How to design and implement a university-based COVID-19 testing programme? An evaluation of a novel RT-LAMP COVID-19 testing programme in a UK university.

BACKGROUND: Little is known about how asymptomatic testing as a method to control transmission of COVID-19 can be implemented, and the prevalence of asymptomatic infection within university populations. The objective of this study was to investigate how to effectively set-up and implement a COVID-19 testing programme using novel reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) technology and to quantify the scale of asymptomatic infection on a university campus. METHODS: An observational study to describe the set-up and implementation of a novel COVID-19 testing programme on a UK university campus between September and December 2020. RT-LAMP testing was used to identify asymptomatic cases. RESULTS: A total of 1,673 tests were performed using RT-LAMP during the study period, of which 9 were positive for COVID-19, giving an overall positivity rate of 0.54%, equivalent to a rate in the tested population of 538 cases per 100,000 over the duration of testing. All positive tests were found to be positive on RT-PCR testing, giving a false positive rate of 0%. CONCLUSIONS: This study shows that it is possible to rapidly setup a universal university testing programme for COVID-19 in collaboration with local healthcare providers using RT-LAMP testing. Positive results were comparable to those in the local population, though with a different peak of infection. Further research to inform the design of the testing programme includes focus groups of those who underwent testing and further interrogation of the demographics of those opting to be tested to identify potential access problems or inequalities.

A rapid RT-LAMP SARS-CoV-2 screening assay for collapsing asymptomatic COVID-19 transmission.

PURPOSE: To demonstrate the diagnostic performance of rapid SARS-CoV-2 RT-LAMP assays, comparing the performance of genomic versus sub-genomic sequence target with subsequent application in an asymptomatic screening population. METHODS: RT-LAMP diagnostic specificity (DSe) and sensitivity (DSe) was determined using 114 RT-PCR clinically positive and 88 RT-PCR clinically negative swab samples processed through the diagnostic RT-PCR service within the University Hospitals of Leicester NHS Trust. A swab-based RT-LAMP SARS-CoV-2 screening programme was subsequently made available to all staff and students at the University of Leicester (Autumn 2020), implemented to ISO 15189:2012 standards using NHS IT infrastructure and supported by University Hospital Leicester via confirmatory NHS diagnostic laboratory testing of RT-LAMP 'positive' samples. RESULTS: Validation samples reporting a Ct < 20 were detected at 100% DSe and DSp, reducing to 95% DSe (100% DSp) for all samples reporting a Ct < 30 (both genomic dual sub-genomic assays). Advisory screening identified nine positive cases in 1680 symptom free individuals (equivalent to 540 cases per 100,000) with results reported back to participants and feed into national statistics within 48 hours. CONCLUSION: This work demonstrates the utility of a rapid RT-LAMP assay for collapsing transmission of SARS-CoV-2 in an asymptomatic screening population.

SRC/ABL inhibition disrupts CRLF2-driven signaling to induce cell death in B-cell acute lymphoblastic leukemia.

Children with B-cell precursor acute lymphoblastic leukemia (BCP-ALL) overexpressing the CRLF2 gene (hiCRLF2) have poor prognosis. CRLF2 protein overexpression leads to activated JAK/STAT signaling and trials are underway using JAK inhibitors to overcome treatment failure. Pre-clinical studies indicated limited efficacy of single JAK inhibitors, thus additional pathways must be targeted in hiCRLF2 cells. To identify additional activated networks, we used single-cell mass cytometry to examine 15 BCP-ALL primary patient samples. We uncovered a coordinated signaling network downstream of CRLF2 characterized by co-activation of JAK/STAT, PI3K, and CREB pathways. This CRLF2-driven network could be more effectively disrupted by SRC/ABL inhibition than single-agent JAK or PI3K inhibition, and this could be demonstrated even in primary minimal residual disease (MRD) cells. Our study suggests SCR/ABL inhibition as effective in disrupting the cooperative functional networks present in hiCRLF2 BCP-ALL patients, supporting further investigation of this strategy in pre-clinical studies.

Paternal irradiation perturbs the expression of circadian genes in offspring.

The circadian system represents a complex network which influences the timing of many biological processes. Recent studies have established that circadian alterations play an important role in the susceptibility to many human diseases, including cancer. Here we report that paternal irradiation in mice significantly affects the expression of genes involved in rhythmic processes in their first-generation offspring. Using microarrays, the patterns of gene expression were established for brain, kidney, liver and spleen samples from the non-exposed offspring of irradiated CBA/Ca and BALB/c male mice. The most over-represented categories among the genes differentially expressed in the offspring of control and irradiated males were those involved in rhythmic process, circadian rhythm and DNA-dependent regulation of transcription. The results of our study therefore provide a plausible explanation for the transgenerational effects of paternal irradiation, including increased transgenerational carcinogenesis described in other studies.

The effects of maternal irradiation during adulthood on mutation induction and transgenerational instability in mice.

The long-term genetic effects of maternal irradiation remain poorly understood. To establish the effects of radiation exposure on mutation induction in the germline of directly exposed females and the possibility of transgenerational effects in their non-exposed offspring, adult female BALB/c and CBA/Ca mice were given 1 Gy of acute X-rays and mated with control males. The frequency of mutation at expanded simple tandem repeat (ESTR) loci in the germline of directly exposed females did not differ from that of controls. Using a single-molecule PCR approach, ESTR mutation frequency was also established for both germline and somatic tissues in the first-generation offspring of irradiated parents. While the frequency of ESTR mutation in the offspring of irradiated males was significantly elevated, maternal irradiation did not affect stability in their F(1) offspring. Considering these data and the results of our previous study, we propose that, in sharp contrast to paternal exposure to ionising radiation, the transgenerational effects of maternal high-dose acute irradiation are likely to be negligible.

The effects of in utero irradiation on mutation induction and transgenerational instability in mice.

Epidemiological evidence suggests that the deleterious effects of prenatal irradiation can manifest during childhood, resulting in an increased risk of leukaemia and solid cancers after birth. However, the mechanisms underlying the long-term effects of foetal irradiation remain poorly understood. This study was designed to analyse the impact of in utero irradiation on mutation rates at expanded simple tandem repeat (ESTR) DNA loci in directly exposed mice and their first-generation (F(1)) offspring. ESTR mutation frequencies in the germline and somatic tissues of male and female mice irradiated at 12 days of gestation remained highly elevated during adulthood, which was mainly attributed to a significant increase in the frequency of singleton mutations. The prevalence of singleton mutations in directly exposed mice suggests that foetal irradiation results in genomic instability manifested both in utero and during adulthood. The frequency of ESTR mutation in the F(1) offspring of prenatally irradiated male mice was equally elevated across all tissues, which suggests that foetal exposure results in transgenerational genomic instability. In contrast, maternal in utero exposure did not affect the F(1) stability. Our data imply that the passive erasure of epigenetic marks in the maternal genome can diminish the transgenerational effects of foetal irradiation and therefore provide important clues to the still unknown mechanisms of radiation-induced genomic instability. The results of this study offer a plausible explanation for the effects of in utero irradiation on the risk of leukaemia and solid cancers after birth.

The effects of Atm haploinsufficiency on mutation rate in the mouse germ line and somatic tissue.

Using single-molecule polymerase chain reaction, the frequency of spontaneous and radiation-induced mutation at an expanded simple tandem repeat (ESTR) locus was studied in DNA samples extracted from sperm and bone marrow of Atm knockout (Atm(+/-)) heterozygous male mice. The frequency of spontaneous mutation in sperm and bone marrow in Atm(+/-) males did not significantly differ from that in wild-type BALB/c mice. Acute exposure to 1 Gy of gamma-rays did not affect ESTR mutation frequency in bone marrow and resulted in similar increases in sperm samples taken from Atm(+/-) and BALB/c males. Taken together, these results suggest that the Atm haploinsufficiency analysed in our study does not affect spontaneous and radiation-induced ESTR mutation frequency in mice.

Paternal exposure to ethylnitrosourea results in transgenerational genomic instability in mice.

Recent data shows that the effects of ionizing radiation are not restricted to the directly exposed parental germ cells, but can also manifest in their nonexposed offspring, resulting in elevated mutation rates and cancer predisposition. The mechanisms underlying these transgenerational changes remain poorly understood. One of the most important steps in elucidating these mechanisms is to investigate the initial cellular events that trigger genomic instability. Here we have analyzed the effects of paternal treatment by ethylnitrosourea, an alkylating agent which is known to form specific types of DNA adducts, on the transgenerational effects in the first-generation (F1) offspring of exposed CBA/Ca and BALB/c male mice. Mutation rates at two expanded simple tandem repeat loci were significantly elevated in the F1 germline of both strains. Pre and postmeiotic exposures resulted in similar increases in mutation rate in the F1 germline. Within each strain mutation rates were equally elevated in the germline of male and female F1 offspring of the directly exposed males. The results of our study suggest that transgenerational instability is not attributed to a specific sub-set of DNA lesions, such as double strand breaks, and is most probably triggered by a stress-like response to a generalized DNA damage.

The offspring of irradiated parents, are they stable?

Mutation induction in directly exposed cells is currently regarded as the main component of the genetic risk of ionising radiation for humans. However, recent studies showing that exposure to ionising radiation results in elevated mutation rates detectable in the non-irradiated progeny of exposed cells challenge the existing paradigm in radiation biology. This review describes some recent data on radiation-induced genomic instability in vitro and mainly focuses on the in vivo phenomenon of transgenerational instability, where elevated mutation rates are detected in the non-exposed offspring of irradiated parents. The possible mechanisms and implications of transgenerational instability are also discussed.

Germline mutation rates at tandem repeat loci in DNA-repair deficient mice.

Mutation rates at two expanded simple tandem repeat (ESTR) loci were studied in the germline of non-exposed and irradiated severe combined immunodeficient (scid) and poly(ADP-ribose) polymerase (PARP-1-/-) deficient male mice. Non-exposed scid and PARP-/- male mice showed considerably elevated ESTR mutation rates, far higher than those in wild-type isogenic mice and other inbred strains. The irradiated scid and PARP-1-/- male mice did not show any detectable increases in their mutation rate, whereas significant ESTR mutation induction was observed in the irradiated wild-type isogenic males. ESTR mutation spectra in the scid and PARP-1-/- strains did not differ from those in the isogenic wild-type strains. Considering these data and the results of previous studies, we propose that a delay in repair of DNA damage in scid and PARP-1-/- mice could result in replication fork pausing which, in turn, may affect ESTR mutation rate in the non-irradiated males. The lack of mutation induction in irradiated scid and PARP-1-/- can be explained by the high cell killing effects of irradiation on the germline of deficient mice.