The PAX LC Trial: A Decentralized, Phase 2, Randomized, Double-blind Study of Nirmatrelvir/Ritonavir Compared with Placebo/Ritonavir for Long COVID.

INTRODUCTION: Individuals with long COVID lack evidence-based treatments and have difficulty participating in traditional site-based trials. Our digital, decentralized trial investigates the efficacy and safety of nirmatrelvir/ritonavir, targeting viral persistence as a potential cause of long COVID. METHODS: The PAX LC trial (NCT05668091) is a Phase 2, 1:1 randomized, double-blind, superiority, placebo-controlled trial in 100 community-dwelling, highly symptomatic adult participants with long COVID residing in the 48 contiguous US states to determine the efficacy, safety, and tolerability of 15 days of nirmatrelvir/ritonavir compared with placebo/ritonavir. Participants are recruited via patient groups, cultural ambassadors, and social media platforms. Medical records are reviewed through a platform facilitating participant-mediated data acquisition from electronic health records nationwide. During the drug treatment, participants complete daily digital diaries using a web-based application. Blood draws for eligibility and safety assessments are conducted at or near participants' homes. The study drug is shipped directly to participants' homes. The primary endpoint is the PROMIS-29 Physical Health Summary Score difference between baseline and Day 28, evaluated by a mixed model repeated measure analysis. Secondary endpoints include PROMIS-29 (Mental Health Summary Score and all items), Modified GSQ-30 with supplemental symptoms questionnaire, COVID Core Outcome Measures for Recovery, EQ-5D-5L (Utility Score and all items), PGIS 1 and 2, PGIC 1 and 2, and healthcare utilization. The trial incorporates immunophenotyping to identify long COVID biomarkers and treatment responders. CONCLUSION: The PAX LC trial uses a novel decentralized design and a participant-centric approach to test a 15-day regimen of nirmatrelvir/ritonavir for long COVID.

Long COVID Characteristics and Experience: A Descriptive Study from the Yale LISTEN Research Cohort.

OBJECTIVE: To describe the experience of people with long COVID symptomatology and characterize the psychological, social, and financial challenges they experience. BACKGROUND: The experience of people with long COVID needs further amplification, especially with a comprehensive focus on symptomatology, treatments, and impact on daily life and finances. METHODS: We collected data from individuals aged 18 and older reporting long COVID as participants in the Yale Listen to Immune, Symptom and Treatment Experiences Now (LISTEN) Study. The sample population included 441 participants surveyed between May 2022 and July 2023. We evaluated their demographic characteristics, socioeconomic and psychological status, index infection period, health status, quality of life, symptoms, treatments, pre-pandemic comorbidities, and new-onset conditions. RESULTS: Overall, the median age of the participants with long COVID was 46 years (IQR: 38 to 57 years); 74% were women, 86% were Non-Hispanic White, and 93% were from the United States. Participants reported low health status measured by the Euro-QoL visual analogue scale, with a median score of 49 (IQR: 32 to 61). Participants documented a diverse range of symptoms, with all 96 possible symptom choices being reported. Additionally, participants had tried many treatments (median number of treatments: 19, IQR: 12 to 28). They were also experiencing psychological distress, social isolation, and financial stress. CONCLUSIONS: Despite having tried numerous treatments, participants with long COVID continued to experience an array of health and financial challenges-findings that underscore the failure of the healthcare system to address the medical needs of people with long COVID. These insights highlight the need for crucial medical, mental health, financial, and community support services, as well as further scientific investigation, to address the complex impact of long COVID.

Sex differences in symptomatology and immune profiles of Long COVID.

Strong sex differences in the frequencies and manifestations of Long COVID (LC) have been reported with females significantly more likely than males to present with LC after acute SARS-CoV-2 infection 1-7 . However, whether immunological traits underlying LC differ between sexes, and whether such differences explain the differential manifestations of LC symptomology is currently unknown. Here, we performed sex-based multi-dimensional immune-endocrine profiling of 165 individuals 8 with and without LC in an exploratory, cross-sectional study to identify key immunological traits underlying biological sex differences in LC. We found that female and male participants with LC experienced different sets of symptoms, and distinct patterns of organ system involvement, with female participants suffering from a higher symptom burden. Machine learning approaches identified differential sets of immune features that characterized LC in females and males. Males with LC had decreased frequencies of monocyte and DC populations, elevated NK cells, and plasma cytokines including IL-8 and TGF-ß-family members. Females with LC had increased frequencies of exhausted T cells, cytokine-secreting T cells, higher antibody reactivity to latent herpes viruses including EBV, HSV-2, and CMV, and lower testosterone levels than their control female counterparts. Testosterone levels were significantly associated with lower symptom burden in LC participants over sex designation. These findings suggest distinct immunological processes of LC in females and males and illuminate the crucial role of immune-endocrine dysregulation in sex-specific pathology.

Impact of COVID-19 vaccination on symptoms and immune phenotypes in vaccine-naïve individuals with Long COVID.

Background: Long COVID contributes to the global burden of disease. Proposed root cause hypotheses include the persistence of SARS-CoV-2 viral reservoir, autoimmunity, and reactivation of latent herpesviruses. Patients have reported various changes in Long COVID symptoms after COVID-19 vaccinations, leaving uncertainty about whether vaccine-induced immune responses may alleviate or worsen disease pathology. Methods: In this prospective study, we evaluated changes in symptoms and immune responses after COVID-19 vaccination in 16 vaccine-naïve individuals with Long COVID. Surveys were administered before vaccination and then at 2, 6, and 12 weeks after receiving the first vaccine dose of the primary series. Simultaneously, SARS-CoV-2-reactive TCR enrichment, SARS-CoV-2-specific antibody responses, antibody responses to other viral and self-antigens, and circulating cytokines were quantified before vaccination and at 6 and 12 weeks after vaccination. Results: Self-report at 12 weeks post-vaccination indicated 10 out of 16 participants had improved health, 3 had no change, 1 had worse health, and 2 reported marginal changes. Significant elevation in SARS-CoV-2-specific TCRs and Spike protein-specific IgG were observed 6 and 12 weeks after vaccination. No changes in reactivities were observed against herpes viruses and self-antigens. Within this dataset, higher baseline sIL-6R was associated with symptom improvement, and the two top features associated with non-improvement were high IFN-ß and CNTF, among soluble analytes. Conclusions: Our study showed that in this small sample, vaccination improved the health or resulted in no change to the health of most participants, though few experienced worsening. Vaccination was associated with increased SARS-CoV-2 Spike protein-specific IgG and T cell expansion in most individuals with Long COVID. Symptom improvement was observed in those with baseline elevated sIL-6R, while elevated interferon and neuropeptide levels were associated with a lack of improvement.

Post-Vaccination Syndrome: A Descriptive Analysis of Reported Symptoms and Patient Experiences After Covid-19 Immunization.

Introduction: A chronic post-vaccination syndrome (PVS) after covid-19 vaccination has been reported but has yet to be well characterized. Methods: We included 241 individuals aged 18 and older who self-reported PVS after covid-19 vaccination and who joined the online Yale Listen to Immune, Symptom and Treatment Experiences Now (LISTEN) Study from May 2022 to July 2023. We summarized their demographics, health status, symptoms, treatments tried, and overall experience. Results: The median age of participants was 46 years (interquartile range [IQR]: 38 to 56), with 192 (80%) identifying as female, 209 (87%) as non-Hispanic White, and 211 (88%) from the United States. Among these participants with PVS, 127 (55%) had received the BNT162b2 [Pfizer-BioNTech] vaccine, and 86 (37%) received the mRNA-1273 [Moderna] vaccine. The median time from the day of index vaccination to symptom onset was three days (IQR: 1 day to 8 days). The time from vaccination to symptom survey completion was 595 days (IQR: 417 to 661 days). The median Euro-QoL visual analogue scale score was 50 (IQR: 39 to 70). The five most common symptoms were exercise intolerance (71%), excessive fatigue (69%), numbness (63%), brain fog (63%), and neuropathy (63%). In the week before survey completion, participants reported feeling unease (93%), fearfulness (82%), and overwhelmed by worries (81%), as well as feelings of helplessness (80%), anxiety (76%), depression (76%), hopelessness (72%), and worthlessness (49%) at least once. Participants reported a median of 20 (IQR: 13 to 30) interventions to treat their condition. Conclusions: In this study, individuals who reported PVS after covid-19 vaccination had low health status, high symptom burden, and high psychosocial stress despite trying many treatments. There is a need for continued investigation to understand and treat this condition.

Internal tremors and vibrations in long COVID: a cross-sectional study.

Importance: Internal tremors and vibrations symptoms have been described as part of neurologic disorders but not fully described as a part of long COVID. Objective: To compare demographics, socioeconomic characteristics, pre-pandemic comorbidities, new-onset conditions, and long COVID symptoms between people with internal tremors and vibrations as part of their long COVID symptoms and people with long COVID but without these symptoms. Design: A cross-sectional study, Listen to Immune, Symptom and Treatment Experiences Now (LISTEN), of adults with and without long COVID and post-vaccination syndrome, defined by self-report. Setting: Hugo Health Kindred, a decentralized digital research platform hosting a network of English-speaking adults interested in contributing to COVID-related research. No geographic limitation applied. Participants: The study population included 423 participants who enrolled in LISTEN between May 2022 and June 2023, completed the initial and the conditions and symptoms surveys, reported long COVID, and did not report post-vaccination syndrome. Exposure: Long COVID symptoms of internal tremors and vibrations. Main outcomes and Measures: Demographics, pre-pandemic comorbidities, and current conditions, other symptoms, and quality of life at the time of surveys. Results: Of the 423 participants (median age, 46 years [IQR, 38-56]), 74% were female, 87% were Non-Hispanic White, 92% lived in the United States, 46% were infected before the Delta wave, and 158 (37%) reported "internal tremors, or buzzing/vibration" as a long COVID symptom. Before long COVID, the groups had similar comorbidities. Participants with internal tremors were different from others in having worse health as measured by the Euro-QoL visual analogue scale (median: 40 points [IQR, 30-60] vs. 50 points [IQR, 35-62], P = 0.007), having financial difficulties caused by the pandemic (very much financial difficulties, 22% [95% CI, 16-30] vs. 11% [7.3-15], P < 0.001), often feeling socially isolated (43% [95% CI, 35-52] vs. 37% [31-43], P = 0.039), and having higher rates of self-reported new-onset mast cell disorders (11% [95% CI, 7.1-18] vs. 2.6% [1.2-5.6], Bonferroni-adjusted P = 0.008) and neurologic conditions (including but not limited to seizures, dementia, multiple sclerosis, Parkinson's disease, neuropathy, etc.; 22% [95% CI, 16-29] vs. 8.3% [5.4-12], Bonferroni-adjusted P = 0.004). Conclusions and Relevance: Among people with long COVID, those with internal tremors and vibrations have several other associated symptoms and worse health status, despite having similar pre-pandemic comorbidities, suggesting it may reflect a severe phenotype of long COVID. KEY POINTS: Question: Do people with long COVID symptoms of internal tremors and vibrations differ from others with long COVID but without these symptoms?Findings: In this cross-sectional study that included 423 adults with long COVID, 158 (37%) reported having "internal tremors, or buzzing/vibration," had worse quality of life, more financial difficulties, and higher rates of new-onset mast cell disorders and neurologic conditions, compared with others with long COVID but without internal tremors and vibrations.Meaning: Internal tremors and vibrations may reflect a severe phenotype of long COVID.

Distinguishing features of long COVID identified through immune profiling.

Post-acute infection syndromes may develop after acute viral disease1. Infection with SARS-CoV-2 can result in the development of a post-acute infection syndrome known as long COVID. Individuals with long COVID frequently report unremitting fatigue, post-exertional malaise, and a variety of cognitive and autonomic dysfunctions2-4. However, the biological processes that are associated with the development and persistence of these symptoms are unclear. Here 275 individuals with or without long COVID were enrolled in a cross-sectional study that included multidimensional immune phenotyping and unbiased machine learning methods to identify biological features associated with long COVID. Marked differences were noted in circulating myeloid and lymphocyte populations relative to the matched controls, as well as evidence of exaggerated humoral responses directed against SARS-CoV-2 among participants with long COVID. Furthermore, higher antibody responses directed against non-SARS-CoV-2 viral pathogens were observed among individuals with long COVID, particularly Epstein-Barr virus. Levels of soluble immune mediators and hormones varied among groups, with cortisol levels being lower among participants with long COVID. Integration of immune phenotyping data into unbiased machine learning models identified the key features that are most strongly associated with long COVID status. Collectively, these findings may help to guide future studies into the pathobiology of long COVID and help with developing relevant biomarkers.

IL-10/IL-6 ratio from nasal & oral swab samples, acts as an inflammatory indicator for COVID-19 patients infected with the delta variant.

Background: Hyper-inflammatory immune response of SARS-CoV-2 is often characterized by the release of multiple pro-inflammatory cytokines with an impact on the expression of numerous other interleukins (ILs). However, from oral and nasal swab samples the specific quantitative association of the different IL-markers with the disease progression and its relationship with the status of vaccination remains unclear. Materials and methods: Patients' combined oral and nasal swab samples were collected from both non-vaccinated and double-vaccinated individuals with high (Ct value < 25) and low (Ct value > 30) viral loads, along with uninfected donors. None of the patients were critically ill, or needed ICU support. The expression of different cytokines (IL6, IL10, IL1B, IFNG) and mucin (MUC5AC, MUC1) markers were assessed between different groups by qRT-PCR. The important cytokine markers differentiating between vaccinated and non-vaccinated patients were identified by PCA. Conclusion: IL6 expression was higher in non-vaccinated COVID-19 patients infected with delta-variant irrespective of their viral-load compared to uninfected individuals. However, in double-vaccinated patients, only in high viral-load patients (Ct value < 25), IL6 expression increased. In high viral-load patients, irrespective to their vaccination status, IL10 expression was lower compared to the uninfected control group. Surprisingly, IL10 expression was lower in double-vaccinated patients with Ct value > 30. IL1B, and IFNG expression remained unaltered in uninfected and infected individuals. However, MUC5AC expression was lower in non-vaccinated patients with Ct value < 25 compared to control group. Our study unveiled that IL10/IL6 ratio can be used as a biomarker for COVID-19 patients upon proper establishment of it in a clinical setting.

Distinguishing features of Long COVID identified through immune profiling.

SARS-CoV-2 infection can result in the development of a constellation of persistent sequelae following acute disease called post-acute sequelae of COVID-19 (PASC) or Long COVID 1-3 . Individuals diagnosed with Long COVID frequently report unremitting fatigue, post-exertional malaise, and a variety of cognitive and autonomic dysfunctions 1-3 ; however, the basic biological mechanisms responsible for these debilitating symptoms are unclear. Here, 215 individuals were included in an exploratory, cross-sectional study to perform multi-dimensional immune phenotyping in conjunction with machine learning methods to identify key immunological features distinguishing Long COVID. Marked differences were noted in specific circulating myeloid and lymphocyte populations relative to matched control groups, as well as evidence of elevated humoral responses directed against SARS-CoV-2 among participants with Long COVID. Further, unexpected increases were observed in antibody responses directed against non-SARS-CoV-2 viral pathogens, particularly Epstein-Barr virus. Analysis of circulating immune mediators and various hormones also revealed pronounced differences, with levels of cortisol being uniformly lower among participants with Long COVID relative to matched control groups. Integration of immune phenotyping data into unbiased machine learning models identified significant distinguishing features critical in accurate classification of Long COVID, with decreased levels of cortisol being the most significant individual predictor. These findings will help guide additional studies into the pathobiology of Long COVID and may aid in the future development of objective biomarkers for Long COVID.

Influence of Sub-Inhibitory Dosage of Cefotaxime on Multidrug Resistant Staphylococcus haemolyticus Isolated from Sick Neonatal Care Unit.

Staphylococcus haemolyticus has emerged to be a frequently encountered late-onset sepsis pathogen among newborn infants. Critical care of neonates involves substantial usage of antibiotics and these pathogens are often exposed to sub-optimal doses of antibiotics which can augment maintenance of selection determinants and a range of physiological effects, prime among them being biofilm formation. Therefore, in this study, the outcome of a sub-inhibitory dosage of a commonly prescribed third-generation antibiotic, cefotaxime (CTX), on multidrug resistant (MDR) S. haemolyticus, was investigated. A total of 19 CTX-resistant, MDR and 5 CTX-susceptible strains isolated from neonates were included. Biofilm-forming abilities of S. haemolyticus isolates in the presence of sub-optimal CTX (30 µg/mL) were determined by crystal violet assays and extracellular DNA (eDNA) quantitation. CTX was found to significantly enhance biofilm production among the non-susceptible isolates (p-valueWilcoxintest­0.000008) with an increase in eDNA levels (p-valueWilcoxintest­0.000004). Further, in the absence of antibiotic selection in vitro, populations of MDR isolates, JNM56C1 and JNM60C2 remained antibiotic non-susceptible after >500 generations of growth. These findings demonstrate that sub-optimal concentration of CTX induces biofilm formation and short-term non-exposure to antibiotics does not alter non-susceptibility among S. haemolyticus isolates under the tested conditions.

The Species-Specific 282 Residue in the PB2 Subunit of the Polymerase Regulates RNA Synthesis and Replication of Influenza A Viruses Infecting Bat and Nonbat Hosts.

Bat influenza viruses are genetically distant from classical influenza A viruses (IAVs) and show distinct functional differences in their surface antigens. Nevertheless, any comparative analyses between bat and classical IAV RNA polymerases or their specific subunits are yet to be performed. In this work, we have identified signature residues present in the bat influenza virus polymerase which are responsible for its altered fitness in comparison to the classical IAVs. Through comparative sequence and structural analysis, we have identified specific positions in the PB2 subunit of the polymerase, with differential amino acid preferences among bat and nonbat IAVs. Functional screening helped us to focus upon the previously uncharacterized PB2-282 residue, which is serine in bat virus but harbors highly conserved glutamic acid in classical IAVs. Introduction of E282S mutation in the human-adapted PB2 (influenza A/H1N1/WSN/1933) drastically reduces polymerase activity and replication efficiency of the virus in human, bat, and canine cells. Interestingly, this newly identified PB2-282 residue within an evolutionary conserved "S-E-S" motif, present across different genera of influenza viruses and serving as a key regulator of RNA synthesis activity of the polymerase. In contrast, bat influenza viruses harbor an atypical "S-S-T" motif at the same position of PB2, alteration of which with the human-like "S-E-T" motif significantly enhances its (H17N10/Guatemala/164/2009) polymerase activity in human cells. Together, our data indicate that the PB2-S282 residue may serve as an inherent restriction element of the bat virus polymerase, limiting its activity in other host species. IMPORTANCE Influenza A viruses are known for their ability to perform cross-species transmission, facilitated by amino acid alterations either in the surface antigen hemagglutinin (HA) or in the polymerase subunit PB2. Recent isolation of influenza A-like viruses from bats raised concern about their epizootic and zoonotic potential. Here, we identify a novel species-specific signature present within the influenza virus polymerase that may serve as a key factor in adaptation of influenza viruses from bat to nonbat host species. The PB2-282 residue, which harbors a highly conserved glutamic acid for influenza viruses across all genera (A, B, C, and D), encompasses an atypical serine in the case of bat influenza viruses. Our data show that the human-adapted polymerase, harboring a bat-specific signature (PB2-S282,) performs poorly, while bat PB2 protein, harboring a human-specific signature (PB2-E282), shows increased fitness in human cells.

Predominance of genomically defined A lineage of HPV16 over D lineage in Indian patients from eastern India with squamous cell carcinoma of the cervix in association with distinct oncogenic phenotypes.

Human papillomavirus type-16 (HPV16) is classified into lineages, A, B, C and D and 10 sub-lineages portraying variable infectivity, persistence, and cytological outcomes, however, with geographical variations. Our objective was to delineate the distinctive features of lineages among cervical squamous cell carcinoma (SCC) in the eastern region of India. A total of 145 SCC cases and 24 non-malignant specimens, harboring episomal HPV16, were included. The presence of higher proportion of lineage A over D was observed among SCC cases (86.89% A1, 8.97% D1 and 4.14% D2), while only A1 sub-lineage viruses were found among control specimens. Among the A1 viruses, an association of variants in the E5 (Y44L, I65V), E6 (L83V) genes and LCR: C7577T with SCC, with combined Odd's ratio (95% CI) of 20.5(4.61-91.25) was observed. Network analyses revealed the presence of 10 clades of lineage A viruses comprising of 64 HPV16 genomes harboring the risk alleles. High episomal HPV16 DNA copy numbers (adjusted p-value= 0.0271) and E7 mRNA expression (p-value=0.000017) predominated in SCC with lineage A, over D. Our study highlights the distinctive modalities of oncogenicity among different HPV16 lineages.

Association of clade-G SARS-CoV-2 viruses and age with increased mortality rates across 57 countries and India.

Several reports have highlighted the contributions of host factors such as age, gender and co-morbidities such as diabetes, hypertension and coronary heart disease in determining COVID-19 disease severity. However, inspite of initial efforts at understanding the contributions of SARS-CoV-2 variants, most were unable to delineate causality. Hence, in this study we re-visited the contributions of different clades of viruses (G, GR and GH) along with other attributes in explaining the disparity in mortality rates among countries. A total of 26,642 high quality SARS-CoV-2 sequences were included and the A23,403G (S:D614G) variant was found to be in linkage disequilibrium with C14,408 U (RdRp: P323L). Linear regression analyses revealed increase in age [Odds ratio: 1.055 (p-value 0.000358)] and higher frequency of clade-G viruses [Odds ratio: 1.029(p-value 0.000135)] could explain 37.43% of the differences in mortality rates across the 58 countries (Multiple R-squared: 0.3743). Next, Machine-Learning algorithms LogitBoost and AdaboostM1 were applied to determine whether countries belonging to high/low mortality groups could be classified using the same attributes and accurate classification was achieved in 70.69% and 62.07% of the countries, respectively. Further, evolutionary analyses of the Indian viral population (n = 662) were carried out. Allele frequency spectrum, nucleotide diversity (π) values and negative Tajima's D values across ORFs were indicative of population expansion. Network analysis revealed the presence of two major clusters of viral haplotypes, namely, clade-G and a variant of clade L [Lv] having the RdRp:A97V amino acid change. Clade-G genomes were found to be evolving more rapidly and were also found in higher proportions in three states with highest mortality rates namely, Gujarat, Madhya Pradesh and West Bengal. Thus, the findings of this study and results from in vitro studies highlighting the role of these variants in increasing transmissibility and altering response to antivirals reflect the role of viral factors in disease prognosis.

Prevalence of Colistin-Resistant, Carbapenem-Hydrolyzing Proteobacteria in Hospital Water Bodies and Out-Falls of West Bengal, India.

Indiscriminate use of antibiotics has resulted in a catastrophic increase in the levels of antibiotic resistance in India. Hospitals treat critical bacterial infections and thus can serve as reservoirs of multidrug resistant (MDR) bacteria. Hence, this study was conducted to gauge the prevalence patterns of MDR bacteria in hospital wastewater. Water samples collected from 11 hospitals and 4 environmental sources belonging to 5 most-densely populated districts of West Bengal, India were grown on MacConkey and Eosin Methylene Blue agar. A total of 84 (hospital-associated = 70, environmental water sources = 14) isolates were characterized. The predominant species found in water from hospital-associated areas (HAA) were Acinetobacter baumannii (22.9%), Escherichia coli (28.6 %), and Klebsiella pneumoniae (25.7%). Greater than 75% of the HAA isolates were found to be mcr-1 gene negative and colistinresistant. Meropenem non-susceptibility was also high among the HAA isolates at 58.6%, with the presence of the carbapenemase gene and blaNDM in 67.1% of the non-susceptible isolates. Among the three predominant species, significantly higher numbers of E. coli isolates were found to be non-susceptible to meropenem ((80%), p-value = 0.00432) and amikacin (AK (90%), p-value = 0.00037). This study provides evidence for the presence of high numbers of colistin-resistant and carbapenem-hydrolyzing Proteobacteriain hospital wastewater.

Bactericidal Activity of Lactic Acid against Clinical, Carbapenem-Hydrolyzing, Multi-Drug-Resistant Klebsiella pneumoniae Planktonic and Biofilm-Forming Cells.

: Carbapenem resistant Klebsiella pneumoniae has been highlighted to be a critical pathogen by the World Health Organization. The objectives of this study were to assess the efficacy of lactic acid (LA) against planktonic cells and biofilms formed by carbapenem-hydrolyzing K. pneumoniae isolates obtained from the nares of preterm neonates. Time-kill assays with graded percentages of (v/v) LA in water were initially carried out against planktonic cells of a meropenem (MRP)-resistant K. pneumoniae isolate, JNM11.C4. The efficacy parameters such as optimal incubation time and minimum inhibitory concentration were determined by comparing colony-forming unit counts (log(10)CFU). Scanning electron microscopy was used to visualize cell damage. Likewise, JNM11.C4 biofilms were treated with graded series of (v/v) LA. Six carbapenem-hydrolyzing isolates were next used to validate the results. A reduction of 3.6 ± 0.6 log(10) CFU/mL in JNM11.C4 planktonic cells and >3 ± 0.03log(10) CFU/mL in biofilm-forming cells were observed using 0.225% and 2% LA, respectively, after three hours. Similar decreases in viable cell-counts were observed both in the case of planktonic (˃3.6 ± 0.3log(10) CFU/mL) and biofilm-forming cells (3.8 ± 0.3log(10) CFU/mL) across all the six clinical isolates. These results indicate that LA is an effective antimicrobial against planktonic carbapenem-hydrolyzing K. pneumoniae cells and biofilms.

Complete Genome Sequences of Eight Human Papillomavirus Type 16 Asian American and European Variant Isolates from Cervical Biopsies and Lesions in Indian Women.

Human papillomavirus type 16 (HPV16), a member of the Papillomaviridae family, is the primary etiological agent of cervical cancer. Here, we report the complete genome sequences of four HPV16 Asian American variants and four European variants, isolated from cervical biopsies and scrapings in India.

Limits and patterns of cytomegalovirus genomic diversity in humans.

Human cytomegalovirus (HCMV) exhibits surprisingly high genomic diversity during natural infection although little is known about the limits or patterns of HCMV diversity among humans. To address this deficiency, we analyzed genomic diversity among congenitally infected infants. We show that there is an upper limit to HCMV genomic diversity in these patient samples, with ∼ 25% of the genome being devoid of polymorphisms. These low diversity regions were distributed across 26 loci that were preferentially located in DNA-processing genes. Furthermore, by developing, to our knowledge, the first genome-wide mutation and recombination rate maps for HCMV, we show that genomic diversity is positively correlated with these two rates. In contrast, median levels of viral genomic diversity did not vary between putatively single or mixed strain infections. We also provide evidence that HCMV populations isolated from vascular compartments of hosts from different continents are genetically similar and that polymorphisms in glycoproteins and regulatory proteins are enriched in these viral populations. This analysis provides the most highly detailed map of HCMV genomic diversity in human hosts to date and informs our understanding of the distribution of HCMV genomic diversity within human hosts.

RNA and ß-hemolysin of group B Streptococcus induce interleukin-1ß (IL-1ß) by activating NLRP3 inflammasomes in mouse macrophages.

The inflammatory cytokine IL-1ß is critical for host responses against many human pathogens. Here, we define Group B Streptococcus (GBS)-mediated activation of the Nod-like receptor-P3 (NLRP3) inflammasome in macrophages. NLRP3 activation requires GBS expression of the cytolytic toxin, ß-hemolysin, lysosomal acidification, and leakage. These processes allow the interaction of GBS RNA with cytosolic NLRP3. The present study supports a model in which GBS RNA, along with lysosomal components including cathepsins, leaks out of lysosomes and interacts with NLRP3 to induce IL-1ß production.

Rapid intrahost evolution of human cytomegalovirus is shaped by demography and positive selection.

Populations of human cytomegalovirus (HCMV), a large DNA virus, are highly polymorphic in patient samples, which may allow for rapid evolution within human hosts. To understand HCMV evolution, longitudinally sampled genomic populations from the urine and plasma of 5 infants with symptomatic congenital HCMV infection were analyzed. Temporal and compartmental variability of viral populations were quantified using high throughput sequencing and population genetics approaches. HCMV populations were generally stable over time, with ~88% of SNPs displaying similar frequencies. However, samples collected from plasma and urine of the same patient at the same time were highly differentiated with approximately 1700 consensus sequence SNPs (1.2% of the genome) identified between compartments. This inter-compartment differentiation was comparable to the differentiation observed in unrelated hosts. Models of demography (i.e., changes in population size and structure) and positive selection were evaluated to explain the observed patterns of variation. Evidence for strong bottlenecks (>90% reduction in viral population size) was consistent among all patients. From the timing of the bottlenecks, we conclude that fetal infection occurred between 13-18 weeks gestational age in patients analyzed, while colonization of the urine compartment followed roughly 2 months later. The timing of these bottlenecks is consistent with the clinical histories of congenital HCMV infections. We next inferred that positive selection plays a small but measurable role in viral evolution within a single compartment. However, positive selection appears to be a strong and pervasive driver of evolution associated with compartmentalization, affecting ≥ 34 of the 167 open reading frames (~20%) of the genome. This work offers the most detailed map of HCMV in vivo evolution to date and provides evidence that viral populations can be stable or rapidly differentiate, depending on host environment. The application of population genetic methods to these data provides clinically useful information, such as the timing of infection and compartment colonization.