Correlates of infectiousness
The role that individuals with asymptomatic or mildly symptomatic severe acute respiratory syndrome coronavirus 2 have in transmission of the virus is not well ...understood. Jones
et al.
investigated viral load in patients, comparing those showing few, if any, symptoms with hospitalized cases. Approximately 400,000 individuals, mostly from Berlin, were tested from February 2020 to March 2021 and about 6% tested positive. Of the 25,381 positive subjects, about 8% showed very high viral loads. People became infectious within 2 days of infection, and in hospitalized individuals, about 4 days elapsed from the start of virus shedding to the time of peak viral load, which occurred 1 to 3 days before the onset of symptoms. Overall, viral load was highly variable, but was about 10-fold higher in persons infected with the B.1.1.7 variant. Children had slightly lower viral loads than adults, although this difference may not be clinically significant.
Science
, abi5273, this issue p.
eabi5273
Analysis of thousands of people who tested positive in Germany reveals that many were asymptomatic and a minority exhibited high viral loads.
INTRODUCTION
Although post facto studies have revealed the importance of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) transmission from presymptomatic, asymptomatic, and mildly symptomatic (PAMS) cases, the virological basis of their infectiousness remains largely unquantified. The reasons for the rapid spread of variant lineages of concern, such as B.1.1.7, have yet to be fully determined.
RATIONALE
Viral load (viral RNA concentration) in patient samples and the rate of isolation success of virus from clinical specimens in cell culture are the clinical parameters most directly relevant to infectiousness and hence to transmission. To increase our understanding of the infectiousness of SARS-CoV-2, especially in PAMS cases and those infected with the B.1.1.7 variant, we analyzed viral load data from 25,381 German cases, including 9519 hospitalized patients, 6110 PAMS cases from walk-in test centers, 1533 B.1.1.7 variant infections, and the viral load time series of 4434 (mainly hospitalized) patients. Viral load results were then combined with estimated cell culture isolation probabilities, producing a clinical proxy estimate of infectiousness.
RESULTS
PAMS subjects had, at the first positive test, viral loads and estimated infectiousness only slightly less than hospitalized patients. Similarly, children were found to have mean viral loads only slightly lower (0.5 log
10
units
or less) than those of adults and ~78% of the adult peak cell culture isolation probability. Eight percent of first-positive viral loads were 10
9
copies per swab or higher, across a wide age range (mean 37.6 years, standard deviation 13.4 years), representing a likely highly infectious minority, one-third of whom were PAMS. Relative to non-B.1.1.7 cases, patients with the B.1.1.7 variant had viral loads that were higher by a factor of 10 and estimated cell culture infectivity that was higher by a factor of 2.6. Similar ranges of viral loads from B.1.1.7 and B.1.177 samples were shown to be capable of causing infection in Caco-2 cell culture. A time-course analysis estimates that a peak viral load of 10
8.1
copies per swab is reached 4.3 days after onset of shedding and shows that, across the course of infection, hospitalized patients have slightly higher viral loads than nonhospitalized cases, who in turn have viral loads slightly higher than PAMS cases. Higher viral loads are observed in first-positive tests of PAMS subjects, likely as a result of systematic earlier testing. Mean culture isolation probability declines to 0.5 at 5 days after peak viral load and to 0.3 at 10 days after peak viral load. We estimate a rate of viral load decline of 0.17 log
10
units per day, which, combined with reported estimates of incubation time and time to loss of successful cell culture isolation, suggests that viral load peaks 1 to 3 days before onset of symptoms (in symptomatic cases).
CONCLUSION
PAMS subjects who test positive at walk-in test centers can be expected to be approximately as infectious as hospitalized patients. The level of expected infectious viral shedding of PAMS people is of high importance because they are circulating in the community at the time of detection of infection. Although viral load and cell culture infectivity cannot be translated directly to transmission probability, it is likely that the rapid spread of the B.1.1.7 variant is partly attributable to higher viral load in these cases. Easily measured virological parameters can be used, for example, to estimate transmission risk from different groups (by age, gender, clinical status, etc.), to quantify variance, to show differences in virus variants, to highlight and quantify overdispersion, and to inform quarantine, containment, and elimination strategies.
Viral load and cell culture infectivity in 25,381 SARS-CoV-2 infections.
(
A
) Viral loads in presymptomatic, asymptomatic, and mildly symptomatic cases (PAMS; red), hospitalized patients (blue), and other subjects (black). (
B
) Expected first-positive viral load and cell culture isolation probability, colored as in (A). (
C
) Temporal estimation with lines representing patients, colored as in (A). (
D
) As in (C), but colored by age.
Two elementary parameters for quantifying viral infection and shedding are viral load and whether samples yield a replicating virus isolate in cell culture. We examined 25,381 cases of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in Germany, including 6110 from test centers attended by presymptomatic, asymptomatic, and mildly symptomatic (PAMS) subjects, 9519 who were hospitalized, and 1533 B.1.1.7 lineage infections. The viral load of the youngest subjects was lower than that of the older subjects by 0.5 (or fewer) log
10
units, and they displayed an estimated ~78% of the peak cell culture replication probability; in part this was due to smaller swab sizes and unlikely to be clinically relevant. Viral loads above 10
9
copies per swab were found in 8% of subjects, one-third of whom were PAMS, with a mean age of 37.6 years. We estimate 4.3 days from onset of shedding to peak viral load (10
8.1
RNA copies per swab) and peak cell culture isolation probability (0.75). B.1.1.7 subjects had mean log
10
viral load 1.05 higher than that of non-B.1.1.7 subjects, and the estimated cell culture replication probability of B.1.1.7 subjects was higher by a factor of 2.6.
The ongoing outbreak of the recently emerged novel coronavirus (2019-nCoV) poses a challenge for public health laboratories as virus isolates are unavailable while there is growing evidence that the ...outbreak is more widespread than initially thought, and international spread through travellers does already occur.
We aimed to develop and deploy robust diagnostic methodology for use in public health laboratory settings without having virus material available.
Here we present a validated diagnostic workflow for 2019-nCoV, its design relying on close genetic relatedness of 2019-nCoV with SARS coronavirus, making use of synthetic nucleic acid technology.
The workflow reliably detects 2019-nCoV, and further discriminates 2019-nCoV from SARS-CoV. Through coordination between academic and public laboratories, we confirmed assay exclusivity based on 297 original clinical specimens containing a full spectrum of human respiratory viruses. Control material is made available through European Virus Archive - Global (EVAg), a European Union infrastructure project.
The present study demonstrates the enormous response capacity achieved through coordination of academic and public laboratories in national and European research networks.
Antigen point-of-care tests (AgPOCTs) can accelerate SARS-CoV-2 testing. As some AgPOCTs have become available, interest is growing in their utility and performance. Here we aimed to compare the ...analytical sensitivity and specificity of seven commercially available AgPOCT devices.
In a single-centre, laboratory evaluation study, we compared AgPOCT products from seven suppliers: the Abbott Panbio COVID-19 Ag Rapid Test, the RapiGEN BIOCREDIT COVID-19 Ag, the Healgen Coronavirus Ag Rapid Test Cassette (Swab), the Coris BioConcept COVID-19 Ag Respi-Strip, the R-Biopharm RIDA QUICK SARS-CoV-2 Antigen, the nal von minden NADAL COVID-19 Ag Test, and the Roche-SD Biosensor SARS-CoV Rapid Antigen Test. Tests were evaluated on recombinant SARS-CoV-2 nucleoprotein, cultured endemic and emerging coronaviruses, stored respiratory samples with known SARS-CoV-2 viral loads, stored samples from patients with respiratory pathogens other than SARS-CoV-2, and self-sampled swabs from healthy volunteers. We estimated analytical sensitivity in terms of approximate viral concentrations (quantified by real-time RT-PCR) that yielded positive AgPOCT results, and specificity in terms of propensity to generate false-positive results.
In 138 clinical samples with quantified SARS-CoV-2 viral load, the 95% limit of detection (concentration at which 95% of test results were positive) in six of seven AgPOCT products ranged between 2·07 × 106 and 2·86 × 107 copies per swab, with an outlier (RapiGEN) at 1·57 × 1010 copies per swab. The assays showed no cross-reactivity towards cell culture or tissue culture supernatants containing any of the four endemic human coronaviruses (HCoV‑229E, HCoV‑NL63, HCoV‑OC43, or HCoV‑HKU1) or MERS-CoV, with the exception of the Healgen assay in one repeat test on HCoV-HKU1 supernatant. SARS-CoV was cross-detected by all assays. Cumulative specificities among stored clinical samples with non-SARS-CoV-2 infections (n=100) and self-samples from healthy volunteers (n=35; cumulative sample n=135) ranged between 98·5% (95% CI 94·2–99·7) and 100·0% (97·2–100·0) in five products, with two outliers at 94·8% (89·2–97·7; R-Biopharm) and 88·9% (82·1–93·4; Healgen). False-positive results did not appear to be associated with any specific respiratory pathogen.
The sensitivity range of most AgPOCTs overlaps with SARS-CoV-2 viral loads typically observed in the first week of symptoms, which marks the infectious period in most patients. The AgPOCTs with limit of detections that approximate virus concentrations at which patients are infectious might enable shortcuts in decision making in various areas of health care and public health.
EU's Horizon 2020 research and innovation programme, German Ministry of Research, German Federal Ministry for Economic Affairs and Energy, German Ministry of Health, and Bill & Melinda Gates Foundation.
Background
During the current severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) pandemic, computed tomography (CT) has become widely used in patients with suspected or known ...coronavirus disease 2019 (COVID-19). This prospective observational study in 28 invasively ventilated and 18 non-invasively ventilated patients with confirmed SARS-CoV-2 contamination aims at investigating SARS-CoV-2 contamination of CT scanner surfaces and its infectiousness.
Methods
Swab sampling of the CT table and gantry before and after CT examinations was performed. Additionally, the CT ventilation system air grid was wiped off after each examination. Real-time reverse-transcription polymerase chain reaction (RT-PCR) for SARS-CoV-2 RNA (ribonucleic acid) and viral cell culture were performed in the virology core lab.
Results
After examination of non-invasively ventilated or non-ventilated patients, SARS-CoV-2 RNA was found in 11.1% (4/36) on patient near surfaces (CT table and gantry) and in 16.7% (3/18) on the CT air grid respectively after examination of invasively ventilated patients in 5.4% (3/56) on CT table and gantry and 7.1% (2/28) on the CT air grid. Surface contamination was more common in non-invasively ventilated or non-ventilated patients with a high viral load who were actively coughing. RT-PCR cycle threshold (Ct) was high (35.96–39.31) in all positive samples and no positive viral cell culture was found.
Conclusion
Our study suggests that CT scanner surface contamination with SARS-CoV-2 is considerable and more common after examination of non-invasively ventilated or non-ventilated patients compared to invasively ventilated patients. However, no viral cell culture positivity was found, hence the infectious potential seems low.
Hepatitis A virus (HAV) is a common human pathogen found exclusively in primates. In a molecular and serologic study of 64 alpacas in Bolivia, we detected RNA of distinct HAV in ≈9% of animals and ...HAV antibodies in ≈64%. Complete-genome analysis suggests a long association of HAV with alpacas.
Enteroviruses (EVs) belong to the family Picornaviridae and are responsible for mild to severe diseases in mammals including humans and non-human primates (NHP). Simian EVs were first discovered in ...the 1950s in the Old World Monkeys and recently in wild chimpanzee, gorilla and mandrill in Cameroon. In the present study, we screened by PCR EVs in 600 fecal samples of wild apes and monkeys that were collected at four sites in Gabon. A total of 32 samples were positive for EVs (25 from mandrills, 7 from chimpanzees, none from gorillas). The phylogenetic analysis of VP1 and VP2 genes showed that EVs identified in chimpanzees were members of two human EV species, EV-A and EV-B, and those identified in mandrills were members of the human species EV-B and the simian species EV-J. The identification of two novel enterovirus types, EV-B112 in a chimpanzee and EV-B113 in a mandrill, suggests these NHPs could be potential sources of new EV types. The identification of EV-B107 and EV90 that were previously found in humans indicates cross-species transfers. Also the identification of chimpanzee-derived EV110 in a mandrill demonstrated a wide host range of this EV. Further research of EVs in NHPs would help understanding emergence of new types or variants, and evaluating the real risk of cross-species transmission for humans as well for NHPs populations.
Enteroviruses, members of the Picornaviridae family, are ubiquitous viruses responsible for mild to severe infections in human populations around the world. In 2010 Pointe-Noire, Republic of Congo ...recorded an outbreak of acute flaccid paralysis (AFP) in the humans, caused by wild poliovirus type 1 (WPV1). One month later, in the Tchimpounga sanctuary near Pointe-Noire, a chimpanzee developed signs similar to AFP, with paralysis of the lower limbs. In the present work, we sought to identify the pathogen, including viral and bacterial agents, responsible for this illness. In order to identify the causative agent, we evaluated a fecal specimen by PCR and sequencing. A Human enterovirus C, specifically of the EV-C99 type was potentially responsible for the illness in this chimpanzee. To rule out other possible causative agents, we also investigated the bacteriome and the virome using next generation sequencing. The majority of bacterial reads obtained belonged to commensal bacteria (95%), and the mammalian virus reads matched mainly with viruses of the Picornaviridae family (99%), in which enteroviruses were the most abundant (99.6%). This study thus reports the first identification of a chimpanzee presenting AFP most likely caused by an enterovirus and demonstrates once again the cross-species transmission of a human pathogen to an ape.
The Middle East respiratory syndrome coronavirus (MERS-CoV) is a lethal zoonotic pathogen endemic to the Arabian Peninsula. Dromedary camels are a likely source of infection and the virus probably ...originated in Africa. We studied the genetic diversity, geographical structure, infection prevalence, and age-associated prevalence among camels at the largest entry port of camels from Africa into the Arabian Peninsula.
In this prospective genomic study, we took nasal samples from camels imported from Sudan and Djibouti into the Port of Jeddah in Jeddah, Saudi Arabia, over an almost 2-year period and local Arabian camels over 2 months in the year after surveillance of the port. We determined the prevalence of MERS-CoV infection, age-associated patterns of infection, and undertook phylogeographical and migration analyses to determine intercountry virus transmission after local lineage establishment. We compared all virological characteristics between the local and imported cohorts. We compared major gene deletions between African and Arabian strains of the virus. Reproductive numbers were inferred with Bayesian birth death skyline analyses.
Between Aug 10, 2016, and May 3, 2018, we collected samples from 1196 imported camels, of which 868 originated from Sudan and 328 from Djibouti, and between May 1, and June 25, 2018, we collected samples from 472 local camels, of which 189 were from Riyadh and 283 were from Jeddah, Saudi Arabia. Virus prevalence was higher in local camels than in imported camels (224 47·5% of 472 vs 157 13·1% of 1196; p<0·0001). Infection prevalence peaked among camels older than 1 year and aged up to 2 years in both groups, with 255 (66·9%) of 381 positive cases in this age group. Although the overall geographical distribution of the virus corresponded with the phylogenetic tree topology, some virus exchange was observed between countries corresponding with trade routes in the region. East and west African strains of the virus appear to be geographically separated, with an origin of west African strains in east Africa. African strains of the virus were not re-sampled in Saudi Arabia despite sampling approximately 1 year after importation from Africa. All local Arabian samples contained strains of the virus that belong to a novel recombinant clade (NRC) first detected in 2014 in Saudi Arabia. Reproduction number estimates informed by the sequences suggest sustained endemicity of NRC, with a mean Re of 1·16.
Despite frequent imports of MERS-CoV with camels from Africa, African lineages of MERS-CoV do not establish themselves in Saudi Arabia. Arabian strains of the virus should be tested for changes in virulence and transmissibility.
German Ministry of Research and Education, EU Horizon 2020, and Emerging Diseases Clinical Trials Partnership.
International travel is a major driver of the introduction and spread of SARS-CoV-2.
To investigate SARS-CoV-2 genetic diversity in the region of a major transport hub in Germany, we characterized ...the viral sequence diversity of the SARS-CoV-2 variants circulating in Frankfurt am Main, the city with the largest airport in Germany, from the end of October to the end of December 2020.
In total, we recovered 136 SARS-CoV-2 genomes from nasopharyngeal swab samples. We isolated 104 isolates that were grown in cell culture and RNA from the recovered viruses and subjected them to full-genome sequence analysis. In addition, 32 nasopharyngeal swab samples were directly sequenced.
We found 28 different lineages of SARS-CoV-2 circulating during the study period, including the variant of concern B.1.1.7 (Δ69/70, N501Y). Six of the lineages had not previously been observed in Germany. We detected the spike protein (S) deletion Δ69/Δ70 in 15% of all sequences, a four base pair (bp) deletion (in 2.9% of sequences) and a single bp deletion (in 0.7% of sequences) in ORF3a, leading to ORF3a truncations. In four sequences (2.9%), an amino acid deletion at position 210 in S was identified. In a single sample (0.7%), both a 9 bp deletion in ORF1ab and a 7 bp deletion in ORF7a were identified. One sequence in lineage B.1.1.70 had an N501Y substitution while lacking the Δ69/70 in S. The high diversity of sequences observed over two months in Frankfurt am Main highlights the persisting need for continuous SARS-CoV-2 surveillance using full-genome sequencing, particularly in cities with international airport connections.
Coronavirus disease 2019 (COVID-19) is an acute infection of the respiratory tract that emerged in late 2019
. Initial outbreaks in China involved 13.8% of cases with severe courses, and 6.1% of ...cases with critical courses
. This severe presentation may result from the virus using a virus receptor that is expressed predominantly in the lung
; the same receptor tropism is thought to have determined the pathogenicity-but also aided in the control-of severe acute respiratory syndrome (SARS) in 2003
. However, there are reports of cases of COVID-19 in which the patient shows mild upper respiratory tract symptoms, which suggests the potential for pre- or oligosymptomatic transmission
. There is an urgent need for information on virus replication, immunity and infectivity in specific sites of the body. Here we report a detailed virological analysis of nine cases of COVID-19 that provides proof of active virus replication in tissues of the upper respiratory tract. Pharyngeal virus shedding was very high during the first week of symptoms, with a peak at 7.11 × 10
RNA copies per throat swab on day 4. Infectious virus was readily isolated from samples derived from the throat or lung, but not from stool samples-in spite of high concentrations of virus RNA. Blood and urine samples never yielded virus. Active replication in the throat was confirmed by the presence of viral replicative RNA intermediates in the throat samples. We consistently detected sequence-distinct virus populations in throat and lung samples from one patient, proving independent replication. The shedding of viral RNA from sputum outlasted the end of symptoms. Seroconversion occurred after 7 days in 50% of patients (and by day 14 in all patients), but was not followed by a rapid decline in viral load. COVID-19 can present as a mild illness of the upper respiratory tract. The confirmation of active virus replication in the upper respiratory tract has implications for the containment of COVID-19.
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