Molecular Studies on Plasmodium During Development in the Mosquito

Among the factors that regulate transmission of malaria are host-derived immune factors which can inhibit the progression of Plasmodium through the mosquito. This thesis addresses whether members of a family of LCCL/lectin Adhesive–like Proteins (LAPs) could act as feasible targets to elicit an antibody-mediated blockade in parasite transmission. The LAP family comprises six putatively secreted multidomain proteins which appear conserved among Plasmodium species and other apicomplexan parasites. Studies utilise the Plasmodium berghei rodent malaria parasite as an experimental system to evaluate the potential transmission-blocking activity of antibodies raised against LAPs. Regions of the proteins were selected for heterologous expression in Escherichia coli. Eight recombinant proteins were successfully expressed and were used as antigens to generate antisera in mice. Following immunogenicity tests, antisera against regions of LAP1, LAP3, LAP4 and LAP6 were selected for further characterisation and tested for antimalarial transmission-blocking activity. A combination of in vitro and in vivo assays revealed that the presence of anti-LAP antibodies did not inhibit parasite development. Whilst not conclusively excluding their potential, this work provided no further evidence to support the inclusion of LAPs as candidates for transmission-blocking vaccines. Immunolocalisation studies using anti-LAP1 antibodies revealed that the protein is expressed in cytoplasmic regions of female gametocytes and to a lesser extent in female gametes. Although it has been proposed that LAP expression may cease following fertilisation, PbLAP1 was detected in the cytoplasm of developing zygotes and intriguingly was subsequently found to concentrate in compartments of the P. berghei ookinete corresponding to the crystalloids. Antisera against regions of LAP1, LAP3, LAP4 and LAP6 were all found to similarly label the distinct electron dense cellular compartments. Furthermore, observation of Pblap mutant ookinetes by light microscopy indicated deficiencies in the formation of crystalloids. A transient cellular compartment, formed in the ookinete and subsequently fragmenting during early stages of oocyst development, roles of the crystalloid remain unknown. The association between LAPs and the crystalloids however leads to potential insights into the biological roles of the LAP family, the crystalloids, and the cellular processes of Plasmodium sporogony. Through microarray analysis, a comparison of the transcriptional profiles of P. berghei Δlap1 and wild-type ookinetes was made, detecting 274 differentially expressed genes and thereby indicating that the absence of PbLAP1 and resulting deficiency in crystalloid formation may have several knock-on effects on cellular processes important to Plasmodium development in the mosquito

Vital functions of the malarial ookinete protein, CTRP, reside in the A domains.

The transformation of malaria ookinetes into oocysts occurs in the mosquito midgut and is a major bottleneck for parasite transmission. The secreted ookinete surface protein, circumsporozoite- and thrombospondin-related adhesive protein (TRAP)-related protein (CTRP), is essential for this transition and hence constitutes a potential target for malaria transmission blockade. CTRP is a modular multidomain protein containing six tandem von Willebrand factor A-like (A) domains and seven tandem thrombospondin type I repeat-like (TS) domains. Here we present, to our knowledge, the first structure-function analysis of CTRP using genetically modified Plasmodium berghei parasites expressing mutant versions of the ctrp gene. Our data show that the A domains of CTRP are critical for ookinete gliding motility and oocyst formation whilst, unexpectedly, its TS domains are fully redundant. These results may have important implications for the design of CTRP-based transmission blocking strategies

A highly effective reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assay for the rapid detection of SARS-CoV-2 infection

The COVID-19 pandemic has illustrated the importance of simple, rapid and accurate diagnostic testing. This study describes the validation of a new rapid SARS-CoV-2 RT-LAMP assay for use on extracted RNA or directly from swab offering an alternative diagnostic pathway that does not rely on traditional reagents that are often in short supply during a pandemic. Analytical specificity (ASp) of this new RT-LAMP assay was 100% and analytical sensitivity (ASe) was between 1 × 101 and 1 × 102 copies per reaction when using a synthetic DNA target. The overall diagnostic sensitivity (DSe) and specificity (DSp) of RNA RT-LAMP was 97% and 99% respectively, relative to the standard of care rRT-PCR. When a CT cut-off of 33 was employed, above which increasingly evidence suggests there is a low risk of patients shedding infectious virus, the diagnostic sensitivity was 100%. The DSe and DSp of Direct RT-LAMP (that does not require RNA extraction) was 67% and 97%, respectively. When setting CT cut-offs of ≤33 and ≤25, the DSe increased to 75% and 100%, respectively, time from swab-to-result, CT < 25, was < 15 min. We propose that RNA RT-LAMP could replace rRT-PCR where there is a need for increased sample throughput and Direct RT-LAMP as a near-patient screening tool to rapidly identify highly contagious individuals within emergency departments and care homes during times of increased disease prevalence ensuring negative results still get laboratory confirmation.</p

Preliminary optimisation of a simplified sample preparation method to permit direct detection of SARS-CoV-2 within saliva samples using reverse-transcription loop-mediated isothermal amplification (RT-LAMP)

We describe the optimisation of a simplified sample preparation method which permits rapid and direct detection of SARS-CoV-2 RNA within saliva, using reverse-transcription loop-mediated isothermal amplification (RT-LAMP). Treatment of saliva samples prior to RT-LAMP by dilution 1:1 in Mucolyse™, followed by dilution in 10 % (w/v) Chelex© 100 Resin and a 98 °C heat step for 2 min enabled detection of SARS-CoV-2 RNA in positive saliva samples. Using RT-LAMP, SARS-CoV-2 RNA was detected in as little as 05:43 min, with no amplification detected in 3097 real-time reverse transcription PCR (rRT-PCR) negative saliva samples from staff tested within a service evaluation study, or for other respiratory pathogens tested (n = 22). Saliva samples can be collected non-invasively, without the need for skilled staff and can be obtained from both healthcare and home settings. Critically, this approach overcomes the requirement for, and validation of, different swabs and the global bottleneck in obtaining access to extraction robots and reagents to enable molecular testing by rRT-PCR. Such testing opens the possibility of public health approaches for effective intervention during the COVID-19 pandemic through regular SARS-CoV-2 testing at a population scale, combined with isolation and contact tracing

Diagnostic accuracy of loop mediated isothermal amplification coupled to nanopore sequencing (LamPORE) for the detection of SARS-CoV-2 infection at scale in symptomatic and asymptomatic populations.

OBJECTIVES Rapid, high throughput diagnostics are a valuable tool, allowing the detection of SARS-CoV-2 in populations, in order to identify and isolate people with asymptomatic and symptomatic infections. Reagent shortages and restricted access to high throughput testing solutions have limited the effectiveness of conventional assays such as reverse transcriptase quantitative PCR (RT-qPCR), particularly throughout the first months of the COVID-19 pandemic. We investigated the use of LamPORE, where loop mediated isothermal amplification (LAMP) is coupled to nanopore sequencing technology, for the detection of SARS-CoV-2 in symptomatic and asymptomatic populations. METHODS In an asymptomatic prospective cohort, for three weeks in September 2020 health care workers across four sites (Birmingham, Southampton, Basingstoke and Manchester) self-swabbed with nasopharyngeal swabs weekly and supplied a saliva specimen daily. These samples were tested for SARS-CoV-2 RNA using the Oxford Nanopore LamPORE system and a reference RT-qPCR assay on extracted sample RNA. A second retrospective cohort of 848 patients with influenza like illness from March 2020 - June 2020, were similarly tested from nasopharyngeal swabs. RESULTS In the asymptomatic cohort a total of 1200 participants supplied 23,427 samples (3,966 swab, 19,461 saliva) over a three-week period. The incidence of SARS-CoV-2 detection using LamPORE was 0.95%. Diagnostic sensitivity and specificity of LamPORE was >99.5% (reducing to ∼ 98% when clustered estimation was used) in both swab and saliva asymptomatic samples when compared to the reference RT-qPCR test. In the retrospective symptomatic cohort, the incidence was 13.4% and the sensitivity and specificity were 100%. CONCLUSIONS LamPORE is a highly accurate methodology for the detection of SARS-CoV-2 in both symptomatic and asymptomatic population settings and can be used as an alternative to RT-qPCR

Reverse-Transcriptase Loop-Mediated Isothermal Amplification Has High Accuracy for Detecting Severe Acute Respiratory Syndrome Coronavirus 2 in Saliva and Nasopharyngeal/Oropharyngeal Swabs from Asymptomatic and Symptomatic Individuals.

Previous studies have described reverse-transcriptase loop-mediated isothermal amplification (RT-LAMP) method for the rapid detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in nasopharyngeal and oropharyngeal swab and saliva samples. This study describes the validation of an improved sample preparation method for extraction-free RT-LAMP and defines the clinical performance of four different RT-LAMP assay formats for detection of SARS-CoV-2 within a multisite clinical evaluation. Direct RT-LAMP was performed on 559 swabs and 86,760 saliva samples and RNA RT-LAMP on extracted RNA from 12,619 swabs and 12,521 saliva samples from asymptomatic and symptomatic individuals across health care and community settings. For direct RT-LAMP, overall diagnostic sensitivity (DSe) of 70.35% (95% CI, 63.48%-76.60%) on swabs and 84.62% (95% CI, 79.50%-88.88%) on saliva was observed, with diagnostic specificity of 100% (95% CI, 98.98%-100.00%) on swabs and 100% (95% CI, 99.72%-100.00%) on saliva when compared with RT-qPCR; analyzing samples with RT-qPCR ORF1ab C values of ≤25 and ≤33, DSe values of 100% (95% CI, 96.34%-100%) and 77.78% (95% CI, 70.99%-83.62%) for swabs were observed, and 99.01% (95% CI, 94.61%-99.97%) and 87.61% (95% CI, 82.69%-91.54%) for saliva, respectively. For RNA RT-LAMP, overall DSe and diagnostic specificity were 96.06% (95% CI, 92.88%-98.12%) and 99.99% (95% CI, 99.95%-100%) for swabs, and 80.65% (95% CI, 73.54%-86.54%) and 99.99% (95% CI, 99.95%-100%) for saliva, respectively. These findings demonstrate that RT-LAMP is applicable to a variety of use cases, including frequent, interval-based testing of saliva with direct RT-LAMP from asymptomatic individuals who may otherwise be missed using symptomatic testing alone

RT-LAMP has high accuracy for detecting SARS-CoV-2 in saliva and naso/oropharyngeal swabs from asymptomatic and symptomatic individuals

Objectives: To validate an improved sample preparation method for extraction free Direct RT-LAMP and define the clinical performance of four different RT-LAMP assay formats for detection of SARS-CoV-2 with a multisite clinical evaluation.Method: We describe Direct RT-LAMP on 559 swabs and 86,760 saliva samples and RNA RT-LAMP on extracted RNA from 12,619 swabs and 12,521 saliva samples collected from asymptomatic and symptomatic individuals across multiple healthcare and community settings.Results: For Direct RT-LAMP, we found a diagnostic sensitivity (DSe) of 70.35% (95% CI 63.48-76.60%) on swabs and 84.62% (79.50-88.88%) on saliva, with diagnostic specificity (DSp) of 100% (98.98-100.00%) on swabs and 100% (99.72-100.00%) on saliva when compared to RT-qPCR. Analysing samples with RT-qPCR ORF1ab CT values of≤25 and ≤33 (high and medium-high viral copy number, respectively), we found DSe of 100% (96.34-100%) and 77.78% (70.99-83.62%) for swabs, and 99.01% (94.61-99.97%) and 87.32% (80.71-92.31%) for saliva. For RNA RT-LAMP DSe and DSp were 95.98% (92.74-98.06%) and 99.99% (99.95-100%) for swabs, and 80.65% (73.54-86.54%) and 99.99% (99.95-100%) for saliva, respectively.Conclusions: The findings from these evaluations demonstrate that RT-LAMP testing of swabs and saliva is applicable to a variety of different use-cases, including frequent, interval-based testing of saliva from asymptomatic individuals via Direct RT-LAMP that may otherwise be missed using symptomatic testing alone.<br/

A randomized trial of planned cesarean or vaginal delivery for twin pregnancy

Background: Twin birth is associated with a higher risk of adverse perinatal outcomes than singleton birth. It is unclear whether planned cesarean section results in a lower risk of adverse outcomes than planned vaginal delivery in twin pregnancy.\ud \ud Methods: We randomly assigned women between 32 weeks 0 days and 38 weeks 6 days of gestation with twin pregnancy and with the first twin in the cephalic presentation to planned cesarean section or planned vaginal delivery with cesarean only if indicated. Elective delivery was planned between 37 weeks 5 days and 38 weeks 6 days of gestation. The primary outcome was a composite of fetal or neonatal death or serious neonatal morbidity, with the fetus or infant as the unit of analysis for the statistical comparison.\ud \ud Results: A total of 1398 women (2795 fetuses) were randomly assigned to planned cesarean delivery and 1406 women (2812 fetuses) to planned vaginal delivery. The rate of cesarean delivery was 90.7% in the planned-cesarean-delivery group and 43.8% in the planned-vaginal-delivery group. Women in the planned-cesarean-delivery group delivered earlier than did those in the planned-vaginal-delivery group (mean number of days from randomization to delivery, 12.4 vs. 13.3; P = 0.04). There was no significant difference in the composite primary outcome between the planned-cesarean-delivery group and the planned-vaginal-delivery group (2.2% and 1.9%, respectively; odds ratio with planned cesarean delivery, 1.16; 95% confidence interval, 0.77 to 1.74; P = 0.49).\ud \ud Conclusion: In twin pregnancy between 32 weeks 0 days and 38 weeks 6 days of gestation, with the first twin in the cephalic presentation, planned cesarean delivery did not significantly decrease or increase the risk of fetal or neonatal death or serious neonatal morbidity, as compared with planned vaginal delivery