A Multiplex One-Step RT-qPCR Protocol to Detect SARS-CoV-2 in NP/OP Swabs and Saliva.

Since December 2019, SARS-CoV-2 has spread extensively throughout the world, with more than 117 million reported cases and 2.6 million deaths (Johns Hopkins coronavirus resource center, https://coronavirus.jhu.edu/map.html). Detecting the virus is the first step in diagnosing the infection, followed by quarantine to prevent transmission. Nasopharyngeal/oropharyngeal swabs (NP/OP) and saliva are two specimen types that are most often analyzed to detect SARS-CoV-2 by molecular tests that detect viral RNA or by antigen/antibody tests that detect viral proteins and/or the host immune response against the virus. Compared to antigen/antibody tests, molecular tests are highly sensitive and specific for detecting the virus. A significant drawback is that specimen collection requirements are specific to each test and cannot be interchanged with another test. Some tests are qualified to be used on NP swabs or saliva, but not both specimen types. Even with NP swabs, a test may be qualified to detect the virus only with swabs collected in viral transport medium (VTM) but not in other media. These restrictive pre-analytic steps are disadvantageous in that a lab would have to develop and validate different tests for SARS-CoV-2 depending on the specimen type and collection media, with added setup cost, infrastructure, and training requirements. To overcome these problems, we developed and validated a cost-effective multiplex reverse-transcription real-time PCR assay that can be used to detect SARS-CoV-2 in different specimen types. The assay is highly sensitive and specific, can be used to detect the virus in saliva as well as NP swabs collected in different media such as VTM, saline, and commercial preservative fluid, and serves as one test for all applications. The protocol also describes an optimal laboratory setup and unidirectional workflow for detecting SARS-CoV-2 by RT-qPCR. © 2021 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Manual viral nucleic acid extraction from NP/OP swabs collected in different media, and from saliva Alternate Protocol 1: Low-throughput automated extraction on the Qiagen EZ1 Advanced XL machine (1-14 samples) Alternate Protocol 2: High-throughput automated extraction on the Kingfisher Flex machine (1-96 samples) Basic Protocol 2: Multiplex RT-qPCR protocol to detect SARS-CoV-2 Alternate Protocol 3: Multiplex one-step RT-qPCR protocol to detect SARS-CoV-2 with S and E gene probes labeled with the same fluorochrome.

Development and validation of viral load assays to quantitate SARS-CoV-2.

SARS-CoV-2 has infected more than 30 million persons throughout the world. A subset of patients suffer serious consequences that require hospitalization and ventilator support. Current tests for SARS-CoV-2 generate qualitative results and are vital to make a diagnosis of the infection. However, they are not helpful to follow changes in viral loads after diagnosis. The ability to quantitatively assess viral levels is necessary to determine the effectiveness of therapy with anti-viral or immune agents. Viral load analysis is also necessary to determine the replicative potential of strains with different mutations, emergence of resistance to anti-viral agents and the stability of viral nucleic acid and degree of RT-PCR inhibition in different types of collection media. Quantitative viral load analysis in body fluids, plasma and tissue may be helpful to determine the effects of the infection in various organ systems. To address these needs, we developed two assays to quantitate SARS-CoV-2. The assays target either the S or E genes in the virus, produce comparable viral load results, are highly sensitive and specific and have a wide range of quantitation. We believe that these assays will be helpful to manage the clinical course of infected patients and may also help to better understand the biology of infection with SARS-CoV-2.

NGS Analysis of Clonality and Minimal Residual Disease in a Patient with Concurrent Richter's Transformation and CLL/SLL.

B-cell lymphomas are neoplastic proliferations of clonal B lymphocytes. Clonality is generally determined by PCR amplification of VDJ rearrangements in the IgH heavy chain or VJ rearrangements in Igκ/Igλ light chain genes followed by capillary electrophoresis. More recently, next-generation sequencing (NGS) has been used to detect clonality in B-cell lymphomas because of the exponential amount of information that is obtained beyond just detecting a clonal population. The additional information obtained is useful for diagnostic confirmation, prognosis assessment, and response to therapy. In this study, we utilized NGS analysis to characterize two histologically distinct lymphomas (DLBCL and CLL/SLL) that were detected contemporaneously in an asymptomatic patient. NGS analysis showed that the same VDJ rearrangement was present in nodal (DLBCL) and marrow (CLL/SLL) biopsies confirming that the DLBCL resulted from Richter's transformation of a subclinical CLL/SLL. The V region of the rearrangement remained unmutated without somatic hypermutation. In silico analysis showed that the HCDR3 sequence was heterogeneous and not stereotypic. Minimal residual disease analysis by NGS showed that the tumor clone decreased by 2.84 logs in the bone marrow after R-CHOP therapy. However, a small number of tumor cells were still detected in the peripheral blood after R-CHOP therapy. Subsequent allogeneic transplantation was successful in eradicating the tumor clone and achieving deep molecular remission. We show that NGS analysis facilitated clinical management in our patient by helping to characterize the VDJ rearrangement in detail and by tracking minimal residual disease with high sensitivity and specificity.

Jumping Performance is Preserved but Not Muscle Thickness in Collegiate Volleyball Players After a Taper.

Bazyler, CD, Mizuguchi, S, Sole, CJ, Suchomel, TJ, Sato, K, Kavanaugh, AA, DeWeese, BH, and Stone, MH. Jumping performance is preserved but not muscle thickness in collegiate volleyball players after a taper. J Strength Cond Res 32(4): 1020-1028, 2018-The purpose of this study was to examine changes in muscle architecture and jumping performance in NCAA division I women's volleyball players throughout a competitive season and in preparation for conference championships. Ten women volleyball players were tested at preseason (T1), pretaper (T2), and post-taper (T3) on measures of vastus lateralis muscle thickness (MT), pennation angle (PA) and fascicle length (FL) using ultrasonography, and unloaded and loaded squat jump height (SJH) and peak power allometrically scaled to body mass (SJPPa) on a force platform. Rating of perceived exertion training load and strength training volume load were monitored weekly. Player's MT (p < 0.001, Glass's Δ = 2.8) and PA increased (p = 0.02, Δ = 3.9) after in-season training. However, MT decreased after the taper (p = 0.01, Δ = 0.6) but remained elevated above preseason values (p < 0.001, Δ = 1.7). There were no statistical changes in FL, SJH, or SJPPa. Large-to-very large negative relationships (r = -0.51 to -0.81) were observed between preseason relative maximal strength and changes in SJH and SJPPa with various loads over the season. These findings demonstrate that relatively low volumes of strength training and concurrent sport training during a tapering period are capable of preserving jumping performance, but not MT in women's volleyball players; however, jumping performance changes seem to be related to the player's strength level. Stronger players may benefit from an overreaching microcycle before the taper to preserve previously accrued muscular adaptations and jumping performance.

Long-Term Changes in Jump Performance and Maximum Strength in a Cohort of National Collegiate Athletic Association Division I Women's Volleyball Athletes.

Kavanaugh, AA, Mizuguchi, S, Sands, WA, Ramsey, MW, and Stone, MH. Long-term changes in jump performance and maximum strength in a cohort of NCAA division I women's volleyball Athletes. J Strength Cond Res 32(1): 66-75, 2018-The purpose of this investigation was to quantify the magnitude of change in maximal strength and jumping abilities over approximately 1, 2, and 3 years of supervised sport and resistance training in National Collegiate Athletic Association (NCAA) division I women's volleyball athletes. This was an exploratory study on a cohort of women's volleyball athletes (n = 29) split into 3 groups based on the length of the time spent in a supervised resistance training program: group 1 (n = 11): 0.7 ± 0.3 years, group 2 (n = 9): 1.6 ± 0.2 years, and group 3 (n = 9): 2.4 ± 0.6 years. Monitoring tests consisted of standing height (cm), body mass (kg), body fat (%), static jump height (SJH) and countermovement JH (CMJH) with 0-, 11-, and 20-kg loads (cm), and midthigh clean pull isometric peak force (IPF) and allometrically scaled IPF (IPFa) (N·kg). Increasing trends were observed for all variables from groups 1 to 2 to 3. Statistically greater improvements (p ≤ 0.05) with moderate to large effect sizes were found between groups 1 and 3 for SJH 0 (19.7%, d = 1.35), SJH 11 (23.8%, d = 1.23), SJH 20 (30.6%, d = 1.20), CMJH 11 (22.6%, d = 1.18), IPF (44.4%, d = 1.22), and IPFa (41.2%, d = 1.32). A combination of traditional resistance training exercises and weightlifting variations at various loads, in addition to volleyball practice, seem to be effective at increasing maximal strength by 44% and vertical JH by 20-30% in NCAA division I women's volleyball athletes after about 2.5 years of training. Furthermore, these characteristics can be improved in the absence of additional plyometric training outside normal volleyball-specific practice.

Characteristics of a National Level Female Weightlifter Peaking for Competition: A Case Study.

Bazyler, CD, Mizuguchi, S, Zourdos, MC, Sato, K, Kavanaugh, AA, DeWeese, BH, Breuel, KF, and Stone, MH. Characteristics of a national level female weightlifter peaking for competition: A case study. J Strength Cond Res 32(11): 3029-3038, 2018-This study investigated physiological and performance changes of a national-level 69 kg female weightlifter after 3 competition phases over a 28-week training period. The athlete first trained for a regional championship (weeks 1-12), followed by a local competition (weeks 13-23) and the national championship (weeks 24-28). Body mass, vastus lateralis cross-sectional area (CSA), and unloaded and loaded squat jump performance were assessed weekly during each 4-week competition phase. Serum biomarkers and dynamic midthigh pulls were assessed before and after each competition phase. Weightlifting performance goals were met for the regional championship (total = 200 kg) and the local competition (total = 193 kg), but not the national championship (total = 196 kg). She lost more body mass in preparation for Nationals (-6.0 kg) compared with regionals (-2.5 kg) and the local competition (+2.2 kg). Vastus lateralis CSA very likely decreased after Nationals (precision = 99%, effect size = 2.08). Her testosterone:cortisol ratio likely increased (88%, 2.64), whereas interleukin-6 (79%, 2.47) and tumor necrosis factor-alpha (81%, 3.59) likely decreased after Nationals. Serum myostatin (99%, 1.95) and decorin (99%, 1.96) very likely decreased after the local competition. Unloaded squat jump height likely increased the week of regionals (89%, 0.95) and the local competition (99%, 1.83), whereas unloaded and loaded squat jump height possibly (69%, 0.99) and likely (82%, 1.52) decreased the week of Nationals. Dynamic midthigh pull vertical displacement likely increased after regionals (93%, 0.84) and likely decreased after Nationals (94%, 0.87). These findings indicate that biomarkers of stress, inflammation, and hypertrophy are related to changes in training volume-load; however, performance measures are needed to assess competition preparedness. Considering the reductions in muscle CSA corresponding with the large reductions in body mass and underperformance at the national championship, sport scientists, and coaches should instruct weightlifters to not attempt large losses in body mass (e.g., >3 kg) close to competition (e.g., <1week).

Returners Exhibit Greater Jumping Performance Improvements During a Peaking Phase Compared With New Players on a Volleyball Team.

PURPOSE: To determine if jumping-performance changes during a peaking phase differed among returners and new players on a female collegiate volleyball team and to determine which variables best explained the variation in performance changes. METHODS: Fourteen volleyball players were divided into 2 groups-returners (n = 7) and new players (n = 7)-who completed a 5-wk peaking phase prior to conference championships. Players were tested at baseline before the preseason on measures of the vastus lateralis cross-sectional area using ultrasonography, estimated back-squat 1-repetition maximum, countermovement jump height (JH), and relative peak power on a force platform. Jumping performance, rating of perceived exertion training load, and sets played were recorded weekly during the peaking phase. RESULTS: There were moderate to very large (P < .01, Glass Δ = 1.74) and trivial to very large (P = .07, Δ = 1.09) differences in JH and relative peak power changes in favor of returners over new players, respectively, during the peaking phase. Irrespective of group, 7 of 14 players achieved peak JH 2 wk after the initial overreach. The number of sets played (r = .78, P < .01) and the athlete's preseason relative 1-repetition maximum (r = .54, P = .05) were the strongest correlates of JH changes during the peaking phase. CONCLUSIONS: Returners achieved greater improvements in jumping performance during the peaking phase compared with new players, which may be explained by the returners' greater relative maximal strength, time spent competing, and training experience. Thus, volleyball and strength coaches should consider these factors when prescribing training during a peaking phase to ensure their players are prepared for important competitions.

Injuries in Collegiate Women's Volleyball: A Four-Year Retrospective Analysis.

A four-year retrospective analysis of injury data was conducted on a collegiate (NCAA Division I) women's volleyball team. Twenty athletes (Year 1: age = 19.4 ± 0.9 y, height = 175.2 ± 5.1 cm, body mass = 70.5 ± 10.2 kg; Year 2: age = 20.1 ± 1.0 y, height = 175.7 ± 4.7 cm, body mass = 69.5 ± 10.1 kg; Year 3: age = 20.1 ± 1.4 y, height = 173.8 ± 6.3 cm, body mass = 69.9 ± 10.8 kg; Year 4: age = 19.5 ± 1.4 y, height = 174.4 ± 8.6 cm, body mass = 72.7 ± 10.8 kg) participated in this study, accounting for 1483 total training exposures. Injury was defined as any damage to a body part, incurred during volleyball or strength and conditioning-related activities, which interfered with training and/or competition. Injury rate was normalized to the number of athletes and exposure and expressed as injuries per 1000 exposures. A total of 133 injuries were recorded. The most common injury was to the knee (left = 7.5%, right = 12.0%). Injuries occurred most often in volleyball practice (75.2%), followed by competition (20.3%), and strength and conditioning-related activities (4.5%). Non-contact injuries (upper body = 26.3%, lower body = 53.4%) were more common than contact injuries (upper-body = 13.5%, lower-body = 6.8%). An examination of injury rates relative to the training year revealed patterns in injury occurrence. Specifically, spikes in injury rate were consistently observed during periods of increased training volume that were preceded by breaks in organized training, such as the early pre-season and off-season training periods.

Modern Techniques and Technologies Applied to Training and Performance Monitoring.

Athlete preparation and performance continue to increase in complexity and costs. Modern coaches are shifting from reliance on personal memory, experience, and opinion to evidence from collected training-load data. Training-load monitoring may hold vital information for developing systems of monitoring that follow the training process with such precision that both performance prediction and day-to-day management of training become adjuncts to preparation and performance. Time-series data collection and analyses in sport are still in their infancy, with considerable efforts being applied in "big data" analytics, models of the appropriate variables to monitor, and methods for doing so. Training monitoring has already garnered important applications but lacks a theoretical framework from which to develop further. As such, we propose a framework involving the following: analyses of individuals, trend analyses, rules-based analysis, and statistical process control.

Changes in Muscle Architecture, Explosive Ability, and Track and Field Throwing Performance Throughout a Competitive Season and After a Taper.

The purpose of this study was to examine the effects of an overreach and taper on measures of muscle architecture, jumping, and throwing performance in Division I collegiate throwers preparing for conference championships. Six collegiate track and field throwers (3 hammer, 2 discus, 1 javelin) trained for 12 weeks using a block-periodization model culminating with a 1-week overreach followed by a 3-week taper (ORT). Session rating of perceived exertion training load (RPETL) and strength training volume-load times bar displacement (VLd) were recorded weekly. Athletes were tested pre-ORT and post-ORT on measures of vastus lateralis architecture, unloaded and loaded squat and countermovement jump performance, underhand and overhead throwing performance, and competition throwing performance. There was a statistical reduction in weight training VLd/session (d = 1.21, p ≤ 0.05) and RPETL/session (d = 0.9, p ≤ 0.05) between the in-season and ORT training phases. Five of 6 athletes improved overhead throw and competition throwing performance after the ORT (d = 0.50, p ≤ 0.05). Vastus lateralis muscle thickness statistically increased after the in-season training phase (d = 0.28, p ≤ 0.05) but did not change after the ORT. Unloaded countermovement jump peak force and relative peak power improved significantly after the ORT (d = 0.59, p ≤ 0.05, d = 0.31, p ≤ 0.05, respectively). These findings demonstrate that an overreaching week followed by a 3-week taper is an effective means of improving explosive ability and throwing performance in collegiate track and field throwers despite the absence of detectable changes in muscle architecture.

Effects of Short-Term Free-Weight and Semiblock Periodization Resistance Training on Metabolic Syndrome.

South, MA, Layne, AS, Stuart, CA, Triplett, NT, Ramsey, MW, Howell, ME, Sands, WA, Mizuguchi, S, Hornsby, WG, Kavanaugh, AA, and Stone, MH. Effects of short-term free-weight and semiblock periodization resistance training on metabolic syndrome. J Strength Cond Res 30(10): 2682-2696, 2016-The effects of short-term resistance training on performance and health variables associated with prolonged sedentary lifestyle and metabolic syndrome (MS) were investigated. Resistance training may alter a number of health-related, physiological, and performance variables. As a result, resistance training can be used as a valuable tool in ameliorating the effects of a sedentary lifestyle including those associated with MS. Nineteen previously sedentary subjects (10 with MS and 9 with nonmetabolic syndrome [NMS]) underwent 8 weeks of supervised resistance training. Maximum strength was measured using an isometric midthigh pull and resulting force-time curve. Vertical jump height (JH) and power were measured using a force plate. The muscle cross-sectional area (CSA) and type were examined using muscle biopsy and standard analysis techniques. Aerobic power was measured on a cycle ergometer using a ParvoMedics 2400 Metabolic system. Endurance was measured as time to exhaustion on a cycle ergometer. After training, maximum isometric strength, JH, jump power, and V[Combining Dot Above]O2peak increased by approximately 10% (or more) in both the metabolic and NMS groups (both male and female subjects). Over 8 weeks of training, body mass did not change statistically, but percent body fat decreased in subjects with the MS and in women, and lean body mass increased in all groups (p ≤ 0.05). Few alterations were noted in the fiber type. Men had larger CSAs compared those of with women, and there was a fiber-specific trend toward hypertrophy over time. In summary, 8 weeks of semiblock free-weight resistance training improved several performance variables and some cardiovascular factors associated with MS.