Species distribution and in vitro antifungal susceptibility of oral yeast isolates from Tanzanian HIV-infected patients with primary and recurrent oropharyngeal candidiasis

\ud In Tanzania, little is known on the species distribution and antifungal susceptibility profiles of yeast isolates from HIV-infected patients with primary and recurrent oropharyngeal candidiasis. A total of 296 clinical oral yeasts were isolated from 292 HIV-infected patients with oropharyngeal candidiasis at the Muhimbili National Hospital, Dar es Salaam, Tanzania. Identification of the yeasts was performed using standard phenotypic methods. Antifungal susceptibility to fluconazole, itraconazole, miconazole, clotrimazole, amphotericin B and nystatin was assessed using a broth microdilution format according to the guidelines of the Clinical and Laboratory Standard Institute (CLSI; M27-A2). Candida albicans was the most frequently isolated species from 250 (84.5%) patients followed by C. glabrata from 20 (6.8%) patients, and C. krusei from 10 (3.4%) patients. There was no observed significant difference in species distribution between patients with primary and recurrent oropharyngeal candidiasis, but isolates cultured from patients previously treated were significantly less susceptible to the azole compounds compared to those cultured from antifungal naïve patients. C. albicans was the most frequently isolated species from patients with oropharyngeal candidiasis. Oral yeast isolates from Tanzania had high level susceptibility to the antifungal agents tested. Recurrent oropharyngeal candidiasis and previous antifungal therapy significantly correlated with reduced susceptibility to azoles antifungal agents.\u

Gut dysbiosis associates with cytokine production capacity in viral-suppressed people living with HIV

BACKGROUND: People living with human immunodeficiency virus (PLHIV) are exposed to chronic immune dysregulation, even when virus replication is suppressed by antiretroviral therapy (ART). Given the emerging role of the gut microbiome in immunity, we hypothesized that the gut microbiome may be related to the cytokine production capacity of PLHIV.METHODS: To test this hypothesis, we collected metagenomic data from 143 ART-treated PLHIV and assessed the ex vivo production capacity of eight different cytokines [interleukin-1β (IL-1β), IL-6, IL-1Ra, IL-10, IL-17, IL-22, tumor necrosis factor, and interferon-γ] in response to different stimuli. We also characterized CD4 + T-cell counts, HIV reservoir, and other clinical parameters. RESULTS: Compared with 190 age- and sex-matched controls and a second independent control cohort, PLHIV showed microbial dysbiosis that was correlated with viral reservoir levels (CD4 + T-cell-associated HIV-1 DNA), cytokine production capacity, and sexual behavior. Notably, we identified two genetically different P. copri strains that were enriched in either PLHIV or healthy controls. The control-related strain showed a stronger negative association with cytokine production capacity than the PLHIV-related strain, particularly for Pam3Cys-incuded IL-6 and IL-10 production. The control-related strain is also positively associated with CD4 + T-cell level. CONCLUSIONS: Our findings suggest that modulating the gut microbiome may be a strategy to modulate immune response in PLHIV.</p

A randomized feasibility trial comparing four antimalarial drug regimens to induce Plasmodium falciparum gametocytemia in the controlled human malaria infection model.

Background: Malaria elimination strategies require a thorough understanding of parasite transmission from human to mosquito. A clinical model to induce gametocytes to understand their dynamics and evaluate transmission-blocking interventions (TBI) is currently unavailable. Here, we explore the use of the well-established Controlled Human Malaria Infection model (CHMI) to induce gametocyte carriage with different antimalarial drug regimens. Methods: In a single centre, open-label randomised trial, healthy malaria-naive participants (aged 18–35 years) were infected with Plasmodium falciparum by bites of infected Anopheles mosquitoes. Participants were randomly allocated to four different treatment arms (n = 4 per arm) comprising low-dose (LD) piperaquine (PIP) or sulfadoxine-pyrimethamine (SP), followed by a curative regimen upon recrudescence. Male and female gametocyte densities were determined by molecular assays. Results: Mature gametocytes were observed in all participants (16/16, 100%). Gametocytes appeared 8.5–12 days after the first detection of asexual parasites. Peak gametocyte densities and gametocyte burden was highest in the LD-PIP/SP arm, and associated with the preceding asexual parasite biomass (p=0.026). Male gametocytes had a mean estimated circulation time of 2.7 days (95% CI 1.5–3.9) compared to 5.1 days (95% CI 4.1–6.1) for female gametocytes. Exploratory mosquito feeding assays showed successful sporadic mosquito infections. There were no serious adverse events or significant differences in the occurrence and severity of adverse events between study arms (p=0.49 and p=0.28). Conclusions: The early appearance of gametocytes indicates gametocyte commitment during the first wave of asexual parasites emerging from the liver. Treatment by LD-PIP followed by a curative SP regimen, results in the highest gametocyte densities and the largest number of gametocyte-positive days. This model can be used to evaluate the effect of drugs and vaccines on gametocyte dynamics, and lays the foundation for fulfilling the critical unmet need to evaluate transmission-blocking interventions against falciparum malaria for downstream selection and clinical development. Funding: Funded by PATH Malaria Vaccine Initiative (MVI). Clinical trial number: NCT02836002

Modest heterologous protection after Plasmodium falciparum sporozoite immunization: a double-blind randomized controlled clinical trial.

BACKGROUND: A highly efficacious vaccine is needed for malaria control and eradication. Immunization with Plasmodium falciparum NF54 parasites under chemoprophylaxis (chemoprophylaxis and sporozoite (CPS)-immunization) induces the most efficient long-lasting protection against a homologous parasite. However, parasite genetic diversity is a major hurdle for protection against heterologous strains. METHODS: We conducted a double-blind, randomized controlled trial in 39 healthy participants of NF54-CPS immunization by bites of 45 NF54-infected (n = 24 volunteers) or uninfected mosquitoes (placebo; n = 15 volunteers) against a controlled human malaria infection with the homologous NF54 or the genetically distinct NF135.C10 and NF166.C8 clones. Cellular and humoral immune assays were performed as well as genetic characterization of the parasite clones. RESULTS: NF54-CPS immunization induced complete protection in 5/5 volunteers against NF54 challenge infection at 14 weeks post-immunization, but sterilely protected only 2/10 and 1/9 volunteers against NF135.C10 and NF166.C8 challenge infection, respectively. Post-immunization plasma showed a significantly lower capacity to block heterologous parasite development in primary human hepatocytes compared to NF54. Whole genome sequencing showed that NF135.C10 and NF166.C8 have amino acid changes in multiple antigens targeted by CPS-induced antibodies. Volunteers protected against heterologous challenge were among the stronger immune responders to in vitro parasite stimulation. CONCLUSIONS: Although highly protective against homologous parasites, NF54-CPS-induced immunity is less effective against heterologous parasite clones both in vivo and in vitro. Our data indicate that whole sporozoite-based vaccine approaches require more potent immune responses for heterologous protection. TRIAL REGISTRATION: This trial is registered in clinicaltrials.gov, under identifier NCT02098590

Safety, Immunogenicity, and Protective Efficacy of Intradermal Immunization with Aseptic, Purified, Cryopreserved Plasmodium falciparum Sporozoites in Volunteers Under Chloroquine Prophylaxis

Immunization of volunteers under chloroquine prophylaxis by bites of *Plasmodium falciparum* sporozoite (PfSPZ)–infected mosquitoes induces > 90% protection against controlled human malaria infection (CHMI). We studied intradermal immunization with cryopreserved, infectious PfSPZ in volunteers taking chloroquine (PfSPZ chemoprophylaxis vaccine [CVac]). Vaccine groups 1 and 3 received 3x monthly immunizations with 7.5 x 10^4 PfSPZ. Control groups 2 and 4 received normal saline. Groups 1 and 2 underwent CHMI (#1) by mosquito bite 60 days after the third immunization. Groups 3 and 4 were boosted 168 days after the third immunization and underwent CHMI (#2) 137 days later. Vaccinees (11/20, 55%) and controls (6/10, 60%) had the same percentage of mild to moderate solicited adverse events. After CHMI #1, 8/10 vaccinees (group 1) and 5/5 controls (group 2) became parasitemic by microscopy; the two negatives were positive by quantitative real-time polymerase chain reaction (qPCR). After CHMI #2, all vaccinees in group 3 and controls in group 4 were parasitemic by qPCR. Vaccinees showed weak antibody and no detectable cellular immune responses. Intradermal immunization with up to 3 x 10^5 PfSPZ-CVac was safe, but induced only minimal immune responses and no sterile protection against Pf CHMI. INTRODUCTIO

Etravirine pharmacokinetics in HIV-infected pregnant women

__Background__ The study goal was to describe etravirine pharmacokinetics during pregnancy and postpartum in HIV-infected women. __Methods__ IMPAACT P1026s and PANNA are on-going, non-randomized, open-label, parallel-group, multi-center phase-IV prospective studies in HIV-infected pregnant women. Intensive steady-state 12-h pharmacokinetic profiles were performed from 2nd trimester through postpartum. Etravirine was measured at two labs using validated ultra performance liquid chromatography (detection limits: 0.020 and 0.026 mcg/mL). __Results__ Fifteen women took etravirine 200 mg twice-daily. Etravirine AUC0-12 was higher in the 3rd trimester compared to paired postpartum data by 34% (median 8.3 vs. 5.3 mcg*h/mL, p = 0.068). Etravirine apparent oral clearance was significantly lower in the 3rd trimester of pregnancy compared to paired postpartum data by 52% (median 24 vs. 38 L/h, p = 0.025). The median ratio of cord blood to maternal plasma concentration at delivery was 0.52 (range: 0.19-4.25) and no perinatal transmission occurred. __Conclusion__ Etravirine apparent oral clearance is reduced and exposure increased during the third trimester of pregnancy. Based on prior dose-ranging and safety data, no dose adjustment is necessary for maternal health but the effects of etravirine in utero are unknown. Maternal health and infant outcomes should be closely monitored until further infant safety data are available. __Clinical Trial registration:__ The IMPAACT protocol P1026s and PANNA study are registered at ClinicalTrials.gov under NCT00042289 and NCT00825929

OECD validation study to assess intra- and inter-laboratory reproducibility of the zebrafish embryo toxicity test for acute aquatic toxicity testing

The OECD validation study of the zebrafish embryo acute toxicity test (ZFET) for acute aquatic toxicity testing evaluated the ZFET reproducibility by testing 20 chemicals at 5 different concentrations in 3 independent runs in at least 3 laboratories. Stock solutions and test concentrations were analytically confirmed for 11 chemicals. Newly fertilised zebrafish eggs (20/concentration and control) were exposed for 96 h to chemicals. Four apical endpoints were recorded daily as indicators of acute lethality: coagulation of the embryo, lack of somite formation, non-detachment of the tail bud from the yolk sac and lack of heartbeat. Results (LC50 values for 48/96 h exposure) show that the ZFET is a robust method with a good intra- and inter-laboratory reproducibility (CV 30%) for some very toxic or volatile chemicals, and chemicals tested close to their limit of solubility. The ZFET is now available as OECD Test Guideline 236. Considering the high predictive capacity of the ZFET demonstrated by Belanger et al. (2013) in their retrospective analysis of acute fish toxicity and fish embryo acute toxicity data, the ZFET is ready to be considered for acute fish toxicity for regulatory purposes

Seasonal Variation in Vitamin D3 Levels Is Paralleled by Changes in the Peripheral Blood Human T Cell Compartment

It is well-recognized that vitamin D3 has immune-modulatory properties and that the variation in ultraviolet (UV) exposure affects vitamin D3 status. Here, we investigated if and to what extent seasonality of vitamin D3 levels are associated with changes in T cell numbers and phenotypes. Every three months during the course of the entire year, human PBMC and whole blood from 15 healthy subjects were sampled and analyzed using flow cytometry. We observed that elevated serum 25(OH)D3 and 1,25(OH)2D3 levels in summer were associated with a higher number of peripheral CD4+ and CD8+ T cells. In addition, an increase in naïve CD4+CD45RA+ T cells with a reciprocal drop in memory CD4+CD45RO+ T cells was observed. The increase in CD4+CD45RA+ T cell count was a result of heightened proliferative capacity rather than recent thymic emigration of T cells. The percentage of Treg dropped in summer, but not the absolute Treg numbers. Notably, in the Treg population, the levels of forkhead box protein 3 (Foxp3) expression were increased in summer. Skin, gut and lymphoid tissue homing potential was increased during summer as well, exemplified by increased CCR4, CCR6, CLA, CCR9 and CCR7 levels. Also, in summer, CD4+ and CD8+ T cells revealed a reduced capacity to produce pro-inflammatory cytokines. In conclusion, seasonal variation in vitamin D3 status in vivo throughout the year is associated with changes in the human peripheral T cell compartment and may as such explain some of the seasonal variation in immune status which has been observed previously. Given that the current observations are limited to healthy adult males, larger population-based studies would be useful to validate these findings

Gut dysbiosis associates with cytokine production capacity in viral-suppressed people living with HIV

BackgroundPeople living with human immunodeficiency virus (PLHIV) are exposed to chronic immune dysregulation, even when virus replication is suppressed by antiretroviral therapy (ART). Given the emerging role of the gut microbiome in immunity, we hypothesized that the gut microbiome may be related to the cytokine production capacity of PLHIV.MethodsTo test this hypothesis, we collected metagenomic data from 143 ART-treated PLHIV and assessed the ex vivo production capacity of eight different cytokines [interleukin-1β (IL-1β), IL-6, IL-1Ra, IL-10, IL-17, IL-22, tumor necrosis factor, and interferon-γ] in response to different stimuli. We also characterized CD4+ T-cell counts, HIV reservoir, and other clinical parameters.ResultsCompared with 190 age- and sex-matched controls and a second independent control cohort, PLHIV showed microbial dysbiosis that was correlated with viral reservoir levels (CD4+ T-cell–associated HIV-1 DNA), cytokine production capacity, and sexual behavior. Notably, we identified two genetically different P. copri strains that were enriched in either PLHIV or healthy controls. The control-related strain showed a stronger negative association with cytokine production capacity than the PLHIV-related strain, particularly for Pam3Cys-incuded IL-6 and IL-10 production. The control-related strain is also positively associated with CD4+ T-cell level.ConclusionsOur findings suggest that modulating the gut microbiome may be a strategy to modulate immune response in PLHIV

Detection of amyloid beta aggregates in the brain of BALB/c mice after Chlamydia pneumoniae infection

Neuroinflammation, initiated by cerebral infection, is increasingly postulated as an aetiological factor in neurodegenerative diseases such as Alzheimer’s disease (AD). We investigated whether Chlamydia pneumoniae (Cpn) infection results in extracellular aggregation of amyloid beta (Aβ) in BALB/c mice. At 1 week post intranasal infection (p.i.), Cpn DNA was detected predominantly in the olfactory bulbs by PCR, whereas brains at 1 and 3 months p.i. were Cpn negative. At 1 and 3 months p.i., extracellular Aβ immunoreactivity was detected in the brain of Cpn-infected mice but also in the brain of mock-infected mice and mice that were neither Cpn infected nor mock infected. However, these extracellular Aβ aggregates showed morphological differences compared to extracellular Aβ aggregates detected in the brain of transgenic APP751SL/PS1M146L mice. These data do not unequivocally support the hypothesis that Cpn infection induces the formation of AD-like Aβ plaques in the brain of BALB/c mice, as suggested before. However, future studies are required to resolve these differences and to investigate whether Cpn is indeed an etiological factor in AD pathogenesis