Diagnosis of SARS-CoV-2 Infection with LamPORE, a High-Throughput Platform Combining Loop-Mediated Isothermal Amplification and Nanopore Sequencing.

LamPORE is a novel diagnostic platform for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA combining loop-mediated isothermal amplification with nanopore sequencing, which could potentially be used to analyze thousands of samples per day on a single instrument. We evaluated the performance of LamPORE against reverse transcriptase PCR (RT-PCR) using RNA extracted from spiked respiratory samples and stored nose and throat swabs collected at two UK hospitals. The limit of detection of LamPORE was 10 genome copies/µl of extracted RNA, which is above the limit achievable by RT-PCR, but was not associated with a significant reduction of sensitivity in clinical samples. Positive clinical specimens came mostly from patients with acute symptomatic infection, and among them, LamPORE had a diagnostic sensitivity of 99.1% (226/228; 95% confidence interval [CI], 96.9% to 99.9%). Among negative clinical specimens, including 153 with other respiratory pathogens detected, LamPORE had a diagnostic specificity of 99.6% (278/279; 98.0% to 100.0%). Overall, 1.4% (7/514; 0.5% to 2.9%) of samples produced an indeterminate result on first testing, and repeat LamPORE testing on the same RNA extract had a reproducibility of 96.8% (478/494; 94.8% to 98.1%). LamPORE has a similar performance as RT-PCR for the diagnosis of SARS-CoV-2 infection in symptomatic patients and offers a promising approach to high-throughput testing.

SARS-CoV-2 antibody prevalence, titres and neutralising activity in an antenatal cohort, United Kingdom, 14 April to 15 June 2020.

SARS-CoV-2 IgG screening of 1,000 antenatal serum samples in the Oxford area, United Kingdom, between 14 April and 15 June 2020, yielded a 5.3% seroprevalence, mirroring contemporaneous regional data. Among the 53 positive samples, 39 showed in vitro neutralisation activity, correlating with IgG titre (Pearson's correlation p<0.0001). While SARS-CoV-2 seroprevalence in pregnancy cohorts could potentially inform population surveillance, clinical correlates of infection and immunity in pregnancy, and antenatal epidemiology evolution over time need further study.

Quantitative SARS-CoV-2 anti-spike responses to Pfizer-BioNTech and Oxford-AstraZeneca vaccines by previous infection status.

OBJECTIVES: We investigated determinants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) anti-spike IgG responses in healthcare workers (HCWs) following one or two doses of Pfizer-BioNTech or Oxford-AstraZeneca vaccines. METHODS: HCWs participating in regular SARS-CoV-2 PCR and antibody testing were invited for serological testing prior to first and second vaccination, and 4 weeks post-vaccination if receiving a 12-week dosing interval. Quantitative post-vaccination anti-spike antibody responses were measured using the Abbott SARS-CoV-2 IgG II Quant assay (detection threshold: ≥50 AU/mL). We used multivariable logistic regression to identify predictors of seropositivity and generalized additive models to track antibody responses over time. RESULTS: 3570/3610 HCWs (98.9%) were seropositive >14 days post first vaccination and prior to second vaccination: 2706/2720 (99.5%) were seropositive after the Pfizer-BioNTech and 864/890 (97.1%) following the Oxford-AstraZeneca vaccines. Previously infected and younger HCWs were more likely to test seropositive post first vaccination, with no evidence of differences by sex or ethnicity. All 470 HCWs tested >14 days after the second vaccination were seropositive. Quantitative antibody responses were higher after previous infection: median (IQR) >21 days post first Pfizer-BioNTech 14 604 (7644-22 291) AU/mL versus 1028 (564-1985) AU/mL without prior infection (p < 0.001). Oxford-AstraZeneca vaccine recipients had lower readings post first dose than Pfizer-BioNTech recipients, with and without previous infection, 10 095 (5354-17 096) and 435 (203-962) AU/mL respectively (both p < 0.001 versus Pfizer-BioNTech). Antibody responses >21 days post second Pfizer vaccination in those not previously infected, 10 058 (6408-15 582) AU/mL, were similar to those after prior infection followed by one vaccine dose. CONCLUSIONS: SARS-CoV-2 vaccination leads to detectable anti-spike antibodies in nearly all adult HCWs. Whether differences in response impact vaccine efficacy needs further study.

SARS-CoV-2 within-host diversity and transmission.

Extensive global sampling and sequencing of the pandemic virus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have enabled researchers to monitor its spread and to identify concerning new variants. Two important determinants of variant spread are how frequently they arise within individuals and how likely they are to be transmitted. To characterize within-host diversity and transmission, we deep-sequenced 1313 clinical samples from the United Kingdom. SARS-CoV-2 infections are characterized by low levels of within-host diversity when viral loads are high and by a narrow bottleneck at transmission. Most variants are either lost or occasionally fixed at the point of transmission, with minimal persistence of shared diversity, patterns that are readily observable on the phylogenetic tree. Our results suggest that transmission-enhancing and/or immune-escape SARS-CoV-2 variants are likely to arise infrequently but could spread rapidly if successfully transmitted.

SARS-CoV-2 RNA detected in blood products from patients with COVID-19 is not associated with infectious virus.

Background: Laboratory diagnosis of SARS-CoV-2 infection (the cause of COVID-19) uses PCR to detect viral RNA (vRNA) in respiratory samples. SARS-CoV-2 RNA has also been detected in other sample types, but there is limited understanding of the clinical or laboratory significance of its detection in blood. Methods: We undertook a systematic literature review to assimilate the evidence for the frequency of vRNA in blood, and to identify associated clinical characteristics. We performed RT-PCR in serum samples from a UK clinical cohort of acute and convalescent COVID-19 cases (n=212), together with convalescent plasma samples collected by NHS Blood and Transplant (NHSBT) (n=462 additional samples). To determine whether PCR-positive blood samples could pose an infection risk, we attempted virus isolation from a subset of RNA-positive samples. Results: We identified 28 relevant studies, reporting SARS-CoV-2 RNA in 0-76% of blood samples; pooled estimate 10% (95%CI 5-18%). Among serum samples from our clinical cohort, 27/212 (12.7%) had SARS-CoV-2 RNA detected by RT-PCR. RNA detection occurred in samples up to day 20 post symptom onset, and was associated with more severe disease (multivariable odds ratio 7.5). Across all samples collected ≥28 days post symptom onset, 0/494 (0%, 95%CI 0-0.7%) had vRNA detected. Among our PCR-positive samples, cycle threshold (ct) values were high (range 33.5-44.8), suggesting low vRNA copy numbers. PCR-positive sera inoculated into cell culture did not produce any cytopathic effect or yield an increase in detectable SARS-CoV-2 RNA. Conclusions: vRNA was detectable at low viral loads in a minority of serum samples collected in acute infection, but was not associated with infectious SARS-CoV-2 (within the limitations of the assays used). This work helps to inform biosafety precautions for handling blood products from patients with current or previous COVID-19.

Association of haptoglobin levels with age, parasite density, and haptoglobin genotype in a malaria-endemic area of Gabon.

Haptoglobin (Hp) levels were investigated in relation to host genotype in a malaria-endemic area in Gabon. A cross-sectional study of 1-12-year-old children was conducted in the rainy season, a period of high malaria transmission, to examine this relationship. Variables that influenced Hp levels were Hp genotype, location, and age interacting with parasite density. At low parasite densities, there was a negative correlation between Hp levels and age. At higher densities, there was a positive correlation with age. This suggests that in the presence of greater parasite-induced hemolysis, older children are capable of increased production of Hp. Sickle cell trait and ABO blood group was not associated with Hp levels in this population.

Risk factors for hematogenous complications of intravascular catheter-associated Staphylococcus aureus bacteremia.

BACKGROUND: The role of both host and pathogen characteristics in hematogenous seeding following Staphylococcus aureus bacteremia is incompletely understood. METHODS: Consecutive patients with intravascular catheter-associated Staphylococcus aureus bacteremia were prospectively recruited over a 91-month period. The corresponding bloodstream isolates were examined for the presence of 35 putative virulence determinants. Patient and bacterial characteristics associated with the development of hematogenous complications (HCs) (i.e., septic arthritis, vertebral osteomyelitis, or endocarditis) were defined. RESULTS: HC occurred in 42 (13%) of 324 patients. Patient characteristics at diagnosis that were associated with HC included community onset (relative risk [RR], 2.25; 95% confidence interval [CI], 1.24-4.07; P=.007), increased symptom duration (odds ratio for each day, 1.14; 95% CI, 1.06-1.2; P<.001), presence of a long-term intravascular catheter or noncatheter prosthesis (RR, 4.02; 95% CI, 1.74-9.27; P<.001), hemodialysis dependence (RR, 3.84; 95% CI, 2.08-7.10; P<.001), and higher APACHE II score (P=.02). Bacterial characteristics included sea (RR, 2.03; 95% CI, 1.16-3.55; P=.011) and methicillin-resistant S. aureus (MRSA) (RR, 2.09; 95% CI, 1.19-3.67; P=.015). Subsequent failure to remove a catheter was also associated with HC (RR, 2.28; 95% CI, 1.22-4.27; P=.011). On multivariable analysis, symptom duration, hemodialysis dependence, presence of a long-term intravascular catheter or a noncatheter device, and infection with MRSA remained significantly associated with HC. CONCLUSIONS: This investigation identifies 4 host- and pathogen-related risk factors for hematogenous bacterial seeding and reaffirms the importance of prompt catheter removal.

Determinants of acquisition and carriage of Staphylococcus aureus in infancy.

Nasal carriage of Staphylococcus aureus is a major risk factor for invasive S. aureus disease. The aim of this study was to define factors associated with carriage. We conducted a prospective, longitudinal community-based study of infants and their mothers for a period of 6 months following delivery. The epidemiology of carriage was examined for 100 infant-mother pairs. Infant carriage varied significantly with age, falling from 40 to 50% during the first 8 weeks to 21% by 6 months. Determinants of infant S. aureus carriage included maternal carriage, breastfeeding, and number of siblings. Bacterial typing of S. aureus was performed by pulsed-field gel electrophoresis and multilocus sequence typing. The majority of individuals carried a single strain of S. aureus over time, and the mother was the usual source for colonizing isolates in infants. The effect of other components of the normal nasal flora on the development of S. aureus carriage was examined in 157 consecutive infants. Negative associations (putative bacterial interference) between S. aureus and other species occurred early in infancy but were not sustained. An increasing antistaphylococcal effect observed over time was not attributable to bacterial interference. S. aureus carriage in infants is likely to be determined by a combination of host, environmental, and bacterial factors, but bacterial interference does not appear to be an ultimate determinant of carrier status.

Virulent combinations of adhesin and toxin genes in natural populations of Staphylococcus aureus.

Most cases of severe Staphylococcus aureus disease cannot be explained by the action of a single virulence determinant, and it is likely that a number of factors act in combination during the infective process. This study examined the relationship between disease in humans and a large number of putative virulence determinants, both individually and in combination. S. aureus isolates (n = 334) from healthy blood donors and from patients with invasive disease were compared for variation in the presence of 33 putative virulence determinants. After adjusting for the effect of clonality, seven determinants (fnbA, cna, sdrE, sej, eta, hlg, and ica) were significantly more common in invasive isolates. All seven factors contributed independently to virulence. No single factor predominated as the major predictor of virulence, their effects appearing to be cumulative. No combinations of the seven genes were either more or less likely to cause disease than others with the same number of virulence-associated genes. There was evidence of considerable horizontal transfer of genes on a background of clonality. Our findings also suggested that allelic variants of a polymorphic locus can make different contributions to the disease process, further study of which is likely to expand our understanding of staphylococcal disease pathogenesis.