BackgroundPreterm delivery causes substantial neonatal mortality and morbidity. Unrecognized intra-amniotic infections caused by cultivation-resistant microbes may play a role. Molecular methods can detect, characterize and quantify microbes independently of traditional culture techniques. However, molecular studies that define the diversity and abundance of microbes invading the amniotic cavity, and evaluate their clinical significance within a causal framework, are lacking.Methods and FindingsIn parallel with culture, we used broad-range end-point and real-time PCR assays to amplify, identify and quantify ribosomal DNA (rDNA) of bacteria, fungi and archaea from amniotic fluid of 166 women in preterm labor with intact membranes. We sequenced up to 24 rRNA clones per positive specimen and assigned taxonomic designations to approximately the species level. Microbial prevalence, diversity and abundance were correlated with host inflammation and with gestational and neonatal outcomes. Study subjects who delivered at term served as controls. The combined use of molecular and culture methods revealed a greater prevalence (15% of subjects) and diversity (18 taxa) of microbes in amniotic fluid than did culture alone (9.6% of subjects; 11 taxa). The taxa detected only by PCR included a related group of fastidious bacteria, comprised of Sneathia sanguinegens, Leptotrichia amnionii and an unassigned, uncultivated, and previously-uncharacterized bacterium; one or more members of this group were detected in 25% of positive specimens. A positive PCR was associated with histologic chorioamnionitis (adjusted odds ratio [OR] 20; 95% CI, 2.4 to 172), and funisitis (adjusted OR 18; 95% CI, 3.1 to 99). The positive predictive value of PCR for preterm delivery was 100 percent. A temporal association between a positive PCR and delivery was supported by a shortened amniocentesis-to-delivery interval (adjusted hazard ratio 4.6; 95% CI, 2.2 to 9.5). A dose-response association was demonstrated between bacterial rDNA abundance and gestational age at delivery (r2 = 0.42; P<0.002).ConclusionsThe amniotic cavity of women in preterm labor harbors DNA from a greater diversity of microbes than previously suspected, including as-yet uncultivated, previously-uncharacterized taxa. The strength, temporality and gradient with which these microbial sequence types are associated with preterm delivery support a causal relationship.
The association between nasopharyngeal (NP) SARS-CoV-2 viral loads and clinical outcomes remains debated. Here, we examined the factors that might predict the NP viral load and the role of the viral load as a predictor of clinical outcomes. A convenience sample of 955 positive remnant NP swab eluent samples collected during routine care between 18 November 2020 and 26 September 2021 was cataloged and a chart review was performed. For non-duplicate samples with available demographic and clinical data (i.e., non-employees), an aliquot of eluent was sent for a droplet digital PCR quantification of the SARS-CoV-2 viral load. Univariate and multivariate analyses were performed to identify the clinical predictors of NP viral loads and the predictors of COVID-19-related clinical outcomes. Samples and data from 698 individuals were included in the final analysis. The sample cohort had a mean age of 50 years (range: 19–91); 86.6% were male and 76.3% were unvaccinated. The NP viral load was higher in people with respiratory symptoms (p = 0.0004) and fevers (p = 0.0006). In the predictive models for the clinical outcomes, the NP viral load approached a significance as a predictor for in-hospital mortality. In conclusion, the NP viral load did not appear to be a strong predictor of moderate-to-severe disease in the pre-Delta and Delta phases of the pandemic, but was predictive of symptomatic diseases and approached a significance for in-hospital mortality, providing support to the thesis that early viral control prevents the progression of disease.
Background Droplet digital PCR (ddPCR) has been shown to be more sensitive and precise in the quantification of SARS-CoV-2 when compared to traditional quantitative RT-PCR. Multiple studies have explored associations between SARS-CoV-2 viral load and patient outcomes; however, few have used ddPCR technology. Here we investigated the associations between viral load measured using ddPCR and clinical presentation and outcomes. Methods We performed a retrospective observational study of individuals who tested positive for COVID-19 at the VA San Diego between August 2020 and December 2021. SARS-CoV-2 viral load from nasopharyngeal swabs was determined using ddPCR. Baseline demographics, past medical history, clinical course, and laboratory data were abstracted from the chart. Results A total of 696 individuals were included, 86% (n=603) of whom were male. The average age was 50-years-old [range: 19-98]. Three-quarters of individuals (76%, n=528) were unvaccinated at diagnosis. Frequency of comorbidities are shown in Table 1. The majority of individuals developed symptoms with 75% (n=516) reporting respiratory symptoms, 47% (n=317) fever, 34% (n=230) GI symptoms, and 23% (n=161) loss of taste and/or smell. A total of 24% of veterans were evaluated only in the emergency department, 21% (n=149) were admitted to the hospital; 9% (n=60) required ICU level of care, 33% of these (n=20) required intubation, and 16 individuals died during hospitalization. SARS-CoV-2 log10 viral load was not associated with age, and only a weak correlation was seen with time from onset of symptoms (r2=-0.1, p=0.04). No association was observed between viral load and peak CRP, ferritin, d-dimer, or nadir absolute lymphocyte count. Mean viral load was significantly higher in veterans reporting fever (5.0 vs 5.4, p=0.02) and respiratory symptoms (4.7 vs 5.3, p=0.01). Interestingly, vaccinated veterans also had higher viral loads(5.8 vs 5.0, p< 0.0001). Baseline characteristics of individuals with COVID-19 Histogram of COVID-19 RNA viral load Conclusion Fever and respiratory symptoms were associated with higher viral loads as expected. The association of vaccination with higher viral load may reflect selection bias for infections in the delta wave. Future work will include multivariate analyses to adjust for medical history and timing of sampling. Disclosures Sanjay R. Mehta, MD, Zibdy Health: Advisor/Consultant.
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