking to the cervical mucosa.

In vitro activity of EDTA and TOL-463 against Neisseria gonorrhoeae.

Neisseria gonorrhoeae quickly develops drug resistance. Time-kill curves revealed that EDTA and TOL-463 inhibit growth similar to penicillin, ciprofloxacin, and azithromycin. Furthermore, synergistic and additive antimicrobial interactions occurred when EDTA and TOL-463 were combined with penicillin or azithromycin, respectively, suggesting that further investigations into these unconventional antimicrobials may be advantageous.

Topical tenofovir protects against vaginal simian HIV infection in macaques coinfected with Chlamydia trachomatis and Trichomonas vaginalis.

BACKGROUND: Chlamydia trachomatis and Trichomonas vaginalis, two prevalent sexually transmitted infections, are known to increase HIV risk in women and could potentially diminish preexposure prophylaxis efficacy, particularly for topical interventions that rely on local protection. We investigated in macaques whether coinfection with Chlamydia trachomatis/Trichomonas vaginalis reduces protection by vaginal tenofovir (TFV) gel. METHODS: Vaginal TFV gel dosing previously shown to provide 100 or 74% protection when applied either 30 min or 3 days before simian HIV(SHIV) challenge was assessed in pigtailed macaques coinfected with Chlamydia trachomatis/Trichomonas vaginalis and challenged twice weekly with SHIV162p3 for up to 10 weeks (two menstrual cycles). Three groups of six macaques received either placebo or 1% TFV gel 30 min or 3 days before each SHIV challenge. We additionally assessed TFV and TFV diphosphate concentrations in plasma and vaginal tissues in Chlamydia trachomatis/Trichomonas vaginalis coinfected (n = 4) and uninfected (n = 4) macaques. RESULTS: Chlamydia trachomatis/Trichomonas vaginalis coinfections were maintained during the SHIV challenge period. All macaques that received placebo gel were SHIV infected after a median of seven challenges (one menstrual cycle). In contrast, no infections were observed in macaques treated with TFV gel 30 min before SHIV challenge (P < 0.001). Efficacy was reduced to 60% when TFV gel was applied 3 days before SHIV challenge (P = 0.07). Plasma TFV and TFV diphosphate concentrations in tissues and vaginal lymphocytes were significantly higher in Chlamydia trachomatis/Trichomonas vaginalis coinfected compared with Chlamydia trachomatis/Trichomonas vaginalis uninfected macaques. CONCLUSION: Our findings in this model suggest that Chlamydia trachomatis/Trichomonas vaginalis coinfection may have little or no impact on the efficacy of highly effective topical TFV modalities and highlight a significant modulation of TFV pharmacokinetics.

Recovery of Neisseria gonorrhoeae from 4 commercially available transport systems.

Four commercial transport systems for the recovery of Neisseria gonorrhoeae were evaluated in support of the need to obtain culture isolates for the detection of antimicrobial resistance. Bacterial recovery from the InTray GC system was superior with minimal loss of viability in contrast to non-nutritive transport systems.