The December 2019 outbreak of a novel respiratory virus, SARS-CoV-2, has become an ongoing global pandemic due in part to the challenge of identifying symptomatic, asymptomatic, and pre-symptomatic ...carriers of the virus. CRISPR diagnostics can augment gold-standard PCR-based testing if they can be made rapid, portable, and accurate. Here, we report the development of an amplification-free CRISPR-Cas13a assay for direct detection of SARS-CoV-2 from nasal swab RNA that can be read with a mobile phone microscope. The assay achieved ∼100 copies/μL sensitivity in under 30 min of measurement time and accurately detected pre-extracted RNA from a set of positive clinical samples in under 5 min. We combined crRNAs targeting SARS-CoV-2 RNA to improve sensitivity and specificity and directly quantified viral load using enzyme kinetics. Integrated with a reader device based on a mobile phone, this assay has the potential to enable rapid, low-cost, point-of-care screening for SARS-CoV-2.
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•CRISPR-Cas13a can quantitatively detect SARS-CoV-2 RNA without pre-amplification•Combining crRNAs targeting multiple regions of the viral RNA enhances sensitivity•Cas13a can accurately and rapidly quantify SARS-CoV-2 RNA in patient samples•A mobile phone-based device allows for portable and sensitive readout of the assay
Fozouni et al. devise a way to use CRISPR-Cas13a to detect and quantify SARS-CoV-2 RNA from patient samples without the need for a pre-amplification step. They then show how the assay’s signal can be efficiently detected with a portable, mobile phone-based device.
Background
Belatacept improves long‐term graft survival, but control of some primary viral infections may be impaired. We evaluated the impact of belatacept and tacrolimus on cytomegalovirus (CMV) ...viral control, remission and relapse in CMV high‐risk and moderate‐risk recipients.
Methods
Using a multistate Markov model, we evaluated viral load state transitions of 173 kidney transplant recipients with at least one episode of viremia within 1 year after transplant: state 1, undetectable/low viral load; state 2, moderate viremia; and state 3, severe viremia.
Results
Among high‐risk recipients, belatacept‐treated recipients exhibited a significantly higher probability of entering moderate viremia (.36; 95% CI = .31, .41) than tacrolimus‐treated recipients (.20; 95% CI = .13, .29). The expected number of days in viremic states differed. High‐risk belatacept‐treated recipients persisted in moderate viremia for significantly longer (128 days, 95% CI = 110, 146) than did tacrolimus‐treated recipients (70.0 days, 95% CI = 45.2, 100) and showed a trend of shorter duration in low/undetectable viral load state (172 days, 95% CI = 148, 195) than did tacrolimus‐treated recipients (239 days, 95% CI = 195, 277). Moderate‐risk recipients showed better viral load control and with no differences by immunosuppression.
Conclusion
High‐risk belatacept‐treated recipients showed defects in sustaining viral control relative to tacrolimus‐treated recipients. Avoidance of initial use belatacept in high‐risk recipients or development of modified management protocols should be considered.
Abstract
The 2019 novel coronavirus (2019-nCoV) was detected in the self-collected saliva of 91.7% (11/12) of patients. Serial saliva viral load monitoring generally showed a declining trend. Live ...virus was detected in saliva by viral culture. Saliva is a promising noninvasive specimen for diagnosis, monitoring, and infection control in patients with 2019-nCoV infection.
The UNAIDS 90-90-90 strategy clearly stipulates that 90% of all people on antiretroviral therapy (ART) should have a suppressed viral load. Intensified adherence counselling (IAC) was recently ...recommended by WHO to improve viral suppression among ART-treated paediatric and adolescent clients with virological failure. This paper describes the implementation and outcomes of IAC in the first year of implementation in a public ART program, to inform strategic interventions to reach the "third 90" among children.
A retrospective chart review was conducted for all children aged 9 months to 19 years with HIV viral loads (VL) ≥ 1000 copies/ml at 15 public health facilities from June 2015-December 2016. Data on initial VL test results, IAC sessions, repeat VL test results, and ART regimen switch were abstracted and analysed for completion of IAC and viral suppression after IAC.
A total of 449 children had a detectable viral load above 1000 copies/ml, after an average of 3.5 years (SD 5.8) years of ART. 192 (43%) were 10-20 years of age, and 320 (71%) were receiving Nevirapine-based ART regimen. Out of 345 (77%) who completed the recommended three IAC sessions, 62 (23%) achieved viral suppression following IAC. The mean time from 1st to 3rd IAC session was 113 (SD 153) days and 172 (50%) of the children had completed the three sessions within 200 days.
Suppression rates were low among ART-treated children with virological failure that completed the recommended three IAC sessions. As we move towards having 90% of ART-treated children and adolescents achieve and maintain viral suppression, there is need to re-evaluate the implementation of IAC among children and adolescents to consider both psychosocial and biological factors such as resistance testing for those with multiple detectable viral loads.
Despite immense progress in antiretroviral therapy (ART) scale-up, many people still lack access to basic standards of care, with our ability to meet the Joint United Nations Programme on HIV/AIDS ...90-90-90 treatment targets for HIV/AIDS dependent on dramatic improvements in diagnostics. The World Health Organization recommends routine monitoring of ART effectiveness using viral load (VL) testing at 6 months and every 12 months, to monitor treatment adherence and minimize failure, and will publish its VL toolkit later this year. However, the cost and complexity of VL is preventing scale-up beyond developed contries and there is a lack of awareness among clinicians as to the long-term patient benefits and its role in prolonging the longevity of treatment programs. With developments in this diagnostic field rapidly evolving—including the recent improvements for accurately using dried blood spots and the imminent appearance to the market of point-of-care technologies offering decentralized diagnosis—we describe current barriers to VL testing in resource-limited settings. Effective scale-up can be achieved through health system and laboratory system strengthening and test price reductions, as well as tackling multiple programmatic and funding challenges.
Abstract
Background
Coronavirus disease 2019 (COVID-19) has become a public health emergency. The widely used reverse transcription–polymerase chain reaction (RT-PCR) method has limitations for ...clinical diagnosis and treatment.
Methods
A total of 323 samples from 76 COVID-19–confirmed patients were analyzed by droplet digital PCR (ddPCR) and RT-PCR based 2 target genes (ORF1ab and N). Nasal swabs, throat swabs, sputum, blood, and urine were collected. Clinical and imaging data were obtained for clinical staging.
Results
In 95 samples that tested positive by both methods, the cycle threshold (Ct) of RT-PCR was highly correlated with the copy number of ddPCR (ORF1ab gene, R2 = 0.83; N gene, R2 = 0.87). Four (4/161) negative and 41 (41/67) single-gene positive samples tested by RT-PCR were positive according to ddPCR with viral loads ranging from 11.1 to 123.2 copies/test. The viral load of respiratory samples was then compared and the average viral load in sputum (17 429 ± 6920 copies/test) was found to be significantly higher than in throat swabs (2552 ± 1965 copies/test, P < .001) and nasal swabs (651 ± 501 copies/test, P < .001). Furthermore, the viral loads in the early and progressive stages were significantly higher than that in the recovery stage (46 800 ± 17 272 vs 1252 ± 1027, P < .001) analyzed by sputum samples.
Conclusions
Quantitative monitoring of viral load in lower respiratory tract samples helps to evaluate disease progression, especially in cases of low viral load.
As a public health emergency, quantitative monitoring of coronavirus disease 2019 (COVID-19) in lower respiratory tract samples in infected patients helps to evaluate disease progression, especially in cases of low viral load.
Commercial HIV-1 RNA viral load assays have been routinely used in developed countries to monitor antiretroviral treatment (ART). However, these assays require expensive equipment and reagents, ...well-trained operators, and established laboratory infrastructure. These requirements restrict their use in resource-limited settings where people are most afflicted with the HIV-1 epidemic. Inexpensive alternatives such as the Ultrasensitive p24 assay, the reverse transcriptase (RT) assay and in-house reverse transcription quantitative polymerase chain reaction (RT-qPCR) have been developed. However, they are still time-consuming, technologically complex and inappropriate for decentralized laboratories as point-of-care (POC) tests. Recent advances in microfluidics and nanotechnology offer new strategies to develop low-cost, rapid, robust and simple HIV-1 viral load monitoring systems. We review state-of-the-art technologies used for HIV-1 viral load monitoring in both developed and developing settings. Emerging approaches based on microfluidics and nanotechnology, which have potential to be integrated into POC HIV-1 viral load assays, are also discussed.
HIV viral reservoirs are established very early during infection. Resident memory T cells (T
) are present in tissues such as the lower female genital tract, but the contribution of this subset of ...cells to the pathogenesis and persistence of HIV remains unclear. Here, we show that cervical CD4
T
display a unique repertoire of clusters of differentiation, with enrichment of several molecules associated with HIV infection susceptibility, longevity and self-renewing capacities. These protein profiles are enriched in a fraction of CD4
T
expressing CD32. Cervical explant models show that CD4
T
preferentially support HIV infection and harbor more viral DNA and protein than non-T
. Importantly, cervical tissue from ART-suppressed HIV
women contain high levels of viral DNA and RNA, being the T
fraction the principal contributor. These results recognize the lower female genital tract as an HIV sanctuary and identify CD4
T
as primary targets of HIV infection and viral persistence. Thus, strategies towards an HIV cure will need to consider T
phenotypes, which are widely distributed in tissues.
A central paradigm of immunity is that interferon (IFN)-mediated antiviral responses precede pro-inflammatory ones, optimizing host protection and minimizing collateral damage
. Here, we report that ...for coronavirus disease 2019 (COVID-19) this paradigm does not apply. By investigating temporal IFN and inflammatory cytokine patterns in 32 moderate-to-severe patients with COVID-19 hospitalized for pneumonia and longitudinally followed for the development of respiratory failure and death, we reveal that IFN-λ and type I IFN production were both diminished and delayed, induced only in a fraction of patients as they became critically ill. On the contrary, pro-inflammatory cytokines such as tumor necrosis factor (TNF), interleukin (IL)-6 and IL-8 were produced before IFNs in all patients and persisted for a prolonged time. This condition was reflected in blood transcriptomes wherein prominent IFN signatures were only seen in critically ill patients who also exhibited augmented inflammation. By comparison, in 16 patients with influenza (flu) hospitalized for pneumonia with similar clinicopathological characteristics to those of COVID-19 and 24 nonhospitalized patients with flu with milder symptoms, IFN-λ and type I IFN were robustly induced earlier, at higher levels and independently of disease severity, whereas pro-inflammatory cytokines were only acutely produced. Notably, higher IFN-λ concentrations in patients with COVID-19 correlated with lower viral load in bronchial aspirates and faster viral clearance and a higher IFN-λ to type I IFN ratio correlated with improved outcome for critically ill patients. Moreover, altered cytokine patterns in patients with COVID-19 correlated with longer hospitalization and higher incidence of critical disease and mortality compared to flu. These data point to an untuned antiviral response in COVID-19, contributing to persistent viral presence, hyperinflammation and respiratory failure.
The SARS-CoV-2 Delta variant has spread rapidly worldwide. To provide data on its virological profile, we here report the first local transmission of Delta in mainland China. All 167 infections could ...be traced back to the first index case. Daily sequential PCR testing of quarantined individuals indicated that the viral loads of Delta infections, when they first become PCR-positive, were on average ~1000 times greater compared to lineage A/B infections during the first epidemic wave in China in early 2020, suggesting potentially faster viral replication and greater infectiousness of Delta during early infection. The estimated transmission bottleneck size of the Delta variant was generally narrow, with 1-3 virions in 29 donor-recipient transmission pairs. However, the transmission of minor iSNVs resulted in at least 3 of the 34 substitutions that were identified in the outbreak, highlighting the contribution of intra-host variants to population-level viral diversity during rapid spread.
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