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Metagenomic next-generation sequencing (mNGS) of cerebrospinal fluid (CSF) is an agnostic method for broad-based diagnosis of central nervous system (CNS) infections. Here we analyzed the 7-year performance of clinical CSF mNGS testing of 4,828 samples from June 2016 to April 2023 performed by the University of California, San Francisco (UCSF) clinical microbiology laboratory. Overall, mNGS testing detected 797 organisms from 697 (14.4%) of 4,828 samples, consisting of 363 (45.5%) DNA viruses, 211 (26.4%) RNA viruses, 132 (16.6%) bacteria, 68 (8.5%) fungi and 23 (2.9%) parasites. We also extracted clinical and laboratory metadata from a subset of the samples (n = 1,164) from 1,053 UCSF patients. Among the 220 infectious diagnoses in this subset, 48 (21.8%) were identified by mNGS alone. The sensitivity, specificity and accuracy of mNGS testing for CNS infections were 63.1%, 99.6% and 92.9%, respectively. mNGS testing exhibited higher sensitivity (63.1%) than indirect serologic testing (28.8%) and direct detection testing from both CSF (45.9%) and non-CSF (15.0%) samples (P

Vaginal colonization by fungi may elicit genital inflammation and enhance the risk of adverse reproductive health outcomes, such as HIV acquisition. Cross-sectional studies have linked fungi with an absence of bacterial vaginosis (BV), but it is unclear whether shifts in vaginal bacteria alter the abundance of vaginal fungi. Vaginal swabs collected following topical metronidazole treatment for BV during the phase 2b, placebo-controlled trial of LACTIN-V, a Lactobacillus crispatus-based live biotherapeutic, were assayed with semi-quantitative PCR for the relative quantitation of fungi and key bacterial species and multiplex immunoassay for immune factors. Vaginal fungi increased immediately following metronidazole treatment for BV (adjusted P = 0.0006), with most of this increase attributable to Candida albicans. Vaginal fungi were independently linked to elevated levels of the proinflammatory cytokine interleukin (IL) 17A, although this association did not remain significant after correcting for multiple comparisons. Fungal relative abundance by semi-quantitative PCR returned to baseline levels within 1 month of metronidazole treatment and was not affected by LACTIN-V or placebo administration. Fungal abundance was positively associated with Lactobacillus species, negatively associated with BV-associated bacteria, and positively associated with a variety of proinflammatory cytokines and chemokines, including IL-17A, during and after study product administration. Antibiotic treatment for BV resulted in a transient expanded abundance of vaginal fungi in a subset of women which was unaffected by subsequent administration of LACTIN-V. Vaginal fungi were positively associated with Lactobacillus species and IL-17A and negatively associated with BV-associated bacteria; these associations were most pronounced in the longer-term outcomes.IMPORTANCEVaginal colonization by fungi can enhance the risk of adverse reproductive health outcomes and HIV acquisition, potentially by eliciting genital mucosal inflammation. We show that standard antibiotic treatment for bacterial vaginosis (BV) results in a transient increase in the absolute abundance of vaginal fungi, most of which was identified as Candida albicans. Vaginal fungi were positively associated with proinflammatory immune factors and negatively associated with BV-associated bacteria. These findings improve our understanding of how shifts in the bacterial composition of the vaginal microbiota may enhance proliferation by proinflammatory vaginal fungi, which may have important implications for risk of adverse reproductive health outcomes among women.

Central nervous system (CNS) infections carry a substantial burden of morbidity and mortality worldwide, and accurate and timely diagnosis is required to optimize management. Metagenomic next-generation sequencing (mNGS) has proven to be a valuable tool in detecting pathogens in patients with suspected CNS infection. By sequencing microbial nucleic acids present in a patient's cerebrospinal fluid, brain tissue, or samples collected outside of the CNS, such as plasma, mNGS can detect a wide range of pathogens, including rare, unexpected, and/or fastidious organisms. Furthermore, its target-agnostic approach allows for the identification of both known and novel pathogens. This is particularly useful in cases where conventional diagnostic methods fail to provide an answer. In addition, mNGS can detect multiple microorganisms simultaneously, which is crucial in cases of mixed infections without a clear predominant pathogen. Overall, clinical mNGS testing can help expedite the diagnostic process for CNS infections, guide appropriate management decisions, and ultimately improve clinical outcomes. However, there are key challenges surrounding its use that need to be considered to fully leverage its clinical impact. For example, only a few specialized laboratories offer clinical mNGS due to the complexity of both the laboratory methods and analysis pipelines. Clinicians interpreting mNGS results must be aware of both false negatives-as mNGS is a direct detection modality and requires a sufficient amount of microbial nucleic acid to be present in the sample tested-and false positives-as mNGS detects environmental microbes and their nucleic acids, despite best practices to minimize contamination. Additionally, current costs and turnaround times limit broader implementation of clinical mNGS. Finally, there is uncertainty regarding the best practices for clinical utilization of mNGS, and further work is needed to define the optimal patient population(s), syndrome(s), and time of testing to implement clinical mNGS.

BACKGROUND: Bacterial vaginosis (BV) increases HIV acquisition risk, potentially by eliciting genital inflammation. After BV treatment, the vaginal administration of LACTIN-V, a live biotherapeutic containing the Lactobacillus crispatus strain CTV-05, reduced BV recurrence and vaginal inflammation; however, 3 months after product cessation, CTV-05 colonization was only sustained in 48% of participants. RESULTS: This nested sub-study in 32 participants receiving LACTIN-V finds that 72% (23/32) demonstrate clinically relevant colonization (CTV-05 absolute abundance > 106 CFU/mL) during at least one visit while 28% (9/32) of women demonstrate colonization resistance, even during product administration. Immediately prior to LACTIN-V administration, the colonization-resistant group exhibited elevated vaginal microbiota diversity. During LACTIN-V administration, colonization resistance was associated with elevated vaginal markers of epithelial disruption and reduced chemokines, possibly due to elevated absolute abundance of BV-associated species and reduced L. crispatus. Colonization permissive women were stratified into sustained and transient colonization groups (31% and 41% of participants, respectively) based on CTV-05 colonization after cessation of product administration. These groups also exhibited distinct genital immune profiles during LACTIN-V administration. CONCLUSIONS: The genital immune impact of LACTIN-V may be contingent on the CTV-05 colonization phenotype, which is in turn partially dependent on the success of BV clearance prior to LACTIN-V administration.

Two independent temporal-spatial clusters of hospital-onset Rhizopus infections were evaluated using whole-genome sequencing (WGS). Phylogenetic analysis confirmed that isolates within each cluster were unrelated despite epidemiological suspicion of outbreaks. The ITS1 region alone was insufficient for accurate analysis. WGS has utility for rapid rule-out of suspected nosocomial Rhizopus outbreaks.

BACKGROUND: In 2021, four patients who had received solid organ transplants in the USA developed encephalitis beginning 2-6 weeks after transplantation from a common organ donor. We describe an investigation into the cause of encephalitis in these patients. METHODS: From Nov 7, 2021, to Feb 24, 2022, we conducted a public health investigation involving 15 agencies and medical centres in the USA. We tested various specimens (blood, cerebrospinal fluid, intraocular fluid, serum, and tissues) from the organ donor and recipients by serology, RT-PCR, immunohistochemistry, metagenomic next-generation sequencing, and host gene expression, and conducted a traceback of blood transfusions received by the organ donor. FINDINGS: We identified one read from yellow fever virus in cerebrospinal fluid from the recipient of a kidney using metagenomic next-generation sequencing. Recent infection with yellow fever virus was confirmed in all four organ recipients by identification of yellow fever virus RNA consistent with the 17D vaccine strain in brain tissue from one recipient and seroconversion after transplantation in three recipients. Two patients recovered and two patients had no neurological recovery and died. 3 days before organ procurement, the organ donor received a blood transfusion from a donor who had received a yellow fever vaccine 6 days before blood donation. INTERPRETATION: This investigation substantiates the use of metagenomic next-generation sequencing for the broad-based detection of rare or unexpected pathogens. Health-care workers providing vaccinations should inform patients of the need to defer blood donation for at least 2 weeks after receiving a yellow fever vaccine. Despite mitigation strategies and safety interventions, a low risk of transfusion-transmitted infections remains. FUNDING: US Centers for Disease Control and Prevention (CDC), the Biomedical Advanced Research and Development Authority, and the CDC Epidemiology and Laboratory Capacity Cooperative Agreement for Infectious Diseases.

BackgroundCache Valley virus (CVV) is a mosquito-borne virus that is a rare cause of disease in humans. In the fall of 2020, a patient developed encephalitis 6 weeks following kidney transplantation and receipt of multiple blood transfusions.MethodsAfter ruling out more common etiologies, metagenomic next-generation sequencing (mNGS) of cerebrospinal fluid (CSF) was performed. We reviewed the medical histories of the index kidney recipient, organ donor, and recipients of other organs from the same donor and conducted a blood traceback investigation to evaluate blood transfusion as a possible source of infection in the kidney recipient. We tested patient specimens using reverse-transcription polymerase chain reaction (RT-PCR), the plaque reduction neutralization test, cell culture, and whole-genome sequencing.ResultsCVV was detected in CSF from the index patient by mNGS, and this result was confirmed by RT-PCR, viral culture, and additional whole-genome sequencing. The organ donor and other organ recipients had no evidence of infection with CVV by molecular or serologic testing. Neutralizing antibodies against CVV were detected in serum from a donor of red blood cells received by the index patient immediately prior to transplant. CVV neutralizing antibodies were also detected in serum from a patient who received the co-component plasma from the same blood donation.ConclusionsOur investigation demonstrates probable CVV transmission through blood transfusion. Clinicians should consider arboviral infections in unexplained meningoencephalitis after blood transfusion or organ transplantation. The use of mNGS might facilitate detection of rare, unexpected infections, particularly in immunocompromised patients.

Antimicrobial resistance in urinary tract infections (UTIs) is a major public health concern. This study aims to characterize the phenotypic and genetic basis of multidrug resistance (MDR) among expanded-spectrum cephalosporin-resistant (ESCR) uropathogenic Escherichia coli (UPEC) causing UTIs in California patient populations. Between February and October 2019, 577 ESCR UPEC isolates were collected from patients at 6 clinical laboratory sites across California. Lineage and antibiotic resistance genes were determined by analysis of whole-genome sequence data. The lineages ST131, ST1193, ST648, and ST69 were predominant, representing 46%, 5.5%, 4.5%, and 4.5% of the collection, respectively. Overall, 527 (91%) isolates had an expanded-spectrum β-lactamase (ESBL) phenotype, with blaCTX-M-15, blaCTX-M-27, blaCTX-M-55, and blaCTX-M-14 being the most prevalent ESBL genes. In the 50 non-ESBL phenotype isolates, 40 (62%) contained blaCMY-2, which was the predominant plasmid-mediated AmpC (pAmpC) gene. Narrow-spectrum β-lactamases, blaTEM-1B and blaOXA-1, were also found in 44.9% and 32.1% of isolates, respectively. Among ESCR UPEC isolates, isolates with an ESBL phenotype had a 1.7-times-greater likelihood of being MDR than non-ESBL phenotype isolates (P < 0.001). The cooccurrence of blaCTX-M-15, blaOXA-1, and aac(6')-Ib-cr within ESCR UPEC isolates was strongly correlated. Cooccurrence of blaCTX-M-15, blaOXA-1, and aac(6')-Ib-cr was associated with an increased risk of nonsusceptibility to piperacillin-tazobactam, cefepime, fluoroquinolones, and amikacin as well as MDR. Multivariate regression revealed the presence of blaCTX-M-55, blaTEM-1B, and the ST131 genotype as predictors of MDR. IMPORTANCE The rising incidence of resistance to expanded-spectrum cephalosporins among Escherichia coli strains, the most common cause of UTIs, is threatening our ability to successfully empirically treat these infections. ESCR E. coli strains are often MDR; therefore, UTI caused by these organisms often leads to treatment failure, increased length of hospital stay, and severe complications (D. G. Mark, Y.-Y. Hung, Z. Salim, N. J. Tarlton, et al., Ann Emerg Med 78:357-369, 2021, https://doi.org/10.1016/j.annemergmed.2021.01.003). Here, we performed an in-depth analysis of genetic factors of ESCR E. coli associated with coresistance and MDR. Such knowledge is critical to advance UTI diagnosis, treatment, and antibiotic stewardship.

The effect of vaccination on severity of subsequent COVID-19 in patients with hematologic malignancies (HMs) is unknown. In this single-center retrospective cohort study, we found no difference in severity of COVID-19 disease in vaccinated (n = 16) versus unvaccinated (n = 54) HM patients using an adjusted multiple logistic regression model. Recent anti-B-cell therapy was associated with more severe illness.

BackgroundPreventing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2_ infections in healthcare workers (HCWs) is critical for healthcare delivery. We aimed to estimate and characterize the prevalence and incidence of coronavirus disease 2019 (COVID-19) in a US HCW cohort and to identify risk factors associated with infection.MethodsWe conducted a longitudinal cohort study of HCWs at 3 Bay Area medical centers using serial surveys and SARS-CoV-2 viral and orthogonal serological testing, including measurement of neutralizing antibodies. We estimated baseline prevalence and cumulative incidence of COVID-19. We performed multivariable Cox proportional hazards models to estimate associations of baseline factors with incident infections and evaluated the impact of time-varying exposures on time to COVID-19 using marginal structural models.ResultsA total of 2435 HCWs contributed 768 person-years of follow-up time. We identified 21 of 2435 individuals with prevalent infection, resulting in a baseline prevalence of 0.86% (95% confidence interval [CI], .53%-1.32%). We identified 70 of 2414 incident infections (2.9%), yielding a cumulative incidence rate of 9.11 cases per 100 person-years (95% CI, 7.11-11.52). Community contact with a known COVID-19 case was most strongly correlated with increased hazard for infection (hazard ratio, 8.1 [95% CI, 3.8-17.5]). High-risk work-related exposures (ie, breach in protective measures) drove an association between work exposure and infection (hazard ratio, 2.5 [95% CI, 1.3-4.8). More cases were identified in HCWs when community case rates were high.ConclusionsWe observed modest COVID-19 incidence despite consistent exposure at work. Community contact was strongly associated with infections, but contact at work was not unless accompanied by high-risk exposure.

Laboratory tests for the accurate and rapid identification of SARS-CoV-2 variants can potentially guide the treatment of COVID-19 patients and inform infection control and public health surveillance efforts. Here, we present the development and validation of a rapid COVID-19 variant DETECTR assay incorporating loop-mediated isothermal amplification (LAMP) followed by CRISPR-Cas12 based identification of single nucleotide polymorphism (SNP) mutations in the SARS-CoV-2 spike (S) gene. This assay targets the L452R, E484K/Q/A, and N501Y mutations, at least one of which is found in nearly all major variants. In a comparison of three different Cas12 enzymes, only the newly identified enzyme CasDx1 was able to accurately identify all targeted SNP mutations. An analysis pipeline for CRISPR-based SNP identification from 261 clinical samples yielded a SNP concordance of 97.3% and agreement of 98.9% (258 of 261) for SARS-CoV-2 lineage classification, using SARS-CoV-2 whole-genome sequencing and/or real-time RT-PCR as test comparators. We also showed that detection of the single E484A mutation was necessary and sufficient to accurately identify Omicron from other major circulating variants in patient samples. These findings demonstrate the utility of CRISPR-based DETECTR as a faster and simpler diagnostic method compared with sequencing for SARS-CoV-2 variant identification in clinical and public health laboratories.

BackgroundBacterial vaginosis might increase HIV risk by eliciting genital inflammation and epithelial barrier disruption, whereas vaginal Lactobacillus crispatus is associated with immune quiescence and HIV protection. We investigated the effect of a live biotherapeutic containing L crispatus CTV-05 (LACTIN-V) on genital immunology and key vaginal bacteria.MethodsThis substudy included women aged 18-45 years who participated in the randomised, placebo-controlled, phase 2b trial of LACTIN-V to reduce bacterial vaginosis recurrence, conducted at four universities and hospitals in the USA. Women with negative results for sexually transmitted infection, pregnancy, and urinary tract infection were provided a 5-day course of vaginal metronidazole 0·75% gel. Those who met at least three of four clinical Amsel criteria for bacterial vaginosis and had a Nugent score of 4-10 from Gram staining were eligible. Participants in the LACTIN-V trial were randomly assigned (2:1) to receive either LACTIN-V or placebo, applied vaginally once per day for 5 days during the first week and then twice per week for 10 more weeks. Follow-up visits occurred 4, 8, 12, and 24 weeks after enrolment. Soluble immune factors and the absolute abundance of bacterial taxa were assayed by mutliplex ELISA and quantitative PCR. The primary outcomes were vaginal levels of IL-1α and soluble E-cadherin at 24 weeks (ie, 13 weeks after treatment cessation).FindingsBetween Feb 21, 2020 and March 18, 2021, we characterised genital immune parameters and the vaginal microbiota in a subset of 66 highly adherent participants who were randomly selected, with no exclusion criteria, from those who had attended all study follow-up visits (n=166) in the larger LACTIN-V clinical trial (n=288). 32 (48%) participants received LACTIN-V and 34 (52%) received placebo. LACTIN-V treatment was significantly associated with lower concentrations of the proinflammatory cytokine IL-1α (β coefficient 0·310, SE 0·149; p=0·042) and soluble E-cadherin (0·429, 0·199; p=0·035), a biomarker of epithelial barrier disruption.InterpretationVaginal administration of LACTIN-V following standard bacterial vaginosis therapy resulted in a sustained reduction in genital inflammation and a biomarker of epithelial integrity. The potential of LACTIN-V to reduce HIV susceptibility merits further investigation.FundingCanadian Institutes of Health Research and the National Institutes of Health National Institute of Allergy and Infectious Diseases.

BackgroundBacterial vaginosis (BV) causes genital inflammation and increases HIV risk, whereas a vaginal microbiota dominated by Lactobacillus species is associated with immune quiescence and relative HIV protection. BV treatment reduces genital inflammation, but it is unclear whether this reduction is driven by a decrease in BV-associated bacteria or an increase in Lactobacillus species.METHODSTo evaluate the short-term effect of standard BV treatment on genital immunology and the vaginal microbiota, vaginal swabs were collected immediately before and after metronidazole treatment for BV and analyzed with multiplex ELISA, metagenomic sequencing, and quantitative PCR.RESULTSTopical metronidazole treatment rapidly reduced vaginal levels of proinflammatory cytokines, chemokines, and soluble immune markers of epithelial barrier disruption. Although the vaginal microbiota shifted to dominance by L. iners or L. jensenii, this proportional shift was primarily driven by a 2 to 4 log10-fold reduction in BV-associated bacteria absolute abundance. BV treatment induced no change in the absolute abundance of L. crispatus or L. iners and only minor (

Powassan virus (POWV) is a tickborne flavivirus that causes encephalitis with severe neurologic sequelae (1). In the United States, POWV infections occur primarily in the Northeast and Great Lakes regions [...]

Simultaneous observations of H3+ ${\mathrm{H}}_{3}^{+}$ and H2 ${\mathrm{H}}_{2}$ in Jupiter's northern infrared aurora were conducted on 02 June 2017 using Keck‐NIRSPEC to produce polar projection maps of H3+ ${\mathrm{H}}_{3}^{+}$ radiance, rotational temperature, column density, and H2 ${\mathrm{H}}_{2}$ radiance. The temperature variations within the auroral region are ∼700−1000 ${\sim} 700-1000$ K, generally consistent with previous studies, albeit with some structural differences. Known auroral heating sources including particle precipitation, Joule heating, and ion drag have been examined by studying the correlations between each derived quantity, yet no single dominant mechanism can be identified as the main driver for the energetics in Jupiter's northern auroral region. It appears that a complex interaction exists between the heating driven by various mechanisms and the cooling from the H3+ ${\mathrm{H}}_{3}^{+}$ thermostat effect. Comparisons between the H3+ ${\mathrm{H}}_{3}^{+}$ temperature and the line‐of‐sight ion velocity in the reference frame of (a) the planetary rotation and (b) the neutral atmosphere further suggest that the local thermodynamic equilibrium effect may play an important role in thermospheric heating at Jupiter. Along with previously reported heating events that occurred in both the lower and upper atmosphere, it is speculated that the heating source may originate from an altitude above Jupiter's stratosphere but below the peak altitude of H3+ ${\mathrm{H}}_{3}^{+}$ overtone and H2 ${\mathrm{H}}_{2}$ quadrupole emissions. Key Points: Scanning maps of H3+ ${\mathrm{H}}_{3}^{+}$ radiance, temperature, column density, and H2 ${\mathrm{H}}_{2}$ radiance are produced using Keck‐NIRSPECCorrelations between derived quantities are investigated to study the heating mechanism of Jupiter's auroral thermosphereThe source of heating may originate from an altitude between the stratosphere and H3+ ${\mathrm{H}}_{3}^{+}$ overtone and H2 ${\mathrm{H}}_{2}$ emission peak altitudes [ABSTRACT FROM AUTHOR]

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