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Purpose: This study presents a case of familial transmission of thyroxine-binding globulin (TBG) deficiency. The SERPINA7-gene which codes for TBG is located on the X-chromosome (Xq21-22). More than 45 mutations have been reported to cause TBG- deficiency from various countries, but none from India so far. Genetic analysis of SERPINA7 gene was carried out to determine the cause of low TBG levels in one family., Methods: DNA samples of the propositus and the family members were subjected to Polymerase Chain Reaction (PCR) followed by direct sequencing. Allele-specific PCR and Next-gen sequencing (NGS) were employed to confirm the site of the mutation. Thyroid function tests were estimated by Radioimmunoassay (RIA) and Immunoradiometric assay (IRMA) kits. X-chromosomal inactivation status was analyzed in the female members harboring the mutation., Results: A mutational screening in this family revealed a novel frame-shift mutation S353Q, 354fs3X in the exon 4 of the SERPINA7 gene which will be referred to as TBG-complete deficiency-India (TBG-CD-Ind). One out of four female family members harboring the mutation showed selective X-chromosomal inactivation. The affected family members were clinically euthyroid initially, showed changes in the thyroid function when tested after a long time span. However, the changes in the thyroid function in the affected family members had an autoimmune etiology., Conclusion: This study presents the first report of TBG-CD from India wherein a novel frameshift mutation referred to as TBG-CD-Ind (S353Q, 354fs3X) in the SERPINA7 gene was detected. No apparent association was identified between thyroid function and the TBG-mutation in the affected subjects. A detailed biochemical and genomic testing to determine the exact cause of discordant TFT in the patients would certainly aid in the unequivocal diagnosis of the thyroid function and for the precise individualized treatment., (© 2021. Italian Society of Endocrinology (SIE).)

Background/objectives: Oxylipins are polyunsaturated fatty acid derivatives involved in the regulation of various processes, including chronic inflammation, insulin resistance and hepatic steatosis. They can be synthesized in various tissues, including adipose tissue. There is some evidence that obesity is associated with the deregulation of serum oxylipin levels. The aim of this study was to evaluate the effect of bariatric surgery (one-anastomosis gastric bypass) on the serum levels of selected oxylipins and their fatty acid precursors and to verify the hypothesis that their changes after surgery can contribute to the resolution of inflammation. Moreover, we compared the oxylipin levels (prostaglandin E2, 13-HODE, maresin 1 and resolvin E1), fatty acids and the expression of enzymes that synthesize oxylipins in adipose tissue of lean controls and subjects with severe obesity., Subjects/methods: The study included 50 patients with severe obesity that underwent bariatric surgery and 41 subjects in lean, control group. Fatty acid content was analyzed by GC-MS, oxylipin concentrations were measured with immunoenzymatic assay kits and real-time PCR analysis was used to assess mRNA levels in adipose tissue., Results: Our results show increased expression of some enzymes that synthesize oxylipins in adipose tissue and alterations in the levels of oxylipins in both adipose tissue and serum of subjects with obesity. After bariatric surgery, the levels of anti-inflammatory oxylipins increased, whereas pro-inflammatory oxylipins decreased., Conclusions: In patients with obesity, the metabolism of oxylipins is deregulated in adipose tissue, and their concentrations in serum are altered. Bariatric surgery modulates the serum levels of pro- and anti-inflammatory oxylipins, which may contribute to the resolution of inflammation., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

Background: Obesity increases the risk of developing impaired glucose tolerance (IGT) and type 2 diabetes (T2D) after myocardial infarction (MI). Brown adipose tissue (BAT) is important to combat obesity and T2D, and increasing BAT mass by transplantation improves glucose metabolism and cardiac function. The objective of this study was to determine if BAT had a protective effect on glucose tolerance and cardiac function in high-fat diet (HFD) fed mice subjected to a mild MI., Methods: Male C57BL/6 mice were fed a HFD for eight weeks and then divided into Sham (Sham-operated) and +BAT (mice receiving 0.1 g BAT into their visceral cavity). Sixteen weeks post-transplantation, mice were further subdivided into ±MI (Sham; Sham-MI; +BAT; +BAT-MI) and maintained on a HFD. Cardiac (echocardiography) and metabolic function (glucose and insulin tolerance tests, body composition and exercise tolerance) were assessed throughout 22 weeks post-MI. Quantitative PCR (qPCR) was performed to determine the expression of genes related to metabolic function of perigonadal adipose tissue (pgWAT), subcutaneous white adipose tissue (scWAT), liver, heart, tibialis anterior skeletal muscle (TA); and BAT., Results: +BAT prevented the increase in left ventricle mass (LVM) and exercise intolerance in response to MI. Similar to what is observed in humans, Sham-MI mice developed IGT post-MI, but this was negated in +BAT-MI mice. IGT was independent of changes in body composition. Genes involved in inflammation, insulin resistance, and metabolism were significantly altered in pgWAT, scWAT, and liver in Sham-MI mice compared to all other groups., Conclusions: BAT transplantation prevents IGT, the increase in LVM, and exercise intolerance following MI. MI alters the expression of several metabolic-related genes in WAT and liver in Sham-MI mice, suggesting that these tissues may contribute to the impaired metabolic response. Increasing BAT may be an important intervention to prevent the development of IGT or T2D and cardiac remodeling in obese patients post-MI., (© 2021. The Author(s).)

Background: The O6-methylguanine-DNA methyltransferase (MGMT) gene prevents mismatch in DNA replication and transcription by repairing mutagenic DNA lesions. MGMT is a predictor biomarker of chemotherapy in high-grade and low-grade gliomas based on high-risk clinical conditions. It also can be used for therapeutic decisions to predict hypermutation in recurrence in newly diagnosed low-grade gliomas. The gold standard  examination for the methylation is Polymerase Chain Reaction (PCR). However, this technique is not widely available in Indonesia for daily practice. Thus, an uncomplicated and simpler method such as immunohistochemistry (IHC) is needed as an alternative examination. This study aimed to predict the diagnostic accuracy of immunohistochemistry (IHC) in detecting the methylation status of O6-methylguanine-DNA methyltransferase (MGMT) in glioma., Methods: This research was a cross-sectional study using formalin-fixed paraffin embedded (FFPE) tissue samples of glioma patients, dating between October 2017 until March 2021. Diagnosis of glioma was established based on clinical, radiological, and histopathological findings. MGMT methylation status was investigated using the IHC and PCR techniques. Diagnostic value of IHC was analyzed, with PCR as a gold standard method. Optimum threshold to determine positivity of IHC was determined by the Area Under the Curve (AUC) on Receiver Operating Characteristics (ROC) curve and Youden index., Results: Among 75 samples examined, 29 (38.7%) patients were methylated. IHC detected MGMT methylation with sensitivity of 86.2%, specificity of 63.0%, positive predictive value of 59.5%, negative predictive value of 87.9% and accuracy of 72.0%. The AUC was 0.746, indicating moderate diagnostic value. Optimum positivity threshold of the IHC examination based on Youden Index was 10%., Conclusion: IHC examination can be used to detect MGMT methylation status of glioma patients in limited resources setting, where PCR technique is not available.

In this study, a diabetic kidney disease model was established by placing the test rats on a high-sugar/high-fat diet combined with streptozotocin induction. Histopathological examination (H&E, Masson, and PASM stain) showed pathological changes in the diabetic rat kidneys, in addition to fibrotic symptoms and collagen deposition. Immunohistochemistry and western blot analyses indicated that the diabetic condition significantly increased the expressions of fibrotic markers including collagen, α -SMA, and fibronectin. The levels of cholesterol, triglyceride, and low-density lipoprotein were also increased in diabetic kidney disease (DKD) rat blood, while the level of high-density lipoprotein was decreased. The results of Oil red O staining experiments indicated that the kidneys of diabetic rats exhibited appreciable fat deposition, with high contents of triglyceride and cholesterol. To inhibit fibrosis and reduce fat deposition, low molecular weight fucoidan (LMWF) may be used. Based on PCR and western blot analyses, LMWF can regulate the expression levels of important lipid metabolism regulators, thereby impeding the development of kidney fibrosis. Through the vitro model, it also be indicated that LMWF could inhibit fibrosis process through regulating lipid metabolism which induced by palmitic acid., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Yan Wang et al.)

Liquid biopsies can be used to investigate tumor-derived DNA, circulating in the cell-free DNA (cfDNA) pool in blood. We aimed to develop a droplet digital polymerase chain reaction (ddPCR) assay detecting hypermethylation of tumor suppressor gene RASSF1A as a simple standard test to detect various pediatric tumor types in small volume blood samples and to evaluate this test for monitoring treatment response of patients with high-risk neuroblastoma., Methods: We developed a ddPCR assay to sensitively detect tumor-derived hypermethylated RASSF1A DNA in liquid biopsies. We tested this assay in plasma of 96 patients with neuroblastoma, renal tumors, rhabdomyosarcoma, or Hodgkin lymphoma at diagnosis and in cerebrospinal fluid of four patients with brain tumors. We evaluated the presence of hypermethylated RASSF1A in plasma samples during treatment and follow-up in 47 patients with neuroblastoma treated according to high-risk protocol and correlated results with blood mRNA-based and bone marrow mRNA-based minimal residual disease detection and clinical outcomes., Results: The total cfDNA level was significantly higher in patients with metastatic neuroblastoma and nephroblastoma compared with healthy adult and pediatric controls. Hypermethylated RASSF1A was present in 41 of 42 patients with metastatic neuroblastoma and in all patients with nephroblastoma, with the median percentage of 69% and 21% of total RASSF1A , respectively. Hypermethylated RASSF1A levels decreased during therapy and recurred at relapse., Conclusion: Our findings demonstrate the value of ddPCR-based detection of hypermethylated RASSF1A as a circulating molecular tumor marker in neuroblastoma. Our preliminary investigation of RASSF1A hypermethylation detection in circulating cfDNA of other pediatric tumor entities demonstrates potential as a pan-tumor marker, but requires investigation in larger cohorts to evaluate its use and limitations., Competing Interests: Johannes H. M. Merks Consulting or Advisory Role: Bayer, GlaxoSmithKline No other potential conflicts of interest were reported. Johannes H. M. Merks Consulting or Advisory Role: Bayer, GlaxoSmithKline No other potential conflicts of interest were reported., (© 2021 by American Society of Clinical Oncology.)

Background: The Rh blood group system has considerable clinical importance. The C, c, and E antigens are targets of alloantibodies. Anti-C, anti-c or anti-E alloreactive antibodies produced in pregnant women can cause anemia of a fetus carrying the corresponding antigens., Aims: Based on NGS technology, we have developed a noninvasive diagnostic assay to predict the fetal blood group of C, c or E antigens by sequencing cell-free DNA (cfDNA) during pregnancy., Materials and Methods: The SNVs underlying either the C, c or E antigens were PCR amplified and sequenced using NGS on a MiSeq instrument. The DNA sequences encoding the C, c or E antigen were counted, as were the number of total sequences. Based on the percentage of fetally derived target SNVs inherited from the father, the fetal blood group could be predicted., Results: The results of 55 consecutive RHCE prenatal analyses with postnatal serological blood group determination of 30 newborns showed no discordant results. A threshold discerning positive from negative samples was set at 0.05% specific reads., Discussion: Noninvasive, prenatal prediction of fetal blood groups by sequencing cfDNA for the detection of low-level RHCE*C, RHCE*c and RHCE*E sequences was established as an accurate and robust assay applicable for use in clinical settings., (© 2021 John Wiley & Sons Ltd.)

Background: Zambia continues to advance on the path to elimination with significant reductions in malaria morbidity and mortality. Crucial components that have contributed to progress thus far and are necessary for achieving the national malaria elimination goals include properly identifying and treating all malaria cases through accurate diagnosis. This study sought to compare and assess the diagnostic performance of Rapid Diagnostic Tests (RDT) and Light Microscopy (LM) with photo-induced electron transfer polymerase chain reaction (PET-PCR) as the gold standard using 2018 Malaria Indicator Survey (MIS) data across Zambia to better understand diagnostic accuracy metrics and how these vary across a transmission gradient., Methods: Cross-sectional samples collected in a nationally representative survey from 7 provinces in Zambia were tested for the presence of malaria parasites by light microscopy (LM), rapid diagnostic test (RDT) and the gold standard PET-PCR. Diagnostic performance was assessed including sensitivity, specificity, negative- and positive-predictive values across a wide malaria transmission spectrum. Diagnostic accuracy metrics were measured, and statistically significant differences were calculated between test methods for different outcome variables., Results: From the individuals included in the MIS, the overall prevalence of Plasmodium falciparum malaria was 32.9% by RDT, 19.4% by LM, and 23.2% by PET-PCR. Herein, RDT and LM diagnostic performance was compared against gold standard PET-PCR with LM displaying a higher diagnostic accuracy than RDTs (91.3% vs. 84.6% respectively) across the transmission spectrum in Zambia. However, the performance of both diagnostics was significantly reduced in low parasitaemia samples. Consistent with previous studies, RDT diagnostic accuracy was predominantly affected by a high rate of false positives., Conclusions: RDTs and LM both perform well across a range of transmission intensities within their respective target applications, i.e., in the community, for the former, where ease of use and speed of result is critical, and at the health facility, for the latter, where accuracy is prioritized. However, the performance of both diagnostic methods is adversely affected by low parasitaemia infections. As Zambia moves towards elimination more sensitive tools may be required to identify the last cases., (© 2021. The Author(s).)

Background: Rapid accurate diagnosis followed by effective treatment is very important for malaria control. Light microscopy remains the "golden standard" method for malaria diagnosis. Diagnostic test method must have sufficient level of accuracy for detecting malaria parasites. Therefore, this study aimed to investigate the diagnostic accuracy of rapid diagnostic tests (RDTs), microscopy, loop-mediated isothermal amplification (LAMP) and/or polymerase chain reaction (PCR) for the malaria diagnosis in Ethiopia., Methods: Data bases such as PubMed, PubMed central, Science direct databases, Google scholar, and Scopus were searched from September to October, 2020 for studies assessing the diagnostic accuracy of RDTs, microscopy, LAMP and PCR methods for malaria diagnosis., Results: A total of 29 studies published between 2001 and 2020 were analysed using review manager, Midas (Stata) and Meta-disc. The sensitivity and specificity of studies comparing RDT with microscopy varies from 79%-100% to 80%-100%, respectively. The sensitivity of LAMP (731 tests) was 100% and its specificity was varies from 85 to 99% when compared with microscopy and PCR. Considerable heterogeneity was observed between studies included in this meta-analysis. Meta-regression showed that blinding status and target antigens were the major sources of heterogeneity (P < 0.05). RDT had an excellent diagnostic accuracy (Area under the ROC Curve = 0.99) when compared with microscopy. Its specificity was quite good (93%-100%) except for one outlier (28%), but lower "sensitivity" was observed when PCR is a reference test. This indicates RDT had a good diagnostic accuracy (AUC = 0.83). Microscopy showed a very good diagnostic accuracy when compared with PCR., Conclusions: The present study showed that microscopy and RDTs had high efficiency for diagnosing febrile malaria patients. The diagnostic accuracy of RDT was excellent when compared with microscopy. This indicates RDTs have acceptable sensitivities and specificities to be used in resource poor settings as an alternative for microscopy. In this study, LAMP showed an excellent sensitivities and specificities. Furthermore, the need of minimum equipment and relatively short time for obtaining results can made LAMP one of the best alternatives especially for accurate diagnosis of asymptomatic malaria., (© 2021. The Author(s).)

Background/objectives: Genes involved in the regulation of metabolism, adipose tissue deposition, inflammation, and the appetite-satiety axis may play an important role in fetal development, and possibly induce permanent metabolic changes and fat accumulation. In this study we investigated: (1) obesity-related gene expression in maternal and cord blood of overweight/obese and normal-weight pregnant women; (2) associations between obesity-related gene expression in maternal and cord blood; and (3) associations of gene expression in each of maternal and cord blood with newborn adiposity., Subjects/methods: Twenty-five overweight/obese and 32 normal-weight pregnant women were selected from the Araraquara Cohort Study according to their pre-pregnancy BMI. Maternal and cord blood gene expression of LEPR, STAT3, PPARG, TLR4, IL-6, IL-10, FTO, MC4R, TNF-α, and NFκB were investigated by relative real-time PCR quantification. The body composition of the newborns was assessed by air displacement plethysmography. Associations between maternal and cord blood gene expression and markers of newborn adiposity (weight, BMI, and fat mass%) were explored by linear regression models controlling for maternal age, pre-pregnancy BMI, maternal gestational weight gain, gestational age, and newborn sex., Results: There was higher TLR4, NFκB, and TNF-a expression, and lower IL-6 expression, in overweight/obese pregnant women and their respective newborns compared with normal-weight women and their newborns (p < 0.001). Maternal PPARG gene expression was associated with both weight and fat mass % of the newborns, and cord blood IL-10 expression was associated with BMI and fat mass %, controlling for confounders., Conclusion: To our knowledge, this is the first study to evaluate the relationship of maternal and cord blood gene expression with adiposity markers of the newborn. Our results provide evidence for the contribution of maternal and cord blood gene expression-particularly maternal PPARG and TLR4 expression, and cord blood IL-10 expression-to newborn weight, BMI, and fat mass %., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

Purpose: The study aimed to evaluate the diagnostic accuracy of polymerase chain reaction ‒based high-risk human papillomavirus (HPV) assays on self-collected vaginal and urine samples for detection of precancerous cervical lesions in referral population., Materials and Methods: Women referred for colposcopy following abnormal cytology, were included this study. A total of 314 matched urine, vaginal, and cervical samples were collected. All samples were tested for HPV DNA using the RealTime HR-S HPV and Anyplex II HPV 28 assays. Primary endpoints were sensitivity for cervical intraepithelial neoplasia (CIN) 2+/CIN3+ and specificity for

Introduction: The Public Health Center (PHC)-known as hokenjo in Japan-assume a crucial role in disease control. Coronavirus disease 2019 (COVID-19) is one of many designated infectious diseases monitored by the agency. During the present pandemic, patients who suspected COVID-19 were instructed to call the Coronavirus Consultation Center in the PHC prior to visiting the hospital. The aim of this study was to elucidate the differences in polymerase chain reaction (PCR) positivity between PHC referrals and direct walk-in patients., Methods: The present was a single-center, retrospective cohort study conducted at the Tokyo Metropolitan Hospital from March to September, 2020. Patients who received a PCR test for SARS-CoV-2 were included and categorized into the PHC referral or direct walk-in groups. The outcomes included the total number of patients undergoing PCR tests and the percentage of PCR positivity in each group., Results: We identified 1680 patients (781 PHC referred and 899 direct walk-in groups). The percentage of PCR positivity did not significantly differ between the PHC referral and direct walk-in groups during the first wave (30.5% vs. 29.2%; p = 0.78). PCR positivity was significantly higher in the PHC referral group than the direct walk-in group during the second wave (30.1% vs. 23.1%; p = 0.051) and entire study period (30.2% vs. 24.7%; p = 0.011)., Conclusions: Despite health authority recommendations, the number of direct walk-in patients were higher than PHC referral patients. The percentage of PCR positivity was significantly higher in the PHC referral group than in the direct walk-in group., Competing Interests: Declaration of competing interest None., (Copyright © 2021 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

We sought to understand the sequence of testing and treatment among nine infants offered both conventional and point-of-care testing and diagnosed as HIV-positive by 6 months of age in Kenya. One infant received per protocol testing and treatment. Patient-level (late presentation and disengagement), provider-level (reluctance and error/oversight) and system-level (stock outs, errors) challenges delayed diagnosis and treatment. Early point-of-care testing can streamline testing; however, challenges mitigate benefits., Competing Interests: The authors have no conflicts of interest to disclose. The authors declare that they have no conflict of interest., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

Control strategies that employ real time polymerase chain reaction (RT-PCR) tests for the diagnosis and surveillance of COVID-19 epidemic are inefficient in fighting the epidemic due to high cost, delays in obtaining results, and the need of specialized personnel and equipment for laboratory processing. Cheaper and faster alternatives, such as antigen and paper-strip tests, have been proposed. They return results rapidly, but have lower sensitivity thresholds for detecting virus. To quantify the effects of the tradeoffs between sensitivity, cost, testing frequency, and delay in test return on the overall course of an outbreak, we built a multi-scale immuno-epidemiological model that connects the virus profile of infected individuals with transmission and testing at the population level. We investigated various randomized testing strategies and found that, for fixed testing capacity, lower sensitivity tests with shorter return delays slightly flatten the daily incidence curve and delay the time to the peak daily incidence. However, compared with RT-PCR testing, they do not always reduce the cumulative case count at half a year into the outbreak. When testing frequency is increased to account for the lower cost of less sensitive tests, we observe a large reduction in cumulative case counts, from 55.4% to as low as 1.22% half a year into the outbreak. The improvement is preserved even when the testing budget is reduced by one half or one third. Our results predict that surveillance testing that employs low-sensitivity tests at high frequency is an effective tool for epidemic control.

Background: Reactive malaria case detection involves the screening of those in contact with index cases and is used in countries in the Greater Mekong Sub-region. The yield of reactive case detection, defined here as the percentage of positive malaria cases among potential contacts who were screened, was assessed., Methods: A literature search was conducted on PubMed to identify studies on reactive case detection in the Greater Mekong Sub-region. Eligible published articles were reviewed and pooled estimates from the studies were calculated, by type of malaria test used., Results: Eighty-five publications were retrieved, of which 8 (9.4%) eligible articles were included in the analysis. The yield from reactive case detection ranged from 0.1 to 4.2%, with higher rates from PCR testing compared with microscopy and/or rapid diagnostic test. The overall yield from microscopy and/or rapid diagnostic test was 0.56% (95% CI 0.31-0.88%), while that from PCR was 2.35% (95% CI 1.19-3.87%). The two studies comparing different target groups showed higher yield from co-workers/co-travellers, compared with household contacts., Conclusion: In low malaria transmission settings, the effectiveness of reactive case detection is diminishing. In the Greater Mekong Sub-region, modifying reactive case detection from household contacts to co-workers/co-travellers and from testing to presumptive treatment of targeted contacts, could increase the impact of this approach.

This study aimed to determine if the crossing point of the initial positive SARS-CoV-2 PCR test correlated with patient demographics, subsequent hospitalization, or duration of positivity. Seventy-three patients with two or more positive PCR tests had a median time of 23 days to two consecutive negative results., Competing Interests: Declaration of competing interest The authors have no relevant conflicts of interest to disclose related to the work in this study., (Copyright © 2020. Published by Elsevier B.V.)

Evidence-based diagnostic algorithm is highly recommended for the visceral leishmaniasis (VL). This cross-sectional study was performed in Bangladesh to evaluate VL diagnostic tools including serology, buffy coat smear microscopy for LD body and various DNA-based techniques using buffy coat in 100 confirmed VL cases and 100 controls. The performance of tools against spleen smear (gold standard) was evaluated using kappa coefficient. Diagnostic precision and other inherent indicators were considered for index scoring (IS) of performance of tools using factor analysis. A diagnostic algorithm was formulated based on the IS and availability of the tools at different health care facilities of Bangladesh. A high level of agreement (kappa ≥  0.80) was observed for all the diagnostic tools. The highest kappa coefficients were found for rK39 RDT and rK39 ELISA (0.95), followed by ssuRNA-PCR (0.94), Buffy coat smear (0.93), rK28 ELISA (0.92), rK28 RDT (0.89), LAMP (0.89), Mini-exon PCR (0.86), ITS1 (0.85), and ITS2 PCR (0.80). rK39 RDT was found to be the best diagnostic test (IS: 1.7) followed by rK28 RDT (IS: 1.5), buffy coat smear microscopy (IS: 0.5), rK39 & rK28 ELISA (IS: 0.3), ssuRNA-PCR (IS: -0.7) and LAMP, Mini-exon, ITS1, & ITS2 PCR (IS: -0.9). rK39 RDT has been proposed as the best option for primary health care facilities, while buffy coat smear microscopy was found to be a good adjunct for confirmation of serology-positive cases and proposed for secondary and tertiary facilities. ssuRNA-PCR or LAMP can be an alternate confirmation tool only applicable to the tertiary facilities., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Experts agree that reverse transcription-polymerase chain reaction (PCR) testing is critical in controlling coronavirus disease 2019 (COVID-19), but decision makers disagree on how much testing is optimal. Controlling for interventions and ecological factors, we used linear regression to quantify testing's impact on COVID-19's average reproduction number, which represents transmissibility, in 173 countries and territories (which account for 99 percent of the world's COVID-19 cases) during March-June 2020. Among interventions, PCR testing had the greatest influence: a tenfold increase in the ratio of tests to new cases reported reduced the average reproduction number by 9 percent across a range of testing levels. Our results imply that mobility reductions (for example, shelter-in-place orders) were less effective in developing countries than in developed countries. Our results help explain how some nations achieved near-elimination of COVID-19 and the failure of lockdowns to slow COVID-19 in others. Our findings suggest that the testing benchmarks used by the World Health Organization and other entities are insufficient for COVID-19 control. Increased testing and isolation may represent the most effective, least costly alternative in terms of money, economic growth, and human life for controlling COVID-19.

Background: Plasmodium falciparum histidine-rich protein 2 (HRP2)-based rapid diagnostic tests (RDTs) are exclusively recommended for malaria diagnosis in Uganda; however, their functionality can be affected by parasite-related factors that have not been investigated in field settings., Methods: Using a cross-sectional design, we analysed 219 RDT-/microscopy+ and 140 RDT+/microscopy+ dried blood spots obtained from symptomatic children aged 2-10 years from 48 districts in Uganda between 2017 and 2019. We aimed to investigate parasite-related factors contributing to false RDT results by molecular characterization of parasite isolates. ArcGIS software was used to map the geographical distribution of parasites. Statistical analysis was performed using chi-square or Fisher's exact tests, with P ≤ 0.05 indicating significance. Odds ratios (ORs) were used to assess associations, while logistic regression was performed to explore possible factors associated with false RDT results., Results: The presence of parasite DNA was confirmed in 92.5% (332/359) of the blood samples. The levels of agreement between the HRP2 RDT and PCR assay results in the (RDT+/microscopy+) and (RDT-/microscopy+) sample subsets were 97.8% (137/140) and 10.9% (24/219), respectively. Factors associated with false-negative RDT results in the (RDT-/microscopy+) samples were parasite density (<1,000/μl), pfhrp2/3 gene deletion and non-P. falciparum species (aOR 2.65, 95% CI: 1.62-4.38, P = 0.001; aOR 4.4, 95% CI 1.72-13.66, P = 0.004; and aOR 18.65, 95% CI: 5.3-38.7, P = 0.001, respectively). Overall, gene deletion and non-P. falciparum species contributed to 12.3% (24/195) and 19.0% (37/195) of false-negative RDT results, respectively. Of the false-negative RDTs results, 80.0% (156/195) were from subjects with low-density infections (< 25 parasites per 200 WBCs or <1,000/μl)., Conclusion: This is the first evaluation and report of the contributions of pfhrp2/3 gene deletion, non-P. falciparum species, and low-density infections to false-negative RDT results under field conditions in Uganda. In view of these findings, the use of HRP2 RDTs should be reconsidered; possibly, switching to combination RDTs that target alternative antigens, particularly in affected areas, may be beneficial. Future evaluations should consider larger and more representative surveys covering other regions of Uganda., Competing Interests: No authors have no competing interests.

Background: MicroRNAs [miRNAs] are cell-specific small non-coding RNAs that can regulate gene expression and have been implicated in inflammatory bowel disease [IBD] pathogenesis. Here we define the cell-specific miRNA profiles and investigate its biomarker potential in IBD., Methods: In a two-stage prospective multi-centre case control study, next generation sequencing was performed on a discovery cohort of immunomagnetically separated leukocytes from 32 patients (nine Crohn's disease [CD], 14 ulcerative colitis [UC], eight healthy controls) and differentially expressed signals were validated in whole blood in 294 patients [97 UC, 98 CD, 98 non-IBD, 1 IBDU] using quantitative PCR. Correlations were analysed with phenotype, including need for early treatment escalation as a marker of progressive disease using Cox proportional hazards., Results: In stage 1, each leukocyte subset [CD4+ and CD8+ T-cells and CD14+ monocytes] was analysed in IBD and controls. Three specific miRNAs differentiated IBD from controls in CD4+ T-cells, including miR-1307-3p [p = 0.01], miR-3615 [p = 0.02] and miR-4792 [p = 0.01]. In the extension cohort, in stage 2, miR-1307-3p was able to predict disease progression in IBD (hazard ratio [HR] 1.98, interquartile range [IQR]: 1.20-3.27; logrank p = 1.80 × 10-3), in particular CD [HR 2.81; IQR: 1.11-3.53, p = 6.50 × 10-4]. Using blood-based multimarker miRNA models, the estimated chance of escalation in CD was 83% if two or more criteria were met and 90% for UC if three or more criteria are met., Interpretation: We have identified and validated unique CD4+ T-cell miRNAs that are differentially regulated in IBD. These miRNAs may be able to predict treatment escalation and have the potential for clinical translation; further prospective evaluation is now indicated., (© The Author(s) 2020. Published by Oxford University Press on behalf of European Crohn’s and Colitis Organisation.)

Genetic diseases are driven by aberrations of the human genome. Identification of such aberrations including structural variations (SVs) is key to our understanding. Conventional short-reads whole genome sequencing (cWGS) can identify SVs to base-pair resolution, but utilizes only short-range information and suffers from high false discovery rate (FDR). Linked-reads sequencing (10XWGS) utilizes long-range information by linkage of short-reads originating from the same large DNA molecule. This can mitigate alignment-based artefacts especially in repetitive regions and should enable better prediction of SVs. However, an unbiased evaluation of this technology is not available. In this study, we performed a comprehensive analysis of different types and sizes of SVs predicted by both the technologies and validated with an independent PCR based approach. The SVs commonly identified by both the technologies were highly specific, while validation rate dropped for uncommon events. A particularly high FDR was observed for SVs only found by 10XWGS. To improve FDR and sensitivity, statistical models for both the technologies were trained. Using our approach, we characterized SVs from the MCF7 cell line and a primary breast cancer tumor with high precision. This approach improves SV prediction and can therefore help in understanding the underlying genetics in various diseases., Competing Interests: I have read the journal's policy and the authors of this manuscript have the following competing interests: Ugur Sahin is co-founder and shareholder of TRON, co-founder and CEO of BioNTech SE.

Background: Little is known about the performance of the Roche novel severe acute respiratory syndrome coronavirus 2 antibody (anti-SARS-CoV-2) assay. We provide an extensive evaluation of this fully automated assay on Cobas e801/e602 immunoassay analyzers., Methods: We assessed the linearity, precision, and throughput of the Roche anti-SARS-CoV-2 assay. Sensitivity was calculated from 349 SARS-CoV-2 polymerase chain reaction (PCR) positive samples; specificity was determined from 715 coronavirus disease 2019 (COVID-19)-naive samples. We examined cross-reactivity against other antibody positive samples [syphilis, rheumatoid factor (RF), antinuclear antibody (ANA), double-stranded DNA (ds-DNA), influenza, dengue, hepatitis B (HBV), hepatitis C (HCV)] and the anti-SARS-CoV-2 kinetics., Results: The assay cut-off index (COI) was linear up to 90.8. The interassay precision was 2.9% for a negative control (COI = 0.1) and 5.1% for a positive control (COI = 3.0). Assay time is 18 min and results are available 1 min later; throughput for 300 samples was 76 min. Only 1 case positive for HBsAg tested falsely positive; specificity was 99.9%. The assay has a sensitivity of 97.1% 14 days after PCR positivity (POS) and 100% at ≥21 days POS; 48.2% of cases had anti-SARS-CoV-2 within 6 days POS. In 11 patients in whom serum was available prior to a positive antibody signal (COI ≥1.0) the interval between the last negative and first positive COI (time to "seroconversion") on average is 3 days (range 1-6 days) and 4 more days (range 1-7) for the anti-SARS-CoV-2 to plateau., Conclusion: The Roche anti-SARS-CoV-2 assay shows excellent performance with minimal cross-reactivity from other viral and confounding antibodies. Antibody development and seroconversion appears quite early., (© American Association for Clinical Chemistry 2020. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Background: While molecular techniques remain the gold standard for diagnosis of acute SARS-CoV-2 infection, serological tests have the unique potential to ascertain how much of the population has been exposed to the COVID-19 pathogen. There have been limited published studies to date documenting the performance of SARS-CoV-2 antibody assays., Methods: We compared the DiaSorin Liaison SARS-CoV-2 S1/S2 IgG and Roche Diagnostics Elecsys Anti-SARS-CoV-2 assays using 228 samples spanning patients with positive PCR for SARS-CoV-2, patients with compatible symptoms but negative PCR, pre-COVID specimens, and potential cross-reactives., Results: Both assays detected antibodies in 18/19 samples collected at least one week after a positive PCR result. Neither method consistently detected antibodies in specimens collected within one week of a positive PCR result (sensitivity < 50%), but antibodies were detected by only Roche in four samples in this time frame. Using 139 pre-COVID and 35 PCR-negative samples, the Roche and DiaSorin assays demonstrated specificities of 100.0% and 98.9%, respectively. Neither assay demonstrated cross-reactivity from other coronaviruses (229E, HKU1, NL63, OC43), respiratory pathogens (adenovirus, metapneumovirus, rhinovirus/enterovirus), or antibodies to other viruses (HIV, EBV, CMV, HBV, HCV, HAV)., Discussion: Overall, the qualitative interpretations afforded by the Roche and DiaSorin assays agreed for 97% of samples evaluated. Minor discrepancies in sensitivity and specificity were observed between methods, with the differences in specificity more clinically significant for our low-prevalence population. For the DiaSorin assay, all disagreements with the Roche assay occurred in samples with quantitative signals near the cut-off determining positivity., (© American Association for Clinical Chemistry 2020. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Background: Coronavirus disease 2019 (COVID-19) was formally characterized as a pandemic on March 11, 2020. Since that time, the COVID-19 pandemic has led to unprecedented demand for healthcare resources. The purpose of this study was to identify changes in laboratory test utilization in the setting of increasing local incidence of COVID-19., Methods: We performed a retrospective assessment of laboratory test order and specimen container utilization at a single, urban tertiary care medical center. Data were extracted from the laboratory information system database over a 10-week period, spanning the primordial inflection of COVID-19 incidence in our region. Total testing volumes were calculated during the first 2 and last 2 weeks of the observation period and used as reference points to examine the absolute and relative differences in test order volume between the prepandemic and COVID-19 surge periods., Results: Between February 2, 2020, and April 11, 2020, there were 873 397 tests ordered and final verified. The in-house SARS-CoV-2 PCR positivity rate for admitted patients in the last week of the observation period was 30.8%. Significant increases in workload were observed in the send-out laboratory section and for COVID-19 diagnosis (PCR) and management-related testing. Otherwise, there was a net decrease in overall demand across nearly all laboratory sections. Increases in testing were noted for tests related to COVID-19 management. Viral transport media and citrated blue top containers demonstrated increases in utilization., Conclusion: Increasing local incidence of COVID-19 had a profound impact on laboratory operations. While volume increases were seen for laboratory tests related to COVID-19 diagnostics and management, including some with limited evidence to support their use, overall testing volumes decreased substantially. During events such as COVID-19, monitoring of such patterns can help inform laboratory management, staffing, and test stewardship recommendations for managing resource and supply availability., (© American Association for Clinical Chemistry 2020. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Objectives: To see what the impact of introducing a rapid polymerase chain reaction-based influenza test has on length of stay (LOS) in emergency department, use of imaging, serum or urine testing, antibiotic use, and antiviral use., Methods: Audit of electronic medical health records was performed for all emergency department visits from 2014 to 2018 between November and March, which was defined as peak flu season. Patients were included if they were between 3 months and 18 years of age, presented during peak flu season, and were tested for influenza. The pre-point of care (POC) period was defined as November through March of 2014 to 2017 which was compared with the post-POC group which was defined as November through March of 2017 to 2018., Results: Patients tested for influenza in the pre-POC period were more likely to have complete blood count testing (44.7% vs 25.6% P < 0.01), more likely to have blood cultures performed (30% vs 16.3%, P < 0.01), more likely to have urine testing performed (21.5% vs 12.2%, P < 0.01), and more likely to have a chest radiograph completed (47.5% vs 34.4%, P < 0.01). There was no significant difference in rates of antibiotics used. There was increased rates of oseltamivir used in the post-POC period (21.2% vs 13.3%, P < 0.05. The median LOS decreased from 239 minutes in the pre-POC period to 232 minutes in the post-POC period (P < 0.05)., Conclusions: With the introduction of a polymerase chain reaction-based point-of-care influenza test, there were overall decreased rates of invasive blood work, urine studies, and imaging, and median LOS. There was also increased antiviral administration.

Background: A large COVID-19 outbreak occurred on the cruise ship Diamond Princess in February 2020. Little information has been reported about the clinical characteristics of the patients., Methods: This single-center, retrospective, observational study was conducted in Yokohama, Japan. We included symptomatic patients who were infected on the ship and admitted to our hospital between 5 and 19 February 2020. All the cases were confirmed with SARS-CoV-2 infection by polymerase chain reaction (PCR)., Results: We confirmed 17 cases. The average age was 69 years; 10 patients were Asian and 7 were Caucasian. Eleven patients had one or more chronic diseases. The major symptoms were cough and fever. Chest computed tomography (CT) scans found bilateral ground-glass opacities predominantly in the peripheral area, which were similar to reports from cases in China. C-reactive protein (CRP) levels were higher in severe and critical cases than in mild to moderate cases. The moderate to severe cases reached symptomatic resolution; one of the three critical cases resulted in death due to multiple organ failure. SARS-CoV-2 was detected by PCR at an average of 7 days after symptomatic resolution., Conclusions: Cough and fever, increased blood CRP levels, and CT findings of bilateral ground-glass opacities predominantly in the peripheral lung were characteristic of the COVID-19 cases in this study. These findings were compatible with those of previous reports., Competing Interests: Declaration of Competing Interest None., (Copyright © 2020 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

Recurrence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) positive detection in infected but recovered individuals has been reported. Patients who have recovered from coronavirus disease 2019 (COVID-19) could profoundly impact the health care system. We sought to define the kinetics and relevance of PCR-positive recurrence during recovery from acute COVID-19 to better understand risks for prolonged infectivity and reinfection. A series of 414 patients with confirmed SARS-Cov-2 infection, at The Second Affiliated Hospital of Southern University of Science and Technology in Shenzhen, China from January 11 to April 23, 2020. Statistical analyses were performed of the clinical, laboratory, radiologic image, medical treatment, and clinical course of admission/quarantine/readmission data, and a recurrence predictive algorithm was developed. 16.7% recovered patients with PCR positive recurring one to three times, despite being in strict quarantine. Younger patients with mild pulmonary respiratory syndrome had higher risk of PCR positivity recurrence. The recurrence prediction model had an area under the ROC curve of 0.786. This case series provides characteristics of patients with recurrent SARS-CoV-2 positivity. Use of a prediction algorithm may identify patients at high risk of recurrent SARS-CoV-2 positivity and help to establish protocols for health policy.

The ability to detect SARS-CoV-2 in the upper respiratory tract ceases after 2 to 3 weeks post-symptom-onset in most patients. In contrast, SARS-CoV-2 can be detected in the stool of some patients for greater than 4 weeks, suggesting that stool may hold utility as an additional source for diagnosis. We validated the Cepheid Xpert Xpress SARS-CoV-2 and Hologic Panther Fusion real-time RT-PCR assays for detection of viral RNA in stool specimens and compared performance. We utilized remnant stool specimens ( n  = 79) from 77 patients with gastrointestinal symptoms. Forty-eight patients had PCR-confirmed COVID-19, and 29 either were nasopharyngeal/oropharyngeal PCR negative or presented for reasons unrelated to COVID-19 and were not tested. Positive percent agreement between the Cepheid and Hologic assays was 93% (95% confidence interval [CI]: 81.1% to 98.2%), and negative percent agreement was 96% (95% CI: 89% to 0.99%). Four discrepant specimens (Cepheid positive only, n  = 2; Hologic positive only, n  = 2) exhibited average cycle threshold ( C T ) values of >37 for the targets detected. Of the 48 patients with PCR-confirmed COVID-19, 23 were positive by both assays (47.9%). For the negative patient group, 2/29 were positive by both assays (6.9%). The two stool PCR-positive, nasopharyngeal/oropharyngeal PCR-negative patients were SARS-CoV-2 IgG positive. Our results demonstrate acceptable agreement between two commercially available molecular assays and support the use of stool PCR to confirm diagnosis when SARS-CoV-2 is undetectable in the upper respiratory tract., (Copyright © 2020 American Society for Microbiology.)

Background: Giardia duodenalis is a common cause of chronic diarrhea especially in tropical countries. Diagnosis is based on microscopy (three stool samples) for trophozoites/cysts. Role of stool or duodenal biopsy PCR as a diagnostic method needs to be defined. We conducted a prospective study to determine the diagnostic characteristics of G. duodenalis stool and duodenal biopsy PCR in comparison to stool microscopy (reference standard). Later, we compared other techniques with stool PCR, considering it as new reference standard and characterized the type of Giardia assemblage., Methods: G. duodenalis stool nested PCR was first evaluated using 40 positive controls and 50 negative controls considering stool microscopy as reference standard. Patients with chronic diarrhea (n = 100) were evaluated by stool microscopy and nested PCR. In 30 patients in whom upper gastrointestinal endoscopy was performed, duodenal biopsy samples were obtained and evaluated by histopathology, imprint cytology, and nested PCR. The type of Giardia assemblage was detected by assemblage-specific PCR., Results: Stool nested PCR was found to have sensitivity and specificity of 100% and 94%, respectively, compared to stool microscopy. In patients with chronic diarrhea, 48% had evidence of Giardia infection. Stool microscopy detected 65%, stool PCR detected an additional 27%, and duodenal biopsy PCR detected an additional 8% of cases. The commonest assemblage found was assemblage B. Clinical and demographic characteristics were similar in patients harboring either assemblage A or B., Conclusion: Stool PCR is more sensitive than stool microscopy. By utilizing stool microscopy, stool nested PCR, and duodenal biopsy PCR in sequential manner, diagnostic yield can be increased.

Digital PCR (dPCR) has developed considerably since the publication of the Minimum Information for Publication of Digital PCR Experiments (dMIQE) guidelines in 2013, with advances in instrumentation, software, applications, and our understanding of its technological potential. Yet these developments also have associated challenges; data analysis steps, including threshold setting, can be difficult and preanalytical steps required to purify, concentrate, and modify nucleic acids can lead to measurement error. To assist independent corroboration of conclusions, comprehensive disclosure of all relevant experimental details is required. To support the community and reflect the growing use of dPCR, we present an update to dMIQE, dMIQE2020, including a simplified dMIQE table format to assist researchers in providing key experimental information and understanding of the associated experimental process. Adoption of dMIQE2020 by the scientific community will assist in standardizing experimental protocols, maximize efficient utilization of resources, and further enhance the impact of this powerful technology., (© American Association for Clinical Chemistry 2020.)

Background: Test on the KRAS somatic mutation status is necessary before cetuximab and panitumumab treatments are given to colorectal cancer patients. Metastatic colorectal cancer patients sometimes lack tumor tissue samples, and the testing of KRAS mutation in plasma samples requires highly sensitive methods., Objectives: The aim of this study was to evaluate the accuracy of digital PCR in detecting KRAS mutation in plasma samples of colorectal cancer patients., Data Sources: Literature research was conducted in Pubmed, Embase, and Cochrane Library., Study Eligibility Criteria, Participants, and Interventions: Database searching found 188 relevant studies. After removing duplicates, eligible studies were selected from 151 publications using the following exclusion criteria: STUDY APPRAISAL AND SYNTHESIS METHODS:: Data were extracted from the eligible studies by 2 independent researchers. Pooled accuracy parameters were calculated from those extracted data using Meta-DiSc and STATA software., Results: Twelve eligible studies were selected for the systematic review and meta-analysis. After calculation, the pooled sensitivity and specificity were 0.83 (95% CI: 0.79-0.86) and 0.91 (95%CI: 0.88-0.93), respectively. Pooled positive likelihood ratio, negative likelihood ratio, and diagnostic odds ratio were 7.30 (95%CI: 4.78-11.17), 0.22 (95%CI: 0.15-0.32), and 41.00 (95%CI: 21.07-79.78), respectively. Area under curve of the summarized ROC curve was 0.9322., Limitations: Although no significant bias was identified, number of included studies was still quite small, especially in subgroup analysis., Conclusions and Implication of Key Findings: Digital PCR showed high accuracy and could be a reliable detection method for KRAS mutation in plasma samples. Large-cohort prospective study is required to further confirm the usefulness of digital PCR in KRAS mutation detection.

Background: Babies of women with heterozygous pathogenic glucokinase (GCK) variants causing mild fasting hyperglycemia are at risk of macrosomia if they do not inherit the variant. Conversely, babies who inherit a pathogenic hepatocyte nuclear factor 4α (HNF4A) diabetes variant are at increased risk of high birth weight. Noninvasive fetal genotyping for maternal pathogenic variants would inform pregnancy management., Methods: Droplet digital PCR was used to quantify reference and variant alleles in cell-free DNA extracted from blood from 38 pregnant women heterozygous for a GCK or HNF4A variant and to determine fetal fraction by measurement of informative maternal and paternal variants. Droplet numbers positive for the reference/alternate allele together with the fetal fraction were used in a Bayesian analysis to derive probability for the fetal genotype. The babies' genotypes were ascertained postnatally by Sanger sequencing., Results: Droplet digital PCR assays for GCK or HNF4A variants were validated for testing in all 38 pregnancies. Fetal fraction of ≥2% was demonstrated in at least 1 cell-free DNA sample from 33 pregnancies. A threshold of ≥0.95 for calling homozygous reference genotypes and ≤0.05 for heterozygous fetal genotypes allowed correct genotype calls for all 33 pregnancies with no false-positive results. In 30 of 33 pregnancies, a result was obtained from a single blood sample., Conclusions: This assay can be used to identify pregnancies at risk of macrosomia due to maternal monogenic diabetes variants., (© American Association for Clinical Chemistry 2020.)

Background Group B Streptococcus (GBS) is a common cause of neonatal sepsis. GBS colonization of the newborn gastrointestinal tract (GIT) may be a critical precursor for late-onset infection. Assessment of the rate of neonatal GBS intestinal colonization has generally relied upon culture-based methods. We used polymerase chain reaction (PCR) and culture to determine the rate of GBS transmission to neonates. We hypothesized that PCR may enhance the detection of neonatal GBS colonization of the GIT, and that the rate will be higher when evaluated with PCR as compared to culture. Methods This was a cross-sectional study, in which mothers who were positive for GBS on routine screening and their healthy infants were eligible for recruitment. Newborn stool was collected after 24 h of life and before hospital discharge, and stored at -80°C for culture and PCR targeting the GBS-specific surface immunogenic protein (sip) gene. Results A total of 94 mother-infant pairs were enrolled; of these pairs, stool was collected from 83 infants. Based on PCR, the overall GBS transmission rate was 3.6% (3/83). The transmission rate was 2.4% (1/41) among vaginal deliveries and 4.8% (2/42) among cesarean deliveries. The results of culture-based transmission detection were identical. Conclusion These results indicate that the rate of GBS transmission is low and that detection may not be enhanced by PCR methods.

Background: This study aimed to assess and compare the clinical value of aqueous humor polymerase chain reaction (PCR) and serologic tests in patients diagnosed with suspected infectious uveitis., Methods: In this retrospective observational study, data of 358 patients who were diagnosed with suspected infectious uveitis and who underwent aqueous humor PCR testing were analyzed. PCR and serologic test results were compared with the clinical features., Results: The rates of initial diagnoses for infectious uveitis were higher with PCR (99 patients, 28%) compared to those with serologic tests (38 pateints, 11%). The diagnostic positivity of PCR was 29% for anterior uveitis, 0% for intermediate uveitis, 5% for posterior uveitis, and 30% for panuveitis. In particular, PCR was useful in confirming the diagnosis of cytomegalovirus and varicella-zoster virus infections and Toxoplasma gondii-associated uveitis. For PCR test, the sensitivity was 0.431, specificity was 0.985, and the negative and positive predictive values were 0.506 and 0.980, respectively. For IgM test, the sensitivity was 0.151, specificity was 0.970, and the negative and positive predictive values were 0.403 and 0.895, respectively., Conclusion: Aqueous humor PCR can be a valuable diagnostic tool for confirming the infectious etiology in patients clinically diagnosed with uveitis. PCR had good predictive and diagnostic value for anterior uveitis and panuveitis compared with that for intermediate and posterior uveitis.

Introduction: Preimplantation genetic testing (PGT) is growing in importance and volume internationally. International societies such as the European Society for Human Reproduction and Embryology compile international results and these data are published in scientific journals. We present the first compilation of practices, quality measuress and outcome data from Nordic clinics performing PGT., Material and Methods: We conducted a structured online survey of PGT practices in the Nordic countries to compare clinical and laboratory techniques, outcomes and quality measures applied in Nordic clinics. The survey was designed by the authors and answered by the authors and members of the study group. The outcome data represents results from 2018. Results and details were clarified through iteration with responding clinics while maintaining anonymity. Response rate in the study was 80%, with 8 of 10 clinics performing PGT responding., Results: Most of the PGT cycles in the Nordic countries are funded through the public healthcare system with University Hospitals performing the majority of treatments, 716/848, or 84.4%, of oocyte retrievals in this dataset. The genetic analyses are in five cases performed by the affiliated local genetic laboratory, and the remaining three consult with large international private enterprise laboratories. Genetic counseling is widely used. Results in the Nordic clinics compare well with international data. Systematic quality control procedures are in place and the larger clinics and laboratories utilize ISO certification or accreditation in the quality management. Automatic witnessing with detailed electronic documentation of laboratory processes is not utilized in the responding clinics, although a majority uses manual witnessing procedures in the laboratory. The outcome after PGT in terms of clinical pregnancy per transfer is around 40% per embryo transfer and compares well with international data., Conclusions: Preimplantation genetic testing is organized in rather few clinics in the Nordic countries and most of them use local laboratories for genetic analyses of the biopsies. Laboratory procedures are largely in accordance with international guidelines and the outcome after PGT in terms of clinical pregnancy per transfer is comparable to results in international reports., (© 2020 Nordic Federation of Societies of Obstetrics and Gynecology.)

Highly sensitive detection of HIV-1 nucleic acids is of critical importance for evaluating treatment interventions superimposed on combination antiretroviral therapy (cART) in HIV-1 infected individuals. SIV infection of rhesus macaques models many key aspects of human HIV-1 infection and plays a key role in evaluation of approaches for prevention, treatment and attempted eradication of HIV infection. Here we describe two droplet digital PCR (ddPCR) assays, a ddPCR DNA assay and an RT-ddPCR RNA assay for detecting simian immunodeficiency virus (SIV) on the RainDance platform. We demonstrate that RainDance ddPCR can tolerate significantly higher cell DNA input without inhibition on a per reaction basis (compared to both qPCR and Bio-Rad ddPCR), thus allowing a large quantity of sample to be analyzed in each reaction. In addition, the combination of a high processivity RT enzyme and RainDance ddPCR could overcome inhibition in severely inhibited (99.99% inhibition in qPCR quantification) viral RNA samples. These assays offer valuable tools for assessing low level viral production/replication and strategies for targeting residual virus in the setting of cART suppression of viral replication. The methodologies presented here can be adapted for a broad range of applications where highly sensitive nucleic acid detection is required., Competing Interests: The authors have declared that no competing interests exist.

Objectives: The primary objective was to describe the clinical characteristics and 30-day mortality rates in emergency department patients with coronavirus disease 2019 (COVID-19) in different diagnostic groupings., Material and Methods: Secondary analysis of the COVID-19 registry compiled by the emergency department of Hospital Clínico San Carlos in Madrid, Spain. We selected suspected COVID-19 cases treated in the emergency department between February 28 and March 31, 2020. The cases were grouped as follows: 1) suspected, no polymerase chain reaction (PCR) test (S/no-PCR); 2) suspected, negative PCR (S/PCR-); 3) suspected, positive PCR (S/PCR+); 4) highly suspected, no PCR, or negative PCR (HS/no or PCR-); and 5) highly suspected, positive PCR (HS/PCR+). We collected clinical, radiologic, and microbiologic data related to the emergency visit. The main outcome was 30-day all-cause mortality. Secondary outcomes were hospitalization and clinical severity of the episode., Results: A total of 1993 cases (90.9%) were included as follows: S/no-PCR, 17.2%; S/PCR-, 11.4%; S/PCR+, 22.1%; HS/no PCR or PCR-, 11.7%; and HS/PCR+, 37.6%. Short-term outcomes differed significantly in the different groups according to demographic characteristics; comorbidity and clinical, radiographic, analytical, and therapeutic variables. Thirty-day mortality was 11.5% (56.5% in hospitalized cases and 19.6% in cases classified as severe). The 2 HS categories and the S/PCR+ category had a greater adjusted risk for 30-day mortality and for having a clinically severe episode during hospitalization in comparison with S/PCR- cases. Only the 2 HS categories showed greater risk for hospitalization than the S/PCR- cases., Conclusion: COVID-19 diagnostic groups differ according to clinical and laboratory characteristics, and the differences are associated with the 30-day prognosis.

This review is the second part of the workshop on digital PCR (dPCR) proposed by the working group of the French society of clinical biology. The first part of the paper discusses the advantages and limitations of dPCR for the search of different molecular abnormalities such as point mutations, copy number variants, DNA methylation, RNA analysis and a more innovative application, the single-cell dPCR. This synthesis makes it possible to propose a positioning of the dPCR compared to the other available technologies in a medical laboratory. In a second part, the main current applications of the dPCR will be addressed including the oncology of solid tumors and liquid biopsies, oncohematology and the follow-up of hemopathies treatments by hematopoietic stem cell transplantation. We will also detail non-invasive prenatal diagnosis and diagnosis of mosaic genetic disease, using the example of McCune-Albright syndrome. Several French specialists in the field who have implemented these techniques in their laboratory have written these different examples of applications jointly. In summary, this manuscript offers an up-to-date view of the positioning of dPCR in relation to other existing technologies in order to best meet the expectations of precision medicine.

Background: Accurate diagnosis of malaria infection is essential for successful control and management of the disease. Both microscopy and rapid diagnostic tests (RDTs) are recommended for malaria diagnosis, however, RDTs are more commonly used. The aim of the current study was to assess the performance of microscopy and RDTs in the diagnosis of Plasmodium falciparum infection using a nested polymerase chain reaction (PCR) assay as the gold standard., Methods: A cross-sectional study was carried out in Kassala Hospital, eastern Sudan. A total of 341 febrile participants of all ages were recruited. Blood specimens were collected and malaria testing was performed using an RDT (SD Bioline Malaria Ag Pf), microscopy and nested PCR. The sensitivity, specificity, positive and negative predictive values (PPV and NPV, respectively) of microscopy and the RDT were investigated., Results: The prevalence of P. falciparum malaria infections in this study was 22.9%, 24.3% and 26.7% by PCR, microscopy and RDT, respectively. Compared with microscopy, the RDT had slightly higher sensitivity (80.7% vs 74.3%; p=0.442), equivalent specificity (89.3% vs 90.4%), a similar PPV (69.2% vs 69.8%) and a higher NPV (94.0% vs 92.2%)., Conclusions: The diagnostic performance of the RDT was better than that of microscopy in the diagnosis of P. falciparum malaria when nested PCR was used as the gold standard., (© The Author(s) 2019. Published by Oxford University Press on behalf of Royal Society of Tropical Medicine and Hygiene. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: Few data are published on perianal tuberculosis., Objective: This study aimed to determine the best method to diagnose tuberculosis in patients with fistula-in-ano and to conduct a systematic review to determine the incidence and characteristics of tuberculosis fistula-in-ano., Data Sources: The prospective study data and existing literature were derived from PubMed, Google scholar, and Scopus STUDY SELECTION:: Prospective analysis of patients with tuberculous fistula-in-ano treated between 2014 and 2018 was conducted, and a systematic review of studies describing ≥3 patients with tuberculosis fistula-in-ano was completed., Intervention: Testing of tuberculosis was performed by histopathology or polymerase chain reaction of tissue or pus from the fistula tract., Main Outcome Measures: The primary outcomes measured were the detection rate of various tests to detect tuberculosis in fistula-in-ano and the prevalence rate of tuberculosis in simple versus complex fistulas., Results: In 637 samples (410 patients) tested, tuberculosis was detected in 49 samples (43 patients). Additional samples (n = 106) sent in patients with a high index of suspicion tested positive in 14 more patients. Thus, overall, 63 samples tested positive in 57 patients (total: 743 samples in 410 patients were tested). Tuberculosis was detected in 2 of 181 patients (1.1%) in tissue (histopathology), in 28 of 341 patients (8.2%) in tissue (polymerase chain reaction), and in 19 of 115 patients (16.5%) in pus (polymerase chain reaction) samples. To detect tuberculosis, tissue (polymerase chain reaction) was significantly better than tissue (histopathology) (28/341 vs 2/181, p < 0.00001) and pus (polymerase chain reaction) was significantly better than tissue (polymerase chain reaction) (19/115 vs 28/341, p < 0.0009). Tuberculosis was significantly more common in complex fistulas than in simple fistulas (20.3% vs 7.2%, p = 0.0002). The systematic review (n = 199) highlighted that tubercular fistulas are more common in recurrent and complex fistulas and in tuberculosis endemic regions., Limitations: The true sensitivity and specificity of each testing modality could not be determined because not all patients with tuberculosis fistula-in-ano were tested by every diagnostic modality studied., Conclusions: The tuberculosis detection rate of polymerase chain reaction was significantly higher than histopathology. Among polymerase chain reaction, pus had higher detection rate than tissue. Tuberculosis was associated with more complex and recurrent fistulas.

Objective: The aim of this study was to evaluate diagnostic practices for suspected community-acquired central nervous system (CNS) infection in an urban pediatric population., Methods: This is an observational, retrospective single-center review of cerebrospinal fluid (CSF) studies in children, 1 month to 21 years old, evaluated for suspected CNS infection from 2004 to 2014. Cases of suspected nosocomial meningitis were excluded. The frequency of N-methyl-D-aspartate receptor antibody (NMDAR ab) encephalitis was analyzed from 2010 to 2014., Results: A total of 940 unique patient visits with CSF studies were included in the final analysis. There were 940 bacterial cultures sent; 4 (0.42%) grew suspected CSF bacterial pathogens, and 18 (1.9%) grew organisms that were suspected contaminants. Bacterial pathogens included late-onset group B Streptococcus in 3 infants younger than 3 months and Streptococcus pneumoniae in an unvaccinated 9-year-old child. Viral CNS infection was 7.5 times more frequent than bacterial infection. Enterovirus was the only virus isolated. Five cases positive for NMDAR ab were identified since 2010., Conclusions: Bacterial studies were performed more frequently than viral and other studies. Cerebrospinal fluid bacterial culture was nearly 5 times more likely to yield a contaminant than a pathogen. The frequency of viral infection was likely underestimated as only 20% were tested, mainly by culture, which is suboptimal. These data suggest diagnostic practices for the evaluation of suspected community-acquired CNS infections in children need to be modified to reflect current epidemiology and highlight the need for greater accessibility to polymerase chain reaction for viral diagnostics. Furthermore, NMDAR ab-mediated encephalitis should be considered early in children presenting with suggestive symptoms.

Nucleic acid amplification tests are commonly used to diagnose Clostridioides difficile infection (CDI). Two-step testing with a toxin enzyme immunoassay is recommended to discriminate between infection and colonization but requires additional resources. Prior studies showed that PCR cycle threshold ( C T ) can predict toxin positivity with high negative predictive value. Starting in October 2016, the predicted toxin result (CT-toxin) based on a validated cutoff was routinely reported at our facility. To evaluate the clinical efficacy of this reporting, all adult patients with positive GeneXpert PCR results from October 2016 through October 2017 underwent a chart review to measure the recurrence of or conversion to a CT-toxin + result and 30-day all-cause mortality. There were 482 positive PCR tests in 430 unique patients, 282 CT-toxin + and 200 CT-toxin - Patient characteristics were similar at testing, though CT-toxin + patients had higher white blood cell (WBC) counts (12.5 × 10 3 versus 9.3 × 10 3 cells/μl; P = 0.001). All cases ( n  = 21) of fulminant CDI had a CT-toxin + result. Index CT-toxin + patients were significantly more likely to have a CT-toxin + result within 90 days than CT-toxin - patients (17.4% [ n  = 49] versus 8.0% [ n  = 16], respectively; P = 0.003). Thirty-day all-cause mortality was higher in CT-toxin - patients (11.1% versus 6.8%; P = 0.1), though no deaths in CT-toxin - patients were directly attributable to CDI. Of the 200 CT-toxin - patients, 51.5% ( n  = 103) were treated for CDI. The rates of conversion to a CT-toxin + result (8.8% versus 7.2%; P = 0.8) and all-cause mortality (8.8% versus 13.4%; P = 0.3) were similar between treated and untreated CT-toxin - patients, respectively. C T -based toxin prediction may identify patients at higher risk for CDI-related complications and reduce treatment among CT-toxin - patients., (Copyright © 2019 American Society for Microbiology.)

Consistent differences among melting curves of PCR-amplified DNA fragments are treated by normalizing the relative fluorescence units (RFU) and performing a clustering analysis, but statistically significant differences among curves are not usually determined. In the present study, an analysis based on functional data analysis (FDA) was implemented to evaluate the existence of statistically significant differences between normalized RFU curves obtained from PCR-HRM (high-resolution melting) analysis by using ANOVA for functional data. The effectiveness of the FDA method was analyzed with data from a set of samples of eight animal species of interest in food analysis, as well as mixtures of DNA from these species, analyzed by PCR-HRM to differentiate them. The statistical method described in this study has been demonstrated to be a robust and precise tool to discriminate among melting curves derived from HRM analysis. This method has advantages over the current comparison methods. PRACTICAL APPLICATION: As long as food fraud and mislabeling exist, new techniques for species identification are needed. High-resolution melting (HRM) has been shown to be a rapid, reliable and inexpensive species identification method. In the present study, functional data analysis (FDA) was applied to HRM curves of DNA from eight animal species used for food, as well as to mixtures of these species in different proportions. FDA has advantages over the usual methods, providing a deeper statistical analysis and facilitating the data interpretation as shown by the HRM analysis for a clearer comparison among individual species and mixtures of species., (© 2019 Institute of Food Technologists®.)

Introduction: Medical management of prosthetic joint infections (PJIs) relies on the identification of causative organisms through traditional culture-based approaches to guide therapy. However, diagnosis of many PJIs remains challenging, with many clinically apparent infections remaining culture-negative. Molecular diagnostics have the potential to increase diagnostic yield, particularly among culture-negative PJIs., Methods: Bone, tissue, or synovial fluid from patients with clinically identified PJIs were collected for inclusion in this study. Samples were assessed with traditional cultures and classified as culture-positive or -negative after 48 h. Samples subsequently underwent a Staphylococcus aureus -/ Kingella kingae -specific PCR followed by a 16s rRNA gene PCR., Results: A total of 77 unique patients with clinically identified PJIs contributed a total of 89 samples for inclusion in the study. There were 54 culture-negative and 35 culture-positive samples evaluated. The sensitivity and specificity of S. aureus PCR in culture-positive samples was 57.1% (95% CI, 34.1%-78.1%) and 92.9% (95% CI, 66.1%-98.9%), respectively. Among culture-positive samples, 16s rRNA gene PCR correctly identified 3 of 21 (14.3%) samples with S. aureus and 2 of 5 (40%) samples with Streptococcus spp. All molecular tests were negative in those with clinically identified, culture-negative PJI., Conclusions: Our study suggests that these diagnostic tools have a limited role in PJI diagnosis., (© 2018 American Association for Clinical Chemistry.)