ABSTRACT
Continued waves, new variants, and limited vaccine deployment mean that SARS-CoV-2 tests remain vital to constrain the COVID-19 pandemic. Affordable, point-of-care (PoC) tests allow rapid screening in non-medical settings. Reverse-transcription loop-mediated isothermal amplification (RT-LAMP) is an appealing approach. A crucial step is to optimize testing in low/medium resource settings. Here, we optimized RT-LAMP for SARS-CoV-2 and human {beta}-actin, and tested clinical samples in multiple countries. "TTTT" linker primers did not improve performance, and while guanidine hydrochloride, betaine and/or Igepal-CA-630 enhanced detection of synthetic RNA, only the latter two improved direct assays on nasopharygeal samples. With extracted clinical RNA, a 20 min RT-LAMP assay was essentially as sensitive as RT-PCR. With raw Canadian nasopharygeal samples, sensitivity was 100% (95% CI: 67.6% - 100%) for those with RT-qPCR Ct values [≤] 25, and 80% (95% CI: 58.4% - 91.9%) for those with 25 < Ct [≤] 27.2. Highly infectious, high titer cases were also detected in Colombian and Ecuadorian labs. We further demonstrate the utility of replacing thermocyclers with a portable PoC device (FluoroPLUM). These combined PoC molecular and hardware tools may help to limit community transmission of SARS-CoV-2.
ABSTRACT
The COVID-19 pandemic caused by the SARS-CoV-2 virus has placed extensive strain on RNA isolation and RT-qPCR reagents. Rapid development of new test kits has helped to alleviate these shortages. However, comparisons of these new detection systems are largely lacking. Here, we compare indirect methods that require RNA extraction, and direct RT-qPCR on patient samples. For RNA isolation we compared four different companies (Qiagen, Invitrogen, BGI and Norgen Biotek). For detection we compared two recently developed Taqman-based modules (BGI and Norgen Biotek), a SYBR green-based approach (NEB Luna Universal One-Step Kit) with published and newly-developed primers, and clinical results (Seegene STARMag RNA extraction system and Allplex 2019-nCoV RT-qPCR assay). Most RNA isolation procedures performed similarly, and while all RT-qPCR modules effectively detected purified viral RNA, the BGI system proved most sensitive, generating comparable results to clinical diagnostic data, and identifying samples ranging from 65 copies - 2.1x105 copies of viral Orf1ab/l. However, the BGI detection system is [~]4x more expensive than other options tested here. With direct RT-qPCR we found that simply adding RNase inhibitor greatly improved sensitivity, without need for any other treatments (e.g. lysis buffers or boiling). The best direct methods were [~]10 fold less sensitive than indirect methods, but reduce sample handling, as well as assay time and cost. These studies will help guide the selection of COVID-19 detection systems and provide a framework for the comparison of additional systems.
ABSTRACT
Objective@#To investigate the impact of intensified maintenance therapy on the prognosis of children and adolescents with advanced lymphoblastic lymphoma (LBL) .@*Methods@#Retrospective analysis on the treatment results of children and adolescents with stage Ⅲ and stage Ⅳ LBL who underwent BFM-NHL-90/-95 regimen without prophylactic radiotherapy. The intensified therapy group included the patients admitted from 1998 to 2005, while others were classified as the non-intensified therapy group. Patients in the intensified therapy group were intravenously treated with "etoposide phosphate plus cytrarabine" and high-dose methotrexate alternately per 2.5-3 months in addition to the oral chemotherapy with 6-mercaptopurine and methotrexate during the maintenance phase.@*Results@#A total of 187 LBL patients were enrolled. The rates of 5-year event free survival were (76.9 ± 5.8) % and (77.9 ± 4.3) % (χ2=0.249, P=0.617) respectively, in the intensified therapy (n=52) and the non-intensified therapy groups (n=135) , while the rates of 5-year overall survival of them were (78.8 ± 5.7) % and (79.8±4.1) % (χ2=0.353, P=0.552) , respectively. Stratified by stage, immunological type as well as risk stratification, the rates of long-term survival were similar between the two groups. During the maintenance phase, the rates of grade Ⅲ and Ⅳ myelosuppression in the intensified therapy and the non-intensified maintenance groups were 55.8% and 18.5%, respectively (χ2=25.363, P<0.05) .@*Conclusion@#Intensified maintenance therapy failed to improve the prognosis of patients with advanced LBL.
ABSTRACT
Objective: To investigate the effect of gross total resection on the local control and survival of patients with stage IV neuroblastoma (NB) and analyze the extent of surgical resection of primary tumors that affects patient survival. Methods: A total of 96 patients with stage Ⅳ NB who were admitted to the Sun Yat-Sen University Cancer Center between January 2000 and December 2011 were analyzed. The patients were treated with combined-modality therapy, including chemotherapy, surgery, and/or radiotherapy. The patients were divided according to the extent of surgical resection of primary tumor into the following groups: group A, biopsy or tumor removal of less than 50% of the primary lesion; group B, incomplete resection of more than 50% but less than 90% of the lesion; group C, removal of more than 90% of the lesion; and group D, complete resection with or without macroscopic residual tumors. The survival rates of each group were analyzed. Results: The median age of the 96 patients was 4.4 years, ranging from 1.2-18.8 years. The overall 3-year progression-free survival (PFS) and overall survival (OS) of the total patients were 32.8% and 36.7%, respectively. A total of 24 cases were assigned in group A, 10 in group B, 23 in group C, and 39 in group D. Subgroup analysis revealed that the 3-year PFS rate was 17.5% for group A, 20.0% for group B, 45.1% for group C, and 40.5% for group D. The PFS rates were not statistically significant-ly different between groups A and B (P=0.352) and between groups C and D (P = 0.792). However, the OS was higher in groups C and D than that in groups A and B. The 3-year PFS rates were 42.2% and 17.8% for groups C and D (P<0.001), respectively. Conclu-sion: Resection extension of more than 90% of the primary tumor combined with chemotherapy and (or) radiation therapy can improve the survival of patients with stage Ⅳ NB. However, this treatment modality does not affect the treatment outcomes for minimal gross tu-mor residuals.
ABSTRACT
<p><b>OBJECTIVE</b>To evaluate the long-term survival of children and adolescents with lymphoblastic lymphoma (LBL) treated by a modified NHL-BFM-90 protocol.</p><p><b>METHODS</b>From March 1998 to November 2010, 107 untreated patients with LBL (age <18 years) were enrolled and stratified into three groups (R1, R2 and R3), according to the stage of disease and response to induction chemotherapy. All patients received different intensive chemotherapy regimens based on a modified NHL-BFM-90 protocol. Total treatment duration was 2 years.</p><p><b>RESULTS</b>Of the 107 patients, 79 were boys and 28 were girls, with a median age of 10 years (range 2.5-18 years). Six patients (5.6%) were stage I/II, 101 (94.4%) stage III/IV. The R1, R2 and R3 groups accounted for 5.6%, 71.0% and 23.4%, respectively. 75.7% of the patients had T-LBL, and 24.3% was B-LBL. At a median follow-up duration of 60 months (range 1-186 months), 24 patients died. The 5-year event-free survival (EFS) and overall survival (OS) were 75.5% and 77.8 % for all patients, 100.0% and 100.0% for group R1, 84.5% and 87.5 % for R2, 44.0% and 44.0% for R3, 72% and 73.5% for T-LBL, 86.4% and 88.5% for B-LBL, respectively. Myleosuppression was the major toxicity and need aggressive management.</p><p><b>CONCLUSION</b>The modified NHL-BFM-90 protocol is an effective therapy for children and adolescents with LBL in low and intermediate risk. T-LBL had the similar outcomes as B-LBL did. The patients in high-risk group had a poor survival and new protocols are needed.</p>
