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Background: Cellular immune responses characterized by interferon gamma (IFN-gamma) production enhance clearance and confer protective immunity against Chlamydia trachomatis infection but have not been simultaneously investigated in systemic and mucosal compartments.Methods: With use of the IFN-gamma enzyme-linked immunosorbent spot assay, we investigated immune responses to Chlamydia elementary body (EB) and 3 genotypically variant heat shock protein 60 (CHSP60) antigens using peripheral blood mononuclear cells and endometrial mononuclear cells obtained from a female sex worker cohort with high levels of exposure to C. trachomatis.Results: Although we observed a marginally higher frequency of IFN-gamma responses to EB in peripheral blood mononuclear cells, compared with the frequency in endometrial mononuclear cells, the magnitudes of systemic and mucosal responses were similar except for preferential targeting of CHSP60 type 2 by endometrial mononuclear cells. Systemic and mucosal responses were highly correlated for EB and CHSP60 types 1 and 2 but not type 3. The frequency and magnitude of systemic responses specific for EB and CHSP60 type 1 were greater for CD4+ T cells than they were for CD8+ T cells, whereas preferential targeting by CHSP60 types 2 and 3 was undetectable. IFN-gamma response to CHSP60 type 1 by peripheral blood mononuclear cells was inversely correlated with systemic antibody titers to CHSP60 type 1.Conclusion: Systemic and mucosal IFN-gamma responses are correlated, with preferential systemic targeting of CD4+ T cells. Furthermore, CHSP60 type 1 response is largely CD4+ T cell mediated and follows discrete T helper 1 and T helper 2 pathways. [ABSTRACT FROM AUTHOR]

Background: Respiratory Syncytial Virus (RSV) infections are a cause of significant morbidity and mortality in children and the elderly. Despite the clinical burden of disease, very little is known about the inter- and intra-seasonal genomic variability of RSV. Furthermore, the recent approval of vaccines and monoclonal antibody therapies will likely lead to higher selective pressure on RSV. Genomic surveillance will be essential to monitor viral changes and inform future therapeutic developments and public health responses. Here, we describe the development of an amplicon-based whole-genome sequencing assay for RSV to enable genomic surveillance., Methods: A 750-bp overlapping amplicon design was developed to co-amplify RSV-A/-B directly from patient samples collected during two respiratory illness seasons (2022/23, 2023/24) for whole-genome sequencing. RSV subtype, clade, and F-protein antigenic site sequences were determined with a custom analytical pipeline., Results: Of the 429 specimens included in the study 410 (95.6 %) samples met acceptability. Our data demonstrated co-circulation of both RSV subtypes, with increasing predominance of RSV-A since 2022. There were seven genomic clades of RSV-A, while >95 % of RSV-B belonged to a single clade. 1.5 % of samples had amino acid changes within the binding sites of the current RSV therapeutics Palivizumab or Nirsevimab., Conclusions: Continuous monitoring of RSV genotypes and mutations will be critical for understanding the impact of new therapeutics and vaccines on RSV epidemiology and detecting emergence of vaccine-escape and/or antiviral resistant mutations., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest, (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Background: The gut microbiota (GM) has proven to be essential for both physical health and mental wellbeing, yet the forces that ultimately shape its composition remain opaque. One critical force known to affect the GM is the social environment. Prior work in humans and free-ranging non-human primates has shown that cohabitation and frequent social interaction can lead to changes in GM composition. However, it is difficult to assess the direction of causation in these studies, and interpretations are complicated by the influence of uncontrolled but correlated factors, such as shared diet., Results: We performed a 15-month longitudinal investigation wherein we disentangled the impacts of diet and social living conditions on GM composition in a captive cohort of 13 male cynomolgus macaques. The animals were in single housing for the first 3 months of the study initially with a variable diet. After baseline data collection they were placed on a controlled diet for the remainder of the study. Following this diet shift the animals were moved to paired housing for 6 months, enabling enhanced social interaction, and then subsequently returned to single housing at the end of our study. This structured sequencing of diet and housing changes allowed us to assess their distinct impacts on GM composition. We found that the early dietary adjustments led to GM changes in both alpha and beta diversity, whereas changes in social living conditions only altered beta diversity. With respect to the latter, we found that two particular bacterial families - Lactobacillaceae and Clostridiaceae - demonstrated significant shifts in abundance during the transition from single housing to paired housing, which was distinct from the shifts we observed based on a change in diet. Conversely, we found that other bacteria previously associated with sociality were not altered based on changes in social living conditions but rather only by changes in diet., Conclusions: Together, these findings decouple the influences that diet and social living have on GM composition and reconcile previous observations in the human and animal literatures. Moreover, the results indicate biological alterations of the gut that may, in part, mediate the relationship between sociality and wellbeing., Competing Interests: Declarations Ethics approval and consent to participate All procedures and animal experiments were approved by the Queen’s University Animal Care Committee and were conducted in full compliance with the guidelines and policies of the Canadian Council on Animal Care and the Animals for Research Act (reference number: 2020–2024). Consent for publication Not applicable. Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Background: Globally, Creutzfeldt-Jakob disease (CJD) affects one in one million people annually, but there is a paucity of recent Canadian data. This study summarizes epidemiology trends and diagnostic timelines of laboratory-confirmed CJD cases in three tertiary Ontario hospitals., Method: Using laboratory information systems, we identified 30 patients with a laboratory-confirmed CJD diagnosis between 2012 and 2022 at three major tertiary hospitals in Ontario. Retrospective chart reviews were then completed., Results: Patients had a mean of 2.2 hospital visits (SD, 1.2) prior to being admitted for testing. The most common symptom presentations included loss of coordination (63.3%), behavioral changes (60%), progressive mobility loss (53.4%), memory loss (50.0%), and involuntary movements (50.0%). Magnetic resonance imaging findings showed potential CJD in 76.7% of cases, and 56.7% exhibited periodic sharp wave complexes characteristic of CJD on electroencephalogram. The mean duration from symptom onset to microbiologic testing was 91 days (SD, 90.7). End-point quaking-induced conversion (EP-QuIC) testing of cerebrospinal fluid was positive in 90.0% of patients, while 83.3% tested positive for 14-3-3 on enzyme-linked immunosorbent assay. Elevated cerebrospinal fluid 14-3-3 levels significantly correlated with shorter duration from symptom onset to death ( R 2 = 0.71, F = 19.55, P = .0022). Post-diagnosis, 46.7% of patients were discharged home, 16.6% were transferred to external palliative care or hospice facilities, and 36.7% died during admission. The mean time from symptom onset to death was 121 days (SD, 120.7), and from diagnosis to death 35 days (SD, 83.9)., Conclusions: This study highlights the importance of early CJD consideration and laboratory testing when appropriate neurologic symptoms are present., Competing Interests: Potential conflicts of interest. All authors: No reported conflicts., (© The Author(s) 2024. Published by Oxford University Press on behalf of Infectious Diseases Society of America.)

Current guideline recommends the use of two identification methods for Neisseria gonorrhoeae. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) is now used for primary identification and may be sufficient for definitive identification of N. gonorrhoeae. The performance of three secondary tests (BactiCard, RapID NH and NET test) were compared using 45 bacterial isolates, including 37 Neisseria species. These secondary tests demonstrated diminished specificity (67% - 88%) for N. gonorrhoeae compared with MALDI-TOF. Additionally, data from six clinical microbiology laboratories was used to compare confirmatory test costs and the agreement of results with MALDI-TOF. Discrepancies were documented for 9.4% of isolates, though all isolates (n= 288) identified by MALDI-TOF as N. gonorrhoeae were confirmed by the reference laboratory. These data demonstrate that MALDI-TOF alone is sufficient for N. gonorrhoeae identification, as secondary did not add diagnostic value but do add costs to the testing process., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: In 2022, the global dissemination of mpox virus (MPXV) outside endemic regions prompted the expansion of diagnostic testing worldwide. This study assesses the performance characteristics of 5 real-time polymerase chain reaction (PCR) assays in detecting MPXV during the 2022 outbreak., Methods: Clinical specimens collected from patients across Ontario, Canada, were tested on the following assays: RealStar Orthopoxyvirus PCR and FlexStar Monkeypox virus PCR (Altona Diagnostics), Novaplex MPXV (Seegene), VIASURE Monkeypox virus Real Time PCR Reagents (CerTest Biotec), and a laboratory-developed test. Positive percent agreement (PPA), negative percent agreement (NPA), relative limit of detection (LOD), and precision were evaluated and MPXV lineages were determined using an amplicon-based whole-genome sequencing (WGS) assay., Results: Swabs were collected from various anatomic sites (65 positive and 30 negative). All assays demonstrated 100% NPA (95% confidence interval, 88.4%/88.1%-100.0%), with PPA ranging from 92.2% (82.7%-97.4%) to 96.9% (89.3%-99.6%). LOD and precision were comparable across assays, with coefficient of variations <3%. WGS analysis identified 6 lineages, all belonging to subclade IIb., Conclusions: The assays exhibited excellent PPA, NPA, LOD, and precision. Ongoing performance monitoring is essential to detect assay escape mutants and ensure universal detection of evolving MPXV strains., Competing Interests: Potential conflicts of interest. K. B. and L. M. M. and received PCR kits in kind from Altona Diagnostics Canada, Seegene Canada, and BD Canada. L. M. M. is the president of the board of the Canadian Foundation for Infectious Diseases. R. K. and P. M. S. are recipients of financial support from the University of Toronto EPIC Mpox grant. All other authors report no potential conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed., (© The Author(s) 2024. Published by Oxford University Press on behalf of Infectious Diseases Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Clostridioides difficile is the leading cause of antibiotic-associated infectious diarrhea. The development of C.difficile infection is tied to perturbations of the bacterial community in the gastrointestinal tract, called the gastrointestinal microbiota. Repairing the gastrointestinal microbiota by introducing lab-designed bacterial communities, or defined microbial communities, has recently shown promise as therapeutics against C.difficile infection, however, the mechanisms of action of defined microbial communities remain unclear. Using an antibiotic- C.difficile mouse model, we report the ability of an 18-member community and a refined 4-member community to protect mice from two ribotypes of C.difficile (CD027, CD078; p < 0.05). Furthermore, bacteria-free supernatant delivered orally to mice from the 4-member community proteolyzed C.difficile toxins in vitro and protected mice from C.difficile infection in vivo (p < 0.05). This study demonstrates that bacteria-free supernatant is sufficient to protect mice from C.difficile; and could be further explored as a therapeutic strategy against C.difficile infection., (© 2024. The Author(s).)

Background: In vitro data suggested reduced neutralizing capacity of sotrovimab, a monoclonal antibody, against Omicron BA.2 subvariant. However, limited in vivo data exist regarding clinical effectiveness of sotrovimab for coronavirus disease 2019 (COVID-19) due to Omicron BA.2., Methods: A multicentre, retrospective cohort study was conducted at three Canadian academic tertiary centres. Electronic medical records were reviewed for patients ≥ 18 years with mild COVID-19 (sequencing-confirmed Omicron BA.1 or BA.2) treated with sotrovimab between February 1 to April 1, 2022. Thirty-day co-primary outcomes included hospitalization due to moderate or severe COVID-19; all-cause intensive care unit (ICU) admission, and all-cause mortality. Risk differences (BA.2 minus BA.1 group) for co-primary outcomes were adjusted with propensity score matching (e.g., age, sex, vaccination, immunocompromised status)., Results: Eighty-five patients were included (15 BA.2, 70 BA.1) with similar baseline characteristics between groups. Adjusted risk differences were non-statistically significant between groups for 30-day hospitalization (- 14.3%; 95% confidence interval (CI): - 32.6 to 4.0%), ICU admission (- 7.1%; 95%CI: - 20.6 to 6.3%), and mortality (- 7.1%; 95%CI: - 20.6 to 6.3%)., Conclusions: No differences were demonstrated in hospitalization, ICU admission, or mortality rates within 30 days between sotrovimab-treated patients with BA.1 versus BA.2 infection. More real-world data may be helpful to properly assess sotrovimab's effectiveness against infections due to specific emerging COVID-19 variants., (© 2024. The Author(s).)

Background & Aims: Abdominal pain is a major symptom of diseases that are associated with microbial dysbiosis, including irritable bowel syndrome and inflammatory bowel disease. Germ-free mice are more prone to abdominal pain than conventionally housed mice, and reconstitution of the microbiota in germ-free mice reduces abdominal pain sensitivity. However, the mechanisms underlying microbial modulation of pain remain elusive. We hypothesized that disruption of the intestinal microbiota modulates the excitability of peripheral nociceptive neurons., Methods: In vivo and in vitro assays of visceral sensation were performed on mice treated with the nonabsorbable antibiotic vancomycin (50 μg/mL in drinking water) for 7 days and water-treated control mice. Bacterial dysbiosis was verified by 16s rRNA analysis of stool microbial composition., Results: Treatment of mice with vancomycin led to an increased sensitivity to colonic distension in vivo and in vitro and hyperexcitability of dorsal root ganglion (DRG) neurons in vitro, compared with controls. Interestingly, hyperexcitability of DRG neurons was not restricted to those that innervated the gut, suggesting a widespread effect of gut dysbiosis on peripheral pain circuits. Consistent with this, mice treated with vancomycin were more sensitive than control mice to thermal stimuli applied to hind paws. Incubation of DRG neurons from naive mice in serum from vancomycin-treated mice increased DRG neuron excitability, suggesting that microbial dysbiosis alters circulating mediators that influence nociception. The cysteine protease inhibitor E64 (30 nmol/L) and the protease-activated receptor 2 (PAR-2) antagonist GB-83 (10 μmol/L) each blocked the increase in DRG neuron excitability in response to serum from vancomycin-treated mice, as did the knockout of PAR-2 in NaV1.8-expressing neurons. Stool supernatant, but not colonic supernatant, from mice treated with vancomycin increased DRG neuron excitability via cysteine protease activation of PAR-2., Conclusions: Together, these data suggest that gut microbial dysbiosis alters pain sensitivity and identify cysteine proteases as a potential mediator of this effect., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The production of β-lactamases by bacterial pathogens endangers antimicrobial therapy, and new inhibitors for β-lactamases are urgently needed. We report the development of a luminescent-based biosensor that quantifies β-lactamase inhibition in a cellular context, based on the activation of transcriptional factor AmpR following the exposure of bacterial cells to β-lactams. This rapid method can account for factors like membrane permeability and can be employed to identify new β-lactamase inhibitors.

Background: The loop electrosurgical excision procedure (LEEP) to treat cervical dysplasia (CD) is known to alter the cervical microbiota, the community of bacteria that play a central role in female genital health. Perturbations to the microbiota of the female urogenital tract (FUT), including the urethra, vagina, and cervix, have been linked with symptoms of sexual dysfunction (SD), though correlations among LEEP, the microenvironment, and SD have not yet been described., Aims: To characterize the FUT microbiota before and after LEEP and investigate possible associations with SD., Methods: Females undergoing LEEP for CD were recruited to participate in the study. Urinary samples and vaginal and cervical swabs were collected immediately before and 3 months after treatment. Bacterial communities were characterized by 16S rRNA next-generation sequencing. Self-report surveys assessing demographics, medical history, and sexual function were completed at the same intervals., Outcomes: Microbiota taxonomy and Female Sexual Function Index (FSFI) scores., Results: Alpha diversity revealed a significant decrease in species richness in the FUT microbiota post-LEEP. Beta diversity demonstrated significant differences among the cervical, urinary, and vaginal microenvironments pre- and post-LEEP. Lactobacillus spp were the dominant microbial genus in the cervical microenvironment pre- and post-LEEP. Although the vaginal and urinary microenvironments were characterized by Prevotella pre-LEEP, they were colonized by Lactobacillus post-LEEP. Following LEEP, some participants experienced a significant increase in proinflammatory bacteria, including the genera Gardnerella , Megasphaera , Sneathia , Parvimonas , and Peptostreptococcus . Others experienced significant decreases in inflammatory and protective bacteria post-LEEP, including Butyricicoccus , Terriporobacter , Intestinimonas , and Negativibacillus. Overall there were no significant changes in pre- and post-LEEP FSFI scores. However, post-LEEP FSFI scores were seemingly associated with changes in inflammatory bacteria in some participants., Clinical Implications: There is an overall reduction in FUT microbiota dysbiosis post-LEEP. However, we show variability as some participants experienced persistent dysbiosis of FUT microbiota and elevated FSFI scores, suggesting that therapies to treat dysbiosis of FUT microbiota may reduce FSFI scores, thereby improving SD symptoms., Strengths and Limitations: We demonstrate novel associations among urogenital sites, microbiota changes, LEEP, and SD. The small sample size and inability of species classification are limitations., Conclusion: Diverse inflammatory microbiota characterizes CD in the FUT, and LEEP mostly returns microenvironments to a healthy state. However, some participants have persistent inflammatory bacteria post-LEEP, suggesting a non-uniform healing response. This study provides an impetus for future longitudinal studies to monitor and restore FUT microenvironments post-LEEP, aimed at mitigating postoperative SD symptoms., Competing Interests: None declared., (© The Author(s) 2023. Published by Oxford University Press on behalf of The International Society of Sexual Medicine.)

We report a small-footprint cost-effective isothermal rapid DNA amplification system, with integrated microfluidics for automated sample analysis and detection of SARS-CoV-2 in human and environmental samples. Our system measures low-level fluorescent signals in real-time during amplification, while maintaining the desired assay temperature on a low power, portable system footprint. A unique soft microfluidic chip design was implemented to mitigate thermocapillary effects and facilitate optical alignment for automated image capture and signal analysis. The system-on-board prototype, coupled with the LAMP primers designed by BioCoS, was sensitive enough to detect large variations in viral loads of SARS-CoV-2 corresponding to a threshold cycle range of 16 to 39. Furthermore, tested samples consisted of a broad range of viral strains and lineages identified in Canada during 2021-2022. Clinical specimens were collected and tested at the Kingston Health Science Centre using a clinically validated PCR assay, and variants were determined using whole genome sequencing.

Importance: Nirmatrelvir-ritonavir is an oral antiviral medication that improves outcomes in SARS-CoV-2 infections. However, there is concern that antiviral resistance will develop and that these viruses could be selected for after treatment., Objective: To determine the prevalence of low-frequency SARS-CoV-2 variants in patient samples that could be selected for by nirmatrelvir-ritonavir., Design, Setting, and Participants: This retrospective cohort study was conducted at 4 laboratories that serve community hospitals, academic tertiary care centers, and COVID-19 assessment centers in Ontario, Canada. Participants included symptomatic or asymptomatic patients who tested positive for SARS-CoV-2 virus and submitted virus samples for diagnostic testing between March 2020 and January 2023., Exposure: SARS-CoV-2 infection., Main Outcomes and Measures: Samples with sufficient viral load underwent next-generation genome sequencing to identify low-frequency antiviral resistance variants that could not be identified through conventional sequencing., Results: This study included 78 866 clinical samples with next-generation whole-genome sequencing data for SARS-CoV-2. Low-frequency variants in the viral nsp5 gene were identified in 128 isolates (0.16%), and no single variant associated with antiviral resistance was predominate., Conclusions and Relevance: This cohort study of low-frequency variants resistant to nirmatrelvir-ritonavir found that these variants were very rare in samples from patients with SARS-CoV-2, suggesting that selection of these variants by nirmatrelvir-ritonavir following the initiation of treatment may also be rare. Surveillance efforts that involve sequencing of viral isolates should continue to monitor for novel resistance variants as nirmatrelvir-ritonavir is used more broadly.

The novel coronavirus disease of 2019 (COVID-19) pandemic has severely impacted the training of health care professional students because of concerns of potential asymptomatic transmission to colleagues and vulnerable patients. From May 27th, 2020, to June 23rd 2021; at a time when B.1.1.7 (alpha) and B.1.617.2 (delta) were the dominant circulating variants, PCR testing was conducted on 1,237 nasopharyngeal swabs collected from 454 asymptomatic health care professional students as they returned to their studies from across Canada to Kingston, ON, a low prevalence area during that period for COVID-19. Despite 46.7% of COVID-19 infections occurring in the 18-29 age group in Kingston, severe-acute-respiratory coronavirus-2 was not detected in any of the samples suggesting that negligible asymptomatic infection occurred in this group and that PCR testing in this setting may not be warranted as a screening tool., (© 2023. The Author(s).)

Importance: Infection transmission following endoscopic retrograde cholangiopancreatography (ERCP) can occur due to persistent contamination of duodenoscopes despite high-level disinfection to completely eliminate microorganisms on the instrument., Objective: To determine (1) contamination rates after high-level disinfection and (2) technical performance of duodenoscopes with disposable elevator caps compared with those with standard designs., Design, Setting, and Participants: In this parallel-arm multicenter randomized clinical trial at 2 tertiary ERCP centers in Canada, all patients 18 years and older and undergoing ERCP for any indication were eligible., Intervention: The intervention was use of duodenoscopes with disposable elevator caps compared with duodenoscopes with a standard design., Main Outcomes and Measures: Coprimary outcomes were persistent microbial contamination of the duodenoscope elevator or channel, defined as growth of at least 10 colony-forming units of any organism or any growth of gram-negative bacteria following high-level disinfection (superiority outcome), and technical success of ERCP according to a priori criteria (noninferiority outcome with an a priori noninferiority margin of 7%), assessed by blinded reviewers., Results: From December 2019 to February 2022, 518 patients were enrolled (259 disposable elevator cap duodenoscopes, 259 standard duodenoscopes). Patients had a mean (SD) age of 60.7 (17.0) years and 258 (49.8%) were female. No significant differences were observed between study groups, including in ERCP difficulty. Persistent microbial contamination was detected in 11.2% (24 of 214) of standard duodenoscopes and 3.8% (8 of 208) of disposable elevator cap duodenoscopes (P = .004), corresponding to a relative risk of 0.34 (95% CI, 0.16-0.75) and number needed to treat of 13.6 (95% CI, 8.1-42.7) to avoid persistent contamination. Technical success using the disposable cap scope was noninferior to that of the standard scope (94.6% vs 90.7%, P = .13). There were no differences between study groups in adverse events and other secondary outcomes., Conclusions and Relevance: In this randomized clinical trial, disposable elevator cap duodenoscopes exhibited reduced contamination following high-level disinfection compared with standard scope designs, without affecting the technical performance and safety of ERCP., Trial Registration: ClinicalTrials.gov Identifier: NCT04040504.

Lower viral loads were observed in the upper respiratory tract of patients infected with BA.1, whereas patients infected with BA.2 and BA.5 had comparable viral loads to those seen with Alpha or Delta. This suggests that viral loads are likely not responsible for the increased transmission of the Omicron lineages., (© The Author(s) 2022. Published by Oxford University Press on behalf of Infectious Diseases Society of America.)

The emergence of Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV-2) was met with rapid development of robust molecular-based detection assays. Many SARS-CoV-2 molecular tests target multiple genetic regions of the virus to maximize detection and protect against diagnostic escape. Despite the relatively moderate mutational rate of SARS-CoV-2, numerous mutations with known negative impact on diagnostic assays have been identified. In early 2021, we identified four samples positive for SARS-CoV-2 with a nucleocapsid (N) gene drop out on Cepheid Xpert® Xpress SARS-CoV-2 assay. Sequencing revealed a single common mutation in the N gene C29200T. Spatiotemporal analysis showed that the mutation was found in at least six different Canadian provinces from May 2020 until May 2021. Phylogenetic analysis showed that this mutation arose multiple times in Canadian samples and is present in six different variants of interest and of concern. The Cepheid testing platform is commonly used in Canada including in remote regions. As such, the existence of N gene mutation dropouts required further investigation. While commercial SARS-CoV-2 molecular detection assays have contributed immensely to the response effort, many vendors are reluctant to make primer/probe sequences publicly available. Proprietary primer/probe sequences create diagnostic 'blind spots' for global SARS-CoV-2 sequence monitoring and limits the ability to detect and track the presence and prevalence of diagnostic escape mutations. We hope that our industry partners will seriously consider making primer/probe sequences available, so that diagnostic escape mutants can be identified promptly and responded to appropriately to maintain diagnostic accuracy., (© 2022. The Author(s).)

Respiratory viruses are transmitted and acquired via the nasal mucosa, and thereby may influence the nasal metabolome composed of biochemical products produced by both host cells and microbes. Studies of the nasal metabolome demonstrate virus-specific changes that sometimes correlate with viral load and disease severity. Here, we evaluate the nasopharyngeal metabolome of COVID-19 infected individuals and report several small molecules that may be used as potential therapeutic targets. Specimens were tested by qRT-PCR with target primers for three viruses: Influenza A (INFA), respiratory syncytial virus (RSV), and SARS-CoV-2, along with unaffected controls. The nasopharyngeal metabolome was characterized using an LC-MS/MS-based screening kit capable of quantifying 141 analytes. A machine learning model identified 28 discriminating analytes and correctly categorized patients with a viral infection with an accuracy of 96% (R 2  = 0.771, Q 2  = 0.72). A second model identified 5 analytes to differentiate COVID19-infected patients from those with INFA or RSV with an accuracy of 85% (R 2  = 0.442, Q 2  = 0.301). Specifically, Lysophosphatidylcholines-a-C18:2 (LysoPCaC18:2) concentration was significantly increased in COVID19 patients (P < 0.0001), whereas beta-hydroxybutyric acid, Methionine sulfoxide, succinic acid, and carnosine concentrations were significantly decreased (P < 0.0001). This study demonstrates that COVID19 infection results in a unique nasopharyngeal metabolomic signature with carnosine and LysoPCaC18:2 as potential therapeutic targets., (© 2022. The Author(s).)

Acinetobacter baumannii and Klebsiella pneumoniae currently rank amongst the most antibiotic-resistant pathogens, responsible for millions of infections each year. In the wake of this crisis, anti-virulence therapeutics targeting bacterial polyphosphate (polyP) homeostasis have been lauded as an attractive alternative to traditional antibiotics. In this work, we show that the small molecule gallein, a known G-protein βγ subunit modulator, also recently proven to have dual-specificity polyphosphate kinase (PPK) inhibition in Pseudomonas aeruginosa , in turn exhibits broad-spectrum PPK inhibition in other priority pathogens. Gallein treatment successfully attenuated virulence factors of K. pneumoniae and A. baumannii including biofilm formation, surface associated motility, and offered protection against A. baumannii challenge in a Caenorhabditis elegans model of infection. This was highlighted most importantly in the critically understudied A. baumannii , where gallein treatment phenocopied a ppk1 knockout strain of a previously uncharacterized PPK1. Subsequent analysis revealed a unique instance of two functionally and phenotypically distinct PPK1 isoforms encoded by a single bacterium. Finally, gallein was administered to a defined microbial community comprising over 30 commensal species of the human gut microbiome, demonstrating the non-disruptive properties characteristic of anti-virulence treatments as microbial biodiversity was not adversely influenced. Together, these results emphasize that gallein is a promising avenue for the development of broad-spectrum anti-virulence therapeutics., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Roberge, Neville, Douchant, Noordhof, Boev, Sjaarda, Sheth and Jia.)

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is currently spreading and mutating with increasing speed worldwide. Therefore, there is an urgent need for a simple, sensitive, and high-throughput (HTP) assay to quantify virus-host interactions in order to quickly evaluate the infectious ability of mutant viruses and to develop or validate virus-inhibiting drugs. Here, we developed an ultrasensitive bioluminescent biosensor to evaluate virus-cell interactions by quantifying the interaction between the SARS-CoV-2 receptor binding domain (RBD) and its cellular receptor angiotensin-converting enzyme 2 (ACE2) both in living cells and in vitro. We have successfully used this novel biosensor to analyze SARS-CoV-2 RBD mutants and evaluated candidate small molecules (SMs), antibodies, and peptides that may block RBD:ACE2 interaction. This simple, rapid, and HTP biosensor tool will significantly expedite the detection of viral mutants and the anti-COVID-19 drug discovery process.

Genome-wide variation in SARS-CoV-2 reveals evolution and transmission dynamics which are critical considerations for disease control and prevention decisions. Here, we review estimates of the genome-wide viral mutation rates, summarize current COVID-19 case load in the province of Ontario, Canada (5 January 2021), and analyze published SARS-CoV-2 genomes from Ontario (collected prior to 24 November 2020) to test for more infectious genetic variants or lineages. The reported mutation rate (∼10 -6  nucleotide [nt] -1  cycle -1 ) for SARS-CoV-2 is typical for coronaviruses. Analysis of published SARS-CoV-2 genomes revealed that the G614 spike protein mutation has dominated infections in Ontario and that SARS-CoV-2 lineages present in Ontario have not differed significantly in their rate of spread. These results suggest that the SARS-CoV-2 population circulating in Ontario has not changed significantly to date. However, ongoing genome monitoring is essential for identification of new variants and lineages that may contribute to increased viral transmission., (Copyright © 2021 Sjaarda et al.)

Background: Carnobacterium species are lactic acid-producing Gram-positive bacteria that have been approved by the US Food and Drug Administration and Health Canada for use as a food bio-preservative. The use of live bacteria as a food additive and its potential risk of infections in immunocompromised patients are not well understood., Case Presentation: An 81-year-old male with a history of metastatic prostate cancer on androgen deprivation therapy and chronic steroids presented to our hospital with a 2-week history of productive cough, dyspnea, altered mentation, and fever. Extensive computed tomography imaging revealed multifocal pneumonia without other foci of infection. He was diagnosed with pneumonia and empirically treated with ceftriaxone and vancomycin. Blood cultures from admission later returned positive for Carnobacterium inhibens. He achieved clinical recovery with step-down to oral amoxicillin/clavulanic acid for a total 7-day course of antibiotics., Conclusions: This is the fourth reported case of bacteremia with Carnobacterium spp. isolated from humans. This case highlights the need to better understand the pathogenicity and disease spectrum of bacteria used in the food industry for bio-preservation, especially in immunocompromised patients.

The emergence and rapid global spread of SARS-CoV-2 demonstrates the importance of infectious disease surveillance, particularly during the early stages. Viral genomes can provide key insights into transmission chains and pathogenicity. Nasopharyngeal swabs were obtained from thirty-two of the first SARS-CoV-2 positive cases (March 18-30) in Kingston Ontario, Canada. Viral genomes were sequenced using Ion Torrent (n = 24) and MinION (n = 27) sequencing platforms. SARS-CoV-2 genomes carried forty-six polymorphic sites including two missense and three synonymous variants in the spike protein gene. The D614G point mutation was the predominate viral strain in our cohort (92.6%). A heterozygous variant (C9994A) was detected by both sequencing platforms but filtered by the ARTIC network bioinformatic pipeline suggesting that heterozygous variants may be underreported in the SARS-CoV-2 literature. Phylogenetic analysis with 87,738 genomes in the GISAID database identified global origins and transmission events including multiple, international introductions as well as community spread. Reported travel history validated viral introduction and transmission inferred by phylogenetic analysis. Molecular epidemiology and evolutionary phylogenetics may complement contact tracing and help reconstruct transmission chains of emerging diseases. Earlier detection and screening in this way could improve the effectiveness of regional public health interventions to limit future pandemics.

Spinal cord injury (SCI) is a devastating injury to the central nervous system in which 60 to 80% of patients experience chronic pain. Unfortunately, this pain is notoriously difficult to treat, with few effective options currently available. Patients are also commonly faced with various compounding injuries and medical challenges, often requiring frequent hospitalization and antibiotic treatment. Change in the gut microbiome from the "normal" state to one of imbalance, referred to as gut dysbiosis, has been found in both patients and rodent models following SCI. Similarities exist in the bacterial changes observed after SCI and other diseases with chronic pain as an outcome. These changes cause a shift in the regulation of inflammation, causing immune cell activation and secretion of inflammatory mediators that likely contribute to the generation/maintenance of SCI pain. Therefore, correcting gut dysbiosis may be used as a tool towards providing patients with effective pain management and improved quality of life., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2020 Published by Elsevier Inc.)

Objective: The aim of the study was to determine whether exclusive breastfeeding or exclusive formula feeding is more cost-effective when a Canadian mother with HIV is adherent to antiretroviral therapy and has full virologic suppression., Design: Current Canadian guidelines recommend that mothers with HIV practice exclusive formula feeding. This contradicts the updated World Health Organization (WHO) guidelines which recommend that mothers with HIV should breastfeed for ≥12 months while receiving support for antiretroviral therapy adherence. Due to the economic and health risks and benefits associated with each modality, there remains expert disagreement on whether the WHO recommendations should be adopted in high-income countries., Methods: A microsimulation model was developed to estimate lifetime costs and effectiveness (i.e., infant's quality-adjusted life years) of a hypothetical group of 1,000,000 initially healthy, HIV-negative infants, if the mother with HIV was on antiretroviral therapy with full virologic suppression and either exclusive breastfeeding or exclusive formula feeding. The model was developed from the economic perspective of the Ontario Ministry of Health, taking into account direct costs associated with infant feeding modality as well as related indirect costs born out of the child's lifetime health outcomes. Uncertainties related to model parameters were evaluated using one-way and probabilistic sensitivity analyses., Results: In comparison to exclusive formula feeding, exclusive breastfeeding was the dominant feeding modality (i.e., less costly and more effective) yielding cost-savings of $13,812 per additional quality-adjusted life year gained. Neither one-way nor probabilistic sensitivity analyses altered the conclusions., Conclusions: Despite the risk of HIV transmission, exclusive breastfeeding was more cost-effective than exclusive formula feeding. These findings merit review of current infant feeding guidelines for mothers with HIV living in high-income countries.

Background: Clostridium difficile (CD) is the leading cause of infectious health-care associated diarrhea. However, little is known regarding CD carriage and transmission amongst asymptomatic colonizers. We evaluated carriage, characterized strains and examined epidemiologic linkages in asymptomatic colonized CD patients., Methods: Rectal swabs from asymptomatic patients admitted to the general medicine ward from April 1-June 30 2012 were collected. PCR-confirmed CD colonies were ribotyped and characterized by Modified-Multi Locus Variable Number Tandem Repeat Analysis (MMLVA)., Results: 1549-swabs were collected from 474-patients. Overall, 50/474(10.6%) were CD PCR-positive, 24/50 were colonized at admission, while 26/50 were first identified > = 72 hours after admission. Amongst the 50 CD PCR-positive patients, 90% were asymptomatically colonized and 80% of individuals carried toxigenic CD-strains, including ribotype-027 (5/45:11%). MMLVA revealed five-clusters involving 15-patients harboring toxigenic (4/5) and non-toxigenic CD strains (1/5). In two clusters, patients were CD positive on admission while in the other three clusters involving 10 patients, we observed CD transmission from asymptomatically colonized patients to 8 previously CD-negative patients., Conclusions: We identified increasing rates of colonization during admission to medical wards. MMLVA typing effectively discriminated between strains and suggests that 20% of patients with CD colonization acquired their strain(s) from asymptomatically colonized individuals in hospital., Competing Interests: The authors have declared that no competing interests exist.

The study of complex microbial communities has become a major research focus as mounting evidence suggests the pivotal role microbial communities play in host health and disease. Microbial communities of the gastrointestinal tract, known as the gut microbiota, have been implicated in aiding the host with vitamin biosynthesis, regulation of host energy metabolism, immune system development, and resistance to pathogen invasion. Conversely, disruptions of the gut microbiota have been linked to host morbidity, including the development of inflammatory diseases, metabolic disorders, increased cardiovascular risk, and increased risk of infectious diseases. However, studying the gut microbiota in humans and animals is challenging, as many microorganisms are fastidious with unique nutritional or environmental requirements that are often not met using conventional culture techniques. Bioreactors provide a unique solution to overcome some of the limitations of conventional culture techniques. Bioreactors have been used to propagate and establish complex microbial communities in vitro by recapitulating the physiological conditions found in the GI tract. These systems further our understanding of microbial physiology and facilitate our understanding of the impact of medications and xenobiotics on microbial communities. Here, we review the versatility and breadth of bioreactor systems that are currently available and how they are being used to study faecal and defined microbial communities. Bioreactors provide a unique opportunity to study complex microbial interactions and perturbations in vitro in a controlled environment without confounding biotic and abiotic variables., (Copyright © 2018. Published by Elsevier Inc.)

Problem: Semen is the primary medium for sexual transmission of HIV-1 and contains high concentrations of TGF-β1, but its role in regulating HIV-mediated immune activation is unclear., Method of Study: TGF-β1 and sCD14 were compared in blood plasma (BP) and seminal plasma (SP) from HIV-uninfected and infected, antiretroviral therapy (ART)-naive and ART-treated men and in THP-1 cells following exposure to HIV-1. The relationship between TGF-β1 and sCD14 was determined by Spearman correlation., Results: Active and latent forms of TGF-β1 were compartmentalized between BP and SP. Highest active TGF-β1 levels were present in SP of ART-naïve chronic-infected men and decreased following ART treatment. Latent TGF-β1 was upregulated in BP following HIV infection, and highest levels were observed in BP of acute-infected men. Similar expression trends were observed between latent TGF-β1 and sCD14 in BP. A significant negative correlation was observed between active TGF-β1, sCD14, and semen viral load in ART-naive men., Conclusion: TGF-β1 is compartmentalized between blood and semen, possibly co-expressed with sCD14 by activated monocytes/macrophages in BP as a result of HIV infection. Conversion of latent TGF-β1 into its active form could contribute to regulation of viral load and immune activation in the male genital tract, but depends on the stage of infection., (© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Due to the lack of detailed reports of Klebsiella pneumoniae carbapenemase (KPC)-producing enterobacteria in Ontario, Canada, we perform a molecular characterization of KPC-producing Enterobacteriaceae submitted to the provincial reference laboratory from 2008 to 2011. Susceptibility profiles were accessed by E-test. Molecular types of isolates were determined by pulse-field gel electrophoresis (PFGE) and multilocus sequence typing. Screening of ß-lactamase genes was performed by multiplex PCR and alleles were identified by DNA sequencing. The genetic platform of blaKPC gene was analyzed by PCR. Plasmid replicons were typed using PCR-based typing approach. KPC-plasmids were also evaluated by S1 nuclease-PFGE and Southern blot. Thirty unique clinical isolates (26 Klebsiella pneumoniae, 2 Enterobacter cloacae, 1 Citrobacter freundii and 1 Raoultella ornithinolytica) were identified as blaKPC positive: 4 in 2008, 3 in 2009, 10 in 2010 and 13 in 2011. The majority exhibited resistance to carbapenems, cephalosporins and fluoroquinolones and two isolates were also resistant to colistin. The isolates harbored blaKPC-2 (n = 23) or blaKPC-3 (n = 7). blaTEM-1 (n = 27) was commonly detected and occasionally blaOXA-1 (n = 3) and blaCTX-M-15 (n = 1). As expected, all K. pneumoniae isolates carried blaSHV-11. blaKPC genes were identified on Tn4401a (n = 20) or b (n = 10) isoforms, on plasmids of different sizes belonging to the incompatibility groups IncFIIA (n = 19), IncN (n = 3), IncI2 (n = 3), IncFrep (n = 2) and IncA/C (n = 1). The occurrence of KPC ß-lactamase in Ontario was mainly associated with the spread of the K. pneumoniae clone ST258.

Background: The HIV RNA viral load (VL) in vaginal secretions and semen is an independent predictor of HIV transmission. Blood VL is associated with semen VL, and local mucosal factors, such as semen cytomegalovirus (CMV) reactivation, may play an important role., Methods: Twenty-one HIV-CMV-coinfected, antiretroviral-naive men received 900 mg of oral valganciclovir once daily for 2 weeks in an open-label study. Blood and semen were collected at baseline, after 2 weeks of valganciclovir, and 2 months after therapy completion. The primary end point was change in semen HIV levels at 2 weeks, and the secondary end points were change in semen HIV VL at 2 months and change in semen CMV levels., Results: The HIV VLs fell significantly at 2 weeks in semen (median 3.44-3.02 log10 copies/mL, P = 0.02) and blood (median 3.61-3.10 log10 copies/mL, P < 0.01) and returned to baseline after therapy completion (median 3.24 and 3.71 log10 copies/mL in semen and blood, respectively). Semen CMV levels also fell on treatment (median 2.13-1.62 log10 copies/mL, P < 0.01) and continued to fall after therapy completion (median 0.91 log10 copies/mL at week 8, P < 0.001 vs. baseline). The reduced semen CMV VL was associated with decreased semen T-cell activation and enhanced CMV-specific T-cell responses in blood; changes in the semen HIV VL were not associated with immune parameters., Conclusions: Although valganciclovir therapy was associated with reduced HIV and semen CMV levels, these results suggest that the reduced HIV VL was a direct drug effect rather than a CMV antiviral effect or CMV-associated immune alterations.

Background: The Tim-3 receptor has been implicated as a negative regulator of adaptive immune responses and has been linked to T-cell dysfunction in chronic viral infections, such as HIV. Blocking Tim-3 has been proposed as a potential therapeutic intervention in HIV infection. However, a more detailed characterization of Tim-3 expression in the presence of HIV is required before such strategies can be considered., Methods: In this study, we investigate Tim-3 expression on innate immune cell subsets in chronic HIV-infected individuals pretherapy and posttherapy., Results: We report that, pretherapy, HIV infection is associated with elevated levels of Tim-3 on resting innate lymphocytes (NK, NKT, and γδ T cells), but not resting monocytes. In the absence of HIV infection, stimulation with an inflammatory stimulus resulted in decreased Tim-3 on monocytes and increased Tim-3 on NK, NKT, and γδ T cells. However, innate cells from HIV-infected donors were significantly less responsive to stimulation. Six months of combination antiretroviral therapy (cART) restored Tim-3 levels on resting NK cells but not NKT or γδ T cells. The responses of all subsets to inflammatory stimuli were restored to some extent with cART but only reached HIV-negative control levels in monocytes and NK cells., Discussion: These results demonstrate that, during HIV infection, Tim-3 expression on innate cells is dysregulated and that this dysregulation is only partially restored after 6 months of cART. Our findings suggest that Tim-3 is differentially regulated on innate immune effector cells, and have direct implications for strategies designed to block Tim-3-ligand interactions.

Background: Effective antiretroviral therapy (ART) dramatically reduces human immunodeficiency virus (HIV) transmission. However, isolated shedding of HIV type 1 (HIV-1) in semen (IHS) can occur in the absence of detectable viremia or genital infections. We hypothesized that ART intensification with medications active in semen might prevent IHS., Methods: Paired blood and semen samples were collected monthly for 6 months from HIV-infected men starting ART that was intensified (iART) with maraviroc and raltegravir in an open-label fashion. Semen parameters were compared to those of historical controls starting standard ART (sART)., Results: Compared with 25 controls who started sART, the semen HIV-1 load in 13 subjects who started iART was more rapidly suppressed (P = .043). IHS was detected at >1 visit in 2 participants (15%) receiving iART and in 12 controls (48%) receiving sART (P = .040). Among iART recipients, IHS was associated with lower raltegravir concentrations in blood and semen, compared with complete HIV-1 suppression (P = .03). Prolonged, high-level IHS (ie, shedding of >5000 RNA copies/mL) was observed in 1 iART recipient (8%), despite rapid viremia suppression and therapeutic drug levels; for 10 months, this virus remained R5 tropic, drug susceptible, and similar in sequence to virus recovered from blood. IHS was not seen after >3 years of effective ART in a parallel, prospective cohort study., Conclusions: iART transiently reduced the occurrence of IHS early after ART initiation but did not prevent high-level IHS. IHS was not seen after more prolonged sART.

Objective: Malaria and HIV-1 adversely interact, with HIV-positive individuals suffering higher parasite burdens and worse clinical outcomes. However, the mechanisms underlying these disease interactions are unclear. We hypothesized that HIV coinfection impairs the innate immune response to malaria, and that combination antiretroviral therapy (cART) may restore this response. Our aim was to examine the innate inflammatory response of natural killer (NK), natural killer T (NKT), and γδ T-cells isolated from the peripheral blood of HIV-infected therapy-naive donors to malaria parasites, and determine the effect of cART on these responses., Methods: Freshly isolated peripheral blood mononuclear cells from 25 HIV-infected individuals pre-cART (month 0) and post-cART (months 3 and 6), and HIV-negative individuals at matched time-points, were cultured in the presence of Plasmodium falciparum parasitized erythrocytes. Supernatants and cells were collected to assess cytokine production and phenotypic changes., Results: Compared to HIV-negative participants, NKT, NK, and γδ T-cell subsets from participants with chronic HIV infection showed marked differences, including decreased production of interferon γ (IFNγ) and tumor necrosis factor (TNF) in response to malaria parasites. IFNγ production was linked to interleukin-18 receptor (IL-18R) expression in all three cell types studied. Six months of cART provided partial cellular reconstitution but had no effect on IL-18R expression, or IFNγ and TNF production., Conclusion: These data suggest that HIV infection impairs the inflammatory response of innate effector cells to malaria, and that the response is not fully restored within 6 months of cART. This may contribute to higher parasite burdens and ineffective immune responses, and have implications for vaccination initiatives in coinfected individuals.

Effective antiretroviral therapy (ART) suppresses the blood HIV RNA viral load (VL) below the level of detection. However, some individuals intermittently shed HIV RNA in semen despite suppression of viremia, a phenomenon termed "isolated HIV semen shedding (IHS)". In a previously reported clinical study, we collected blood and semen samples from HIV-infected men for 6 months after ART initiation, and documented IHS at ≥1 visit in almost half of the participants, independent of ART regimen or semen drug levels. We now report the mucosal immune associations of IHS in these men. Blood and semen plasma cytokine levels were assayed by multiplex enzyme-linked immunosorbent assay, T-cell populations were evaluated by flow cytometry in freshly isolated blood and semen mononuclear cells, and semen cytomegalovirus (CMV) DNA levels were measured by PCR. Although IHS was not associated with altered blood or semen cytokine levels, the phenomenon was associated with a transient, dramatic increase in CD4+ and CD8+ T-cell activation that was restricted to the semen compartment. All participants were CMV infected, and although semen CMV reactivation was common despite ART, this was not associated with T-cell activation or IHS. Further elucidation of the causes of compartmentalized mucosal T-cell activation and IHS may have important public health implications.

Background: Highly active antiretroviral therapy (HAART) dramatically reduces plasma HIV-1 viremia. However, despite completely suppressive HAART, it has been suggested that low-levels of viral replication may persist in the gut mucosa and elsewhere in individuals on long-term HAART., Objective: We conducted a double-blind randomized, placebo-controlled trial evaluating whether intensification of HAART in long-term virologically suppressed individuals with raltegravir is associated with a reduction in the level of proviral HIV-1 DNA in CD4(+) T cells in blood and the sigmoid colon (gut)., Methods: Long-term (>4 years) virologically suppressed HIV-infected individuals on standard HAART were randomized 1 : 1 in a double-blind fashion to receive raltegravir (400  mg twice/day) or placebo for 48 weeks. After week 48, all participants were treated with raltegravir to week 96. Blood and sigmoid biopsies were sampled and the frequency of CD4(+) T cells carrying HIV-1 proviral DNA was determined., Results: Twenty-four study patients were recruited. At 48 weeks, no difference was apparent between participants receiving raltegravir or placebo in blood HIV-1 proviral levels (P = 0.62), CD4(+) T-cell counts (P = 0.25) and gut proviral loads (P = 0.74). Similarly, prolonged raltegravir intensification up to week 96 had no further effect on both blood and gut HIV-1 proviral loads and blood CD4(+) T-cell counts., Conclusion: In long-term virologically suppressed patients on standard HAART, intensification with raltegravir did not result in further decay of CD4(+) T cells carrying HIV-1 proviral DNA in either the blood or gut after 48 or 96 weeks of therapy, or in any increase in CD4(+) T-cell counts.

Objectives: Although more than 60% of HIV transmission occurs via semen, little is known about the immune impact of seminal plasma on HIV susceptibility. Here, we examined the level of selected immunomodulatory factors in seminal plasma from HIV-uninfected and therapy-naive, HIV-infected men in acute and chronic stages; the cytokine response elicited by seminal plasma in genital epithelial cells (GECs); and whether any GEC response to seminal plasma could drive HIV replication in infected T cells., Methods: A panel of nine cytokines and chemokines was measured in seminal plasma from HIV-uninfected and HIV-infected men and in primary GEC cultures following seminal plasma exposure. HIV-long terminal repeat (LTR) activation was measured in 1G5 T cells exposed to supernatants from seminal plasma-treated GECs., Results: Pro-inflammatory cytokines and chemokines were present at significantly higher levels in seminal plasma from acute men, whereas transforming growth factor (TGF)-β1 was significantly higher in seminal plasma from chronic men. Pro-inflammatory cytokine production by GECs was significantly decreased following incubation with seminal plasma from chronic men. Blocking the TGF-β1 receptor in GECs prior to seminal plasma exposure enhanced pro-inflammatory cytokine production. Exposure to seminal plasma activated nuclear factor (NF)-κB in GECs and blocking it significantly reduced pro-inflammatory cytokine production. GEC responses to seminal plasma, especially from acute men, significantly activated HIV-LTR activation in 1G5 T cells., Conclusion: Immunomodulatory factors in seminal plasma vary, depending on presence and stage of HIV infection. Exposure to seminal plasma leads to NF-κB activation and pro-inflammatory cytokine production, whereas TGF-β in seminal plasma may suppress pro-inflammatory cytokine production by GECs. GEC responses to seminal plasma can activate HIV-LTR in infected CD4(+) T cells.

Recent data suggest that CD8+ T-cell effector activity is an important component in the control of HIV replication in elite controllers (ECs). One critical element of CD8+ T-cell effector function and differentiation is the T-box transcription factor T-bet. In the present study, we assessed T-bet expression, together with the effector proteins perforin, granzyme A (Grz A), granzyme B (Grz B), and granulysin, in HIV-specific CD8+ T cells from ECs (n = 20), chronically infected progressors (CPs; n = 18), and highly active antiretroviral therapy (HAART)-suppressed individuals (n = 19). Compared with the other cohort groups, HIV-specific CD8+ T cells among ECs demonstrated a superior ability to express perforin and Grz B, but with no detectable difference in the levels of Grz A or granulysin. We also observed higher levels of T-bet in HIV-specific CD8+ T cells from ECs, with an ensuing positive correlation between T-bet and levels of both perforin and Grz B. Moreover, HIV-specific CD8+ T cells in ECs up-regulated T-bet to a greater extent than CPs after in vitro expansion, with concomitant up-regulation of perforin and Grz B. These results suggest that T-bet may play an important role in driving effector function, and its modulation may lead to enhanced effector activity against HIV.

Objective: Th17 cells play an important role in mucosal defence and repair and are highly susceptible to infection by HIV. Antiretroviral therapy (ART) suppresses HIV viremia and can restore CD4(+) numbers in the blood and gastrointestinal mucosa, but the resolution of systemic inflammation and gut microbial translocation is often incomplete. We hypothesized that this might relate to persistent dysregulation of gut CD4(+) Th17 subsets., Methods: Blood and sigmoid biopsies were collected from HIV-uninfected men, chronically HIV-infected, ART-naive men, and men on effective ART for more than 4 years. Sigmoid provirus levels were assayed blind to participant status, as were CD4(+) Th17 subsets, systemic markers of microbial translocation, and cellular immune activation., Results: There was minimal CD4(+) Th17 dysregulation in the blood until later stage HIV infection, but gastrointestinal Th17 depletion was apparent much earlier, along with increased plasma markers of microbial translocation. Plasma lipopolysaccharide (LPS) remained elevated despite overall normalization of sigmoid Th17 populations on long-term ART, although there was considerable interindividual variability in Th17 reconstitution. An inverse correlation was observed between plasma LPS levels and gut Th17 frequencies, and higher plasma LPS levels correlated with an increased gut HIV proviral reservoir., Conclusion: Sigmoid Th17 populations were preferentially depleted during HIV infection. Despite overall CD4(+) T-cell reconstitution, sigmoid Th17 frequencies after long-term ART were heterogeneous and higher frequencies were correlated with reduced microbial translocation.

The immunologic mechanisms underlying the faster progression of hepatitis C virus (HCV) disease in the presence of human immunodeficiency virus (HIV) coinfection are not clearly understood. T-cell cross-reactivity between HCV and influenza virus-specific epitopes has been associated with rapid progression of HCV disease (S. Urbani, B. Amadei, P. Fisicaro, M. Pilli, G. Missale, A. Bertoletti, and C. Ferrari, J. Exp. Med. 201:675-680, 2005). We asked whether T-cell cross-reactivity between HCV and HIV could exist during HCV/HIV coinfection and affect pathogenesis. Our search for amino acid sequence homology between the HCV and HIV proteomes revealed two similar HLA-A2-restricted epitopes, HIV-Gag (SLYNTVATL [HIV-SL9]) and HCV-NS5b (ALYDVVSKL [HCV-AL9]). We found that 4 out of 20 HLA-A2-positive (HLA-A2(+)) HIV-infected individuals had CD8(+) T cells that recognized both the HIV-SL9 and HCV-AL9 epitopes. However, the AL9 epitope was generally shown to be a weak agonist. Although HCV-monoinfected individuals in our study did not show AL9-specific responses, we found that about half of HCV/HIV-coinfected individuals had dual responses to both epitopes. High dual T-cell recognition among coinfected subjects was usually due to separate T-cell populations targeting each epitope, as determined by pentamer staining. The one individual demonstrating cross-reactive T cells to both epitopes showed the most advanced degree of liver disease. In coinfected individuals, we observed a positive correlation between the magnitudes of T-cell responses to both the SL9 and the AL9 epitopes, which was also positively associated with the clinical parameter of liver damage. Thus, we find that HIV infection induces T cells that can cross-react to heterologous viruses or prime for T cells that are closely related in sequence. However, the induction of cross-reactive T cells may not be associated with control of disease caused by the heterologous virus. This demonstrates that degeneracy of HIV-specific T cells may play a role in the immunopathology of HCV/HIV coinfection.

Background: The blood HIV RNA viral load is the best-defined predictor of HIV transmission, in part due to ease of measurement and the correlation of blood and genital tract (semen or cervico-vaginal) viral load, although recent studies found semen HIV RNA concentration to be a stronger predictor of HIV transmission. There is currently no standardized method for semen collection when measuring HIV RNA concentration. Therefore, we compared two collection techniques in order to study of the impact of antiretroviral therapy on the semen viral load., Methodology/principal Findings: Semen was collected by masturbation from HIV-infected, therapy-naïve men who have sex with men (MSM) either undiluted (Visit 1) or directly into transport medium (Visit 2). Seminal plasma was then isolated, and the HIV RNA concentration obtained with each collection technique was measured and corrected for dilution if necessary. Collection of semen directly into transport medium resulted in a median HIV RNA viral load that was 0.4 log10 higher than undiluted samples., Conclusions/significance: The method of semen collection is an important consideration when quantifying the HIV RNA viral load in this compartment.

Objectives: Sustained suppression of plasma viremia in HIV-infected individuals is attainable with antiretroviral therapy (ART); however, eradication of virus that would allow discontinuation of ART has been hampered by the persistence of HIV reservoirs. It is of great interest to identify individuals who had received ART for prolonged periods of time with extremely low or undetectable HIV reservoirs and monitor plasma viremia following discontinuation of therapy., Methods: We measured the size of HIV reservoirs in CD4(+) T cells of individuals on long-term ART and monitored plasma viremia following cessation of ART in one individual with an exceptionally low viral burden after a decade of therapy., Results: We demonstrated undetectable levels of HIV DNA in the blood of eight of 45 infected individuals on long-term ART. Among those eight individuals, the frequency of cells carrying infectious virus was significantly lower in those who initiated ART during the early versus the chronic phase of infection. One individual with undetectable HIV DNA in both blood and tissue and a profoundly low level of infectious virus experienced plasma viral rebound 50 days following discontinuation of ART., Conclusions: Our data suggest that a significant reduction in the size of viral reservoirs may be achievable in selected individuals who initiate standard ART early in infection. However, given re-emergence of plasma viremia in an individual with an extraordinarily low viral burden, therapeutic strategies aimed at specifically targeting these extremely rare HIV-infected cells with novel interventions may be necessary in order to achieve eradication of virus.

Co-infection of HCV with HIV has been associated with more rapid progression of HCV-related disease. HCV-specific T-cell immune responses, which are essential for disease control, are attenuated in co-infection with HIV. T-cell exhaustion has recently been implicated in the deficient control of chronic viral infections. In the current study, we investigated the role of programmed death-1 (PD-1) and T-cell immunoglobulin and mucin domain-containing molecule-3 (Tim-3) expression in T-cell exhaustion during HCV/HIV co-infection. We show that in HCV/HIV co-infection, both total and HCV-specific T cells co-express Tim-3 and PD-1 in significantly higher frequencies, compared with HCV mono-infection. Co-expression of these two markers on HCV-specific CD8(+) T cells positively correlated with a clinical parameter of liver disease progression. HCV-specific CD8(+) T cells showed greater frequencies of Tim-3/PD-1 co-expression than HIV-specific CD8(+) T cells, which may indicate a greater degree of exhaustion in the former. Blocking Tim-3 or PD-1 pathways restored both HIV- and HCV-specific CD8(+) T-cell expansion in the blood of co-infected individuals. These data demonstrate that co-expression of Tim-3 and PD-1 may play a significant role in HCV-specific T-cell dysfunction, especially in the setting of HIV co-infection.

Effective antiretroviral therapy (ART) may reduce HIV sexual transmission by lowering genital HIV levels. A prospective study of men starting ART (n = 25) demonstrated rapid, substantial reductions in semen HIV RNA. However, despite an undetectable blood viral load, isolated semen HIV shedding was detected at more than one visit in 12 of 25 (48%) participants, with semen HIV RNA levels exceeding 5000 copies/ml in four of 25 (16%). Isolates were drug-sensitive, and this phenomenon was not associated with semen drug levels or regimen.

Progressive loss of T cell functionality is a hallmark of chronic infection with human immunodeficiency virus 1 (HIV-1). We have identified a novel population of dysfunctional T cells marked by surface expression of the glycoprotein Tim-3. The frequency of this population was increased in HIV-1-infected individuals to a mean of 49.4 +/- SD 12.9% of CD8(+) T cells expressing Tim-3 in HIV-1-infected chronic progressors versus 28.5 +/- 6.8% in HIV-1-uninfected individuals. Levels of Tim-3 expression on T cells from HIV-1-infected inviduals correlated positively with HIV-1 viral load and CD38 expression and inversely with CD4(+) T cell count. In progressive HIV-1 infection, Tim-3 expression was up-regulated on HIV-1-specific CD8(+) T cells. Tim-3-expressing T cells failed to produce cytokine or proliferate in response to antigen and exhibited impaired Stat5, Erk1/2, and p38 signaling. Blocking the Tim-3 signaling pathway restored proliferation and enhanced cytokine production in HIV-1-specific T cells. Thus, Tim-3 represents a novel target for the therapeutic reversal of HIV-1-associated T cell dysfunction.

Early and profound CD4+ T-cell depletion in gut-associated lymphoid tissue (GALT) may drive Human Immunodeficiency Virus (HIV) immunopathogenesis, and GALT immune reconstitution on highly active antiretroviral therapy (HAART) may be suboptimal. Blood and sigmoid colon biopsies were collected from HAART-treated individuals with undetectable blood HIV RNA for > or =4 years and from uninfected controls. HIV proviral levels and T-cell phenotype/function were examined in both compartments. CD4+ T-cell reconstitution in the sigmoid, including CD4+ T cells expressing CCR5, exceeded that in blood and did not differ from uninfected controls. Sigmoid HIV proviral load was not correlated with CD4+ reconsitution, but was correlated with the degree of mucosal CD8+ T-cell immune activation. Colonic Gag-specific T-cell responses were common, but were not associated with proviral load or immune activation. In this select study population, long-term HAART was associated with complete CD4+ T-cell reconstitution in sigmoid colon. However, colonic immune activation may drive ongoing HIV replication.

Background: Chronic coinfection with herpes simplex virus type 2 (HSV-2) and human immunodeficiency virus (HIV) has been associated with an increased HIV viral load and more rapid disease progression, perhaps related to HSV-2-associated alterations in host immunity., Methods: Studies were nested within (1) a cross-sectional study of men coinfected with HIV and HSV-2 and (2) women not infected with HIV, both before and after HSV-2 acquisition. HSV-2 infection status was determined by ELISA. HIV-specific CD8(+) T cell epitopes were mapped, and proliferation of HIV-specific cells was also assessed. Systemic inflammatory and regulatory T cell populations were assayed by flow cytometry., Results: The breadth of both the HIV-specific CD8(+) T cell interferon-gamma and proliferative responses was reduced in participants coinfected with HIV and HSV-2, independent of the HIV plasma viral load and CD4(+) T cell count, and the magnitude of the responses was also reduced. HSV-2 infection in this group was associated with increased T cell CD38 expression but not with differences in the proportion of CD4(+) FoxP3(+) regulatory T cells. However, in women not infected with HIV, acquisition of HSV-2 was associated with an increase in the proportion of regulatory T cells., Conclusions: HSV-2 coinfection was associated with reduced HIV-specific T cell responses and systemic inflammation. The immune effects of HSV-2 may underlie the negative impact that this coinfection has on the clinical course of HIV infection.

Background and Aims: Hepatitis C Virus (HCV)-related liver disease progresses more rapidly in individuals co-infected with Human Immunodeficiency Virus-1 (HIV), although the underlying immunologic mechanisms are unknown. We examined whether HIV-specific T-cells are identified in the liver of HCV/HIV co-infected individuals and promote liver inflammation through bystander immune responses., Methods: Ex-vivo intra-hepatic lymphocytes from HCV mono-infected and HCV/HIV co-infected individuals were assessed for immune responses to HIV and HCV antigens by polychromatic flow cytometry., Results: HCV/HIV liver biopsies had similar frequencies of lymphocytes but lower percentages of CD4+ T-cells compared to HCV biopsies. In co-infection, intra-hepatic HIV-specific CD8+ and CD4+ T-cells producing IFN-gamma and TNF-alpha were detected and were comparable in frequency to those that were HCV-specific. In co-infected individuals, viral-specific CD8+ T-cells produced more of the fibrogenic cytokine, TNF-alpha. In both mono- and co-infected individuals, intra-hepatic HCV-specific T-cells were poorly functional compared to HIV-specific T-cells. In co-infection, HAART was not associated with a reconstitution of intra-hepatic CD4+ T-cells and was associated with reduction in both HIV and HCV-specific intra-hepatic cytokine responses., Conclusion: The accumulation of functional HIV-specific T-cells in the liver during HCV/HIV co-infection may represent a bystander role for HIV in inducing faster progression of liver disease.

HIV is generally sexually acquired across the genital or rectal mucosa after exposure to the genital secretions of an HIV-infected partner. Most exposures to HIV do not result in infection, likely due to protection afforded by an intact mucosal epithelium, as well as by innate and adaptive mucosal immune factors present in the genital tract. Another important mucosal determinant of transmission may be the number and activation status of potential HIV target cells, including CCR5/CD4+ T cells and DC-SIGN+ dendritic cells. The simultaneous presence of other genital infections, including classical sexually transmitted infections (STIs), can enhance HIV susceptibility either by breaching the epithelial barrier, recruiting HIV target cells to the genital tract, or by generating a pro-inflammatory local immune milieu. In HIV-infected individuals, genital co-infections increase HIV levels in the genital secretions, thereby increasing secondary sexual transmission. Co-infections that act as important HIV cofactors include human cytomegalovirus (CMV), Herpes simplex virus type 2 (HSV2), Neisseria gonorrhoeae and many others. Strategies focused on genital co-infections, such as vaccines, microbicides and suppressive therapy, are feasible in the short term and have the potential to curb the pandemic.