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Coronavirus disease (COVID-19) caused by SARS-CoV-2 infection has been a global pandemic for more than two years, and it is important to quickly and accurately diagnose and isolate patients with SARS-CoV-2 infection. The BZ COVID-19 NALF Assay could sensitively detect SARS-CoV-2 from a nasopharyngeal swab because it adopts both a loop-mediated isothermal amplification and lateral flow immunochromatography technology. In this study, a total of 389 nasopharyngeal swab samples, of which 182 were SARS-CoV-2 PCR positive and 207 were negative samples, were recruited. Compared to the Allplex™ SARS-CoV-2 Assay, the BZ COVID-19 NALF Assay showed 95.05% sensitivity and 99.03% specificity for detecting SARS-CoV-2. The concordance rate between the BZ COVID-19 NALF Assay and Allplex™ SARS-CoV-2 Assay was 97.69%. The turnaround time of the BZ COVID-19 NALF Assay is only about 40~55 min. The BZ COVID-19 NALF Assay is an accurate, easy, and quick molecular diagnostic test compared to the conventional PCR test for detection of SARS-CoV-2. In addition, the BZ COVID-19 NALF Assay is thought to be very useful in small size medical facilities or developing countries where it is difficult to operate a clinical laboratory.

Chloroquine (CQ) is an important drug used therapeutically for treatment of malaria. However, due to limited number of studies on metabolic targets of chloroquine (CQ), it is difficult to attribute mechanisms underlying resistance associated with usage of this drug. The present study aimed to investigate the metabolic signatures of CQ-resistant Plasmodium falciparum (PfDd2) compared to CQ-sensitive Plasmodium falciparum (Pf3D7). Both Pf3D7 and PfDd2 were treated with CQ at 200 nM for 48 hr; thereafter, the harvested red blood cells (RBCs) and media were subjected to microscopy and high-resolution metabolomics (HRM). Glutathione, γ-L-glutamyl-L-cysteine, spermidine, inosine monophosphate, alanine, and fructose-1,6-bisphosphate were markedly altered in PfDd2 of RBC. In the media, cysteine, cysteic acid, spermidine, phenylacetaldehyde, and phenylacetic acid were significantly altered in PfDd2. These differential metabolic signatures related signaling pathways of PfDd2, such as oxidative stress pathway and glycolysis may provide evidence for understanding the resistance mechanism and pathogenesis of the CQ-resistant parasite.

The computer vision diagnostic approach currently generates several malaria diagnostic tools. It enhances the accessible and straightforward diagnostics that necessary for clinics and health centers in malaria-endemic areas. A new computer malaria diagnostics tool called the malaria scanner was used to investigate living malaria parasites with easy sample preparation, fast and user-friendly. The cultured Plasmodium parasites were used to confirm the sensitivity of this technique then compared to fluorescence-activated cell sorting (FACS) analysis and light microscopic examination. The measured percentage of parasitemia by the malaria scanner revealed higher precision than microscopy and was similar to FACS. The coefficients of variation of this technique were 1.2-6.7% for Plasmodium knowlesi and 0.3-4.8% for P. falciparum. It allowed determining parasitemia levels of 0.1% or higher, with coefficient of variation smaller than 10%. In terms of the precision range of parasitemia, both high and low ranges showed similar precision results. Pearson’s correlation test was used to evaluate the correlation data coming from all methods. A strong correlation of measured parasitemia (r2=0.99, P

As malaria remains a major health problem worldwide, various diagnostic tests have been developed, including microscopy-based and rapid diagnostic tests. LabChip real-time PCR (LRP) is a small and portable device used to diagnose malaria using lab-on-a-chip technology. This study aimed to evaluate the diagnostic performance of LRP for detecting malaria parasites. Two hundred thirteen patients and 150 healthy individuals were enrolled from May 2009 to October 2015. A diagnostic detectability of LRP for malaria parasites was compared to that of conventional RT-PCR. Sensitivity of LRP for Plasmodium vivax, P. falciparum, P. malariae, and P. ovale was 95.5%, 96.0%, 100%, and 100%, respectively. Specificity of LRP for P. vivax, P. falciparum, P. malariae, and P. ovale was 100%, 99.3%, 100%, and 100%, respectively. Cohen’s Kappa coefficients between LRP and CFX96 for detecting P. vivax, P. falciparum, P. malariae, and P. ovale were 0.96, 0.98, 1.00, and 1.00, respectively. Significant difference was not observed between the results of LRP and conventional RT-PCR and microscopic examination. A time required to amplify DNAs using LRP and conventional RT-PCR was 27 min and 86 min, respectively. LRP amplified DNAs 2 times more fast than conventional RT-PCR due to the faster heat transfer. Therefore, LRP could be employed as a useful tool for detecting malaria parasites in clinical laboratories.

Background Histidine-rich protein-2 (HRP2)-based rapid diagnostic tests (RDTs) are the only RDTs recommended for malaria diagnosis in Uganda. However, the emergence of Plasmodium falciparum histidine rich protein 2 and 3 (pfhrp2 and pfhrp3) gene deletions threatens their usefulness as malaria diagnostic and surveillance tools. The pfhrp2 and pfhrp3 gene deletions surveillance was conducted in P. falciparum parasite populations in Uganda. Methods Three-hundred (n = 300) P. falciparum isolates collected from cross-sectional malaria surveys in symptomatic individuals in 48 districts of eastern and western Uganda were analysed for the presence of pfhrp2 and pfhrp3 genes. Presence of parasite DNA was confirmed by PCR amplification of the 18s rRNA gene, msp1 and msp2 single copy genes. Presence or absence of deletions was confirmed by amplification of exon1 and exon2 of pfhrp2 and pfhrp3 using gene specific PCR. Results Overall, pfhrp2 and pfhrp3 gene deletions were detected in 29/300 (9.7%, 95% CI 6.6–13.6%) parasite isolates. The pfhrp2 gene was deleted in 10/300 (3.3%, 95% CI 1.6–6.0%) isolates, pfhrp3 in 9/300 (3.0%, 95% CI 1.4–5.6%) while both pfhrp2 and pfhrp3 were deleted in 10/300 (3.3%, 95% CI 1.6–6.0%) parasite isolates. Proportion of pfhrp2/3 deletions was higher in the eastern 14.7% (95% CI 9.7–20.0%) compared to the western region 3.1% (95% CI 0.8–7.7%), p = 0.001. Geographical location was associated with gene deletions aOR 6.25 (2.02–23.55), p = 0.003. Conclusions This is the first large-scale survey reporting the presence of pfhrp2/3 gene deletions in P. falciparum isolates in Uganda. Roll out of RDTs for malaria diagnosis should take into consideration the existence of pfhrp2/3 gene deletions particularly in areas where they were detected. Periodic pfhrp2/3 surveys are recommended to inform future decisions for deployment of alternative RDTs.

BackgroundHuman parainfluenza virus 3 (HPIV3) is a major respiratory pathogen that causes acute respiratory infections in infants and children. Since September 2021, an out-of-season HPIV3 rebound has been noted in Korea. The objective of this study was to analyze the molecular characteristics of the HPIV3 strains responsible for the outbreak in Seoul, South Korea.MethodsA total of 61 HPIV3-positive nasopharyngeal swab specimens were collected between October and November 2021. Using 33 HPIV3-positive specimens, partial nucleotide sequences of the HPIV3 hemagglutinin-neuraminidase (HN) gene were aligned with previously published HN gene sequences for phylogenetic and genetic distance (p-distance) analyses.ResultsPhylogenetic tree revealed that all Seoul HPIV3 strains grouped within the phylogenetic subcluster C3. However, these strains formed a unique cluster that branched separately from the C3a lineage. This cluster showed 99% bootstrap support with a p-distance < 0.001. Genetic distances within the other C3 lineages ranged from 0.013 (C3a) to 0.023 (C3c). Deduced amino acid sequences of the HN gene revealed four protein substitutions in Seoul HPIV3 strains that have rarely been observed in other reference strains: A22T, K31N, G387S, and E514K.ConclusionsPhylogenetic analysis of Seoul HPIV3 strains revealed that the strain belonged to a separate cluster within subcluster C3. Genetic distances among strains within subcluster C3 suggest the emergence of a new genetic lineage. The emergence of a new genetic lineage could pose a potential risk of a new epidemic. Further monitoring of the circulating HPIV3 strains is needed to understand the importance of newly discovered mutations.

Tuberculosis (TB) is one of the leading causes of infectious mortality from a single infectious agent, Mycobacterium tuberculosis (MTB). This study evaluated the performance of the newly developed BZ TB/NTM NALF assay, which integrated loop-mediated isothermal amplification and lateral flow immunochromatographic assay technologies, for the detection of MTB. A total of 80 MTB-positive samples and 115 MTB-negative samples were collected, all of which were confirmed by TB real-time PCR (RT-PCR) using either AdvanSureTM TB/NTM RT-PCR Kit or Xpert® MTB/RIF Assay. The performance of the BZ TB/NTM NALF assay was evaluated by calculating its sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) in comparison to those of the RT-PCR methods. Compared to the RT-PCR, the sensitivity, specificity, PPV, and NPV of BZ TB/NTM NALF assay were 98.7%, 99.1%, 98.7%, and 99.1%, respectively. The concordance rate between BZ TB/NTM NALF and RT-PCR was 99.0%. Rapid and simple detection of MTB is essential for global case detection and further elimination of TB. The performance of the BZ TB/NTM NALF Assay is acceptable with a high concordance with RT-PCR, indicating that it is reliable for use in a low-resource environment.

Influenza and coronaviruses cause highly contagious respiratory diseases that cause millions of deaths worldwide. Public health measures implemented during the current coronavirus disease (COVID-19) pandemic have gradually reduced influenza circulation worldwide. As COVID-19 measures have relaxed, it is necessary to monitor and control seasonal influenza during this COVID-19 pandemic. In particular, the development of rapid and accurate diagnostic methods for influenza and COVID-19 is of paramount importance because both diseases have significant public health and economic impacts. To address this, we developed a multi-loop-mediated isothermal amplification (LAMP) kit capable of simultaneously detecting influenza A/B and SARS-CoV-2. The kit was optimized by testing various ratios of primer sets for influenza A/B (FluA/FluB) and SARS-CoV-2 and internal control (IC). The FluA/FluB/SARS-CoV-2 multiplex LAMP assay showed 100% specificity for uninfected clinical samples and sensitivities of 90.6%, 86.89%, and 98.96% for LAMP kits against influenza A, influenza B, and SARS-CoV-2 clinical samples, respectively. Finally, the attribute agreement analysis for clinical tests indicated substantial agreement between the multiplex FluA/FluB/SARS-CoV-2/IC LAMP and commercial AllplexTM SARS-CoV-2/FluA/FluB/RSV assays.

Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infections and hospitalization in infants and young children. Here, we analyzed the genetic diversity of RSV using partial G gene sequences in 84 RSV-A and 78 RSV- B positive samples collected in Seoul, South Korea, for 10 consecutive years, from 2010 to 2019. Our phylogenetic analysis revealed that RSV-A strains were classified into either the ON1 (80.9%) or NA1 (19.0%) genotypes. On the other hand, RSV-B strains demonstrated diversified clusters within the BA genotype. Notably, some sequences designated as BA-SE, BA-SE1, and BA-DIS did not cluster with previously identified BA genotypes in the phylogenetic trees. Despite this, they did not meet the criteria for the assignment of a new genotype based on recent classification methods. Selection pressure analysis identified three positive selection sites (amino acid positions 273, 274, and 298) in RSV-A, and one possible positive selection site (amino acid position 296) in RSV-B, respectively. The mean evolutionary rates of Korean RSV-A from 1999 to 2019 and RSV-B strains from 1991 and 2019 were estimated at 3.51 × 10−3 nucleotides (nt) substitutions/site/year and 3.32 × 10−3 nt substitutions/site/year, respectively. The population dynamics in the Bayesian skyline plot revealed fluctuations corresponding to the emergence of dominant strains, including a switch of the dominant genotype from NA1 to ON1. Our study on time-scaled cumulative evolutionary analysis contributes to a better understanding of RSV epidemiology at the local level in South Korea.

An early and accurate diagnosis of Candida albicans is critical for the rapid antifungal treatment of candidemia, a mortal bloodstream infection. This study demonstrates viscoelastic microfluidic techniques for continuous separation, concentration, and subsequent washing of Candida cells in the blood. The total sample preparation system contains two-step microfluidic devices: a closed-loop separation and concentration device and a co-flow cell-washing device. To determine the flow conditions of the closed-loop device, such as the flow rate factor, a mixture of 4 and 13 μm particles was used. Candida cells were successfully separated from the white blood cells (WBCs) and concentrated by 74.6-fold in the sample reservoir of the closed-loop system at 800 μL/min with a flow rate factor of 3.3. In addition, the collected Candida cells were washed with washing buffer (deionized water) in the microchannels with an aspect ratio of 2 at a total flow rate of 100 μL/min. Finally, Candida cells at extremely low concentrations (Ct > 35) became detectable after the removal of WBCs, the additional buffer solution in the closed-loop system (Ct = 30.3 ± 1.3), and further removal of blood lysate and washing (Ct = 23.3 ± 1.6).

The accurate detection of anti-neutralizing SARS-CoV-2 antibodies can aid in the understanding of the development of protective immunity against COVID-19. This study evaluated the diagnostic performance of the RapiSure (EDGC) COVID-19 S1 RBD IgG/Neutralizing Ab Test. Using the 90% plaque reduction neutralization test (PRNT90) as a reference, 200 serum samples collected from 78 COVID-19-positive and 122 COVID-19-negative patients were divided into 76 PRNT90-positive and 124 PRNT90-negative groups. The ability of the RapiSure test to detect antibodies was compared to that of the STANDARD Q COVID-19 IgM/IgG Plus test and that of PRNT90. The positive, negative, and overall percent agreement between the RapiSure and STANDARD Q test was 95.7%, 89.3%, and 91.5%, respectively, with a Cohen’s kappa of 0.82. The RapiSure neutralizing antibody test results revealed a sensitivity of 93.4% and a specificity of 100% compared to the PRNT results, with an overall percent agreement of 97.5% and Cohen’s kappa of 0.95. The diagnostic performance of the RapiSure test was in good agreement with the STANDARD Q COVID-19 IgM/IgG Plus test and comparable to that of the PRNT. The RapiSure S1 RBD IgG/Neutralizing Ab Test was found to be convenient and reliable and, thus, can provide valuable information for rapid clinical decisions during the COVID-19 pandemic.

Currently, SARS-CoV-2 RNA detection using real-time reverse-transcription PCR (rRT-PCR) is the standard diagnostic test for COVID-19 infection. Various rRT-PCR assays are currently used worldwide, targeting different genes of the SARS-CoV-2. Here, we compared the analytical sensitivity and clinical performance (sensitivity and specificity) of Allplex SARS-CoV-2/FluA/FluB/RSV assay (Seegene), Standard M nCoV real-time detection kit (SD Biosensor), and U-TOP COVID-19 detection kit (Seasun Biomaterials) for SARS-CoV-2 detection.Two hundred and forty-nine nasopharyngeal swab samples were evaluated to compare the clinical performance of the rRT-PCR assays. For the analytical performance evaluation, two RNA controls with known viral loads-SARS-CoV-2 RNA control and SARS-COV-2 B.1.351 RNA control-were used to investigate the potential impact of SARS-CoV-2 variants, particularly the B.1.351 lineage.Limits of detection ranged from 650 to 1300 copies/ml for rRT-PCR assays, and the mean differences in cycle threshold (CThe three rRT-PCR assays showed comparable analytical sensitivity and clinical performance. The analytical and clinical sensitivities of each target gene were influenced more by the primer and probe design than the target gene itself.

To date, few studies have investigated the feasibility of the loop-mediated isothermal amplification (LAMP) assay for identifying pathogens in tissue samples. This study aimed to investigate the feasibility of LAMP for the rapid detection of methicillin-susceptible or methicillin-resistantTwenty tissue samples infected with either MSSA (n = 10) or MRSA (n = 10) were obtained from patients who underwent orthopedic surgery for suspected musculoskeletal infection between December 2019 and September 2020. DNA was extracted from the infected tissue samples using the bead-beating method. A multiplex LAMP assay was conducted to identify MSSA and MRSA infections. To recognize theThe LAMP result was positive for samples containing 10The results of this study suggest that the LAMP assay performed with tissue DNA samples can be a useful diagnostic method for the rapid detection of musculoskeletal infections caused by MSSA and MRSA.

Rapid and accurate measurement of SARS-CoV-2 neutralizing antibodies (nAbs) can aid in understanding the development of immunity against COVID-19. This study evaluated the diagnostic performance of a rapid SARS-CoV-2 nAb detection test called the BZ COVID-19 nAb test BZ-nAb (BZ-nAb; BioZentech). Using the 90% plaque-reduction neutralization test (PRNT-90) as a reference, 104 serum specimens collected from COVID-19-positive and -negative patients were grouped into 40 PRNT-90-positive and 64 PRNT-90-negative specimens. The performance of the BZ-nAb was compared with that of the cPass surrogate virus neutralization test (cPass sVNT; Genscript). The BZ-nAb showed a sensitivity ranging from 92.5%–95.0% and specificity ranging from 96.9%–100%, whereas cPass sVNT showed a sensitivity of 100% (95% confidence interval (CI) 90.5%–100%) and specificity of 98.4% (95% CI, 91.6%–100%). The dilution factor obtained with PRNT-90 showed a stronger correlation with the percent inhibition of cPass sVNT (r = 0.8660, p < 0.001) compared with the test and control line ratio (T/C ratio) of the BZ-nAb (r = −0.7089, p < 0.001). An almost perfect agreement was seen between the BZ-nAb and cPass sVNT results, with a strong negative correlation between the BZ-nAb T/C ratio and cPass sVNT percent inhibition (r = −0.8022, p < 0.001). In conclusion, the diagnostic performance of the BZ-nAb was comparable to that of the cPass sVNT, although the BZ-nAb had a slightly lower sensitivity.

We evaluated the performance of the STANDARD Q COVID/FLU Ag Combo test (Q Ag combo test) for the detection of SARS-CoV-2, influenza A, and influenza B using a single point-of-care device compared with real-time PCR. A total of 408 individuals, 55 positives with SARS-CoV-2, 90 with influenza A, 68 with influenza B, and 195 negatives for all viruses, participated. The Q Ag combo test demonstrated a high level of sensitivity of 92.73% and a specificity of 99.49% for the detection of SARS-CoV-2. When the number of days from symptom onset (DSO) was restricted to 0 < DSO ≤ 6, the sensitivity of the Q Ag combo test to detect SARS-CoV-2 was 100%, and when the Ct value of RdRp was ≤20, the sensitivity to detect SARS-CoV-2 was 93.10%. The Q Ag combo test results also demonstrated a sensitivity of 92.22% and a specificity of 100% for influenza A, a sensitivity of 91.18%, and a specificity of 99.49% for influenza B. The agreement analysis of the Q Ag combo test with the RT-PCR results demonstrated excellent outcomes, making it useful and efficient for the detection of SARS-CoV-2, influenza A, and influenza B.

Malaria, caused by the parasite Plasmodium and transmitted by mosquitoes, is an epidemic that mainly occurs in tropical and subtropical regions. As treatments differ across species of malarial parasites, there is a need to develop rapid diagnostic methods to differentiate malarial species. Herein, we developed a multiplex malaria Pan/Pf/Pv/actin beta loop-mediated isothermal amplification (LAMP) to diagnose Plasmodium spp., P. falciparum, and P. vivax, as well as the internal control (IC), within 40 min. The detection limits of the multiplex malaria Pan/Pf/Pv/IC LAMP were 1 × 102, 1 × 102, 1 × 102, and 1 × 103 copies/µL for four vectors, including the 18S rRNA gene (Plasmodium spp.), lactate dehydrogenase gene (P. falciparum), 16S rRNA gene (P. vivax), and human actin beta gene (IC), respectively. The performance of the LAMP assay was compared and evaluated by evaluating 208 clinical samples (118 positive and 90 negative samples) with the commercial RealStar® Malaria S&amp, T PCR Kit 1.0. The developed multiplex malaria Pan/Pf/Pv/IC LAMP assay showed comparable sensitivity (100%) and specificity (100%) with the commercial RealStar® Malaria S&amp, T PCR Kit 1.0 (100%). These results suggest that the multiplex malaria Pan/Pf/Pv/IC LAMP could be used as a point-of-care molecular diagnostic test for malaria.

Background Prompt and accurate identification of carbapenemase production is essential for appropriate treatment and infection control. NG-Test Carba 5 (termed herein “Carba 5”; NG Biotech, Guipry, France) is a multiplex immunochromatographic assay for the rapid phenotypic identification of five major carbapenemases (KPC, NDM, VIM, IMP, and OXA-48-like) from bacterial isolates. This study aimed to evaluate the diagnostic performance of Carba 5 and its impact on the turn-around-time in a clinical microbiology laboratory. Results Carba 5 was retrospectively evaluated using 78 carbapenemase producers and 23 non-carbapenemase producers confirmed by PCR and sequencing. The performance and time required for carbapenemase identification were prospectively evaluated using 47 carbapenem resistant Enterobacteriaceae isolates, and the results were compared to those obtained using Xpert Carba-R (Cepheid, Sunnyvale, CA, USA). For the bacterial isolates included in retrospective and prospective evaluation, the Carba 5 assay correctly identified 147 isolates except one isolate with a sensitivity of 99.13% (95% CI 95.25–99.98%) and specificity of 100% (95% CI 89.42–100%). The Carba 5 assay missed one VIM-1 among 13 VIM producers. The assay showed a sensitivity of 92.31% (95% CI 63.97–99.81%) for detecting VIM and 100% for detecting KPC, NDM, OXA-48-like, and IMP. Compared to the Xpert Carba-R assay, Carba 5 exhibited 100% agreement and was more time-efficient (median time 24 min vs. 1 h 11 min). Conclusions The Carba 5 assay has potential as an alternative to molecular methods for detecting major carbapenemases from bacterial isolates in a clinical microbiology laboratory. Compared to the Xpert Carba-R, Carba 5 turns out to be more affordable and time-efficient while showing a comparable performance, and may accelerate therapeutic and infection control decisions.

We report a case of cellulitis caused by a novel Cupriavidus species identified using whole-genome sequence analysis. Subcutaneous tissue biopsies from the left lower leg of a 67-year-old man who suffered from cellulitis were cultured. Round, convex, gray and non-hemolytic colonies were recovered after 72-h incubation. 16S rRNA sequence analysis showed 98.6% similarity with Cupriavidus basilensis DSM 11853(T) in the NCBI database and 99.9% similarity with C. basilensis KF708 in the EzBioCloud database. Genomic analysis using the MiSeq platform (Illumina, USA) and the TrueBac ID database (ChunLab, Korea) revealed that the average nucleotide identity (ANI) of this strain with C. basilensis DSM 11853(T) was 87.6%. The patient was treated with oral cefditoren pivoxil for 9 weeks. This study is the first to report cellulitis caused by Cupriavidus species strain J1218. (Ann Clin Microbiol 2019;22:105 -109)

Background Malaria rapid diagnostic tests based on histidine-rich protein-2 have played a vital role in improving malaria case management and surveillance particularly in Africa, where Plasmodium falciparum is predominant. However, their usefulness has been threatened by the emergence of gene deletion on P. falciparum histidine rich protein 2 (pfhrp2) and P. falciparum histidine rich protein 3 (pfhrp3). Use of standard and recommended methods is key for accurate investigation, confirmation and reporting of pfhrp2 and pfhrp3 gene deletion. Methods A systematic review was conducted to assess the status, methods and approaches that have been used for investigation, confirmation and reporting of pfhrp2 and pfhrp3 gene deletion in Africa. An online search was done using PubMed and MEDLINE Google Scholar for all articles published in English on pfhrp2/3 gene deletion in Africa. Relevant articles that met the inclusion criteria were summarized and assessed based on the protocol recommended by the World Health Organization for confirmation and reporting of pfhrp2/3 gene deletion. Results The search identified a total of 18 articles out of which 14 (77.7%) fulfilled the criteria for inclusion and were retained for review. The articles were distributed across 12 countries where the pfhrp2 and pfhrp3 gene deletion studies were conducted and reported. The level of pfhrp2/3 gene deletion across selected studies in Africa ranged from the highest 62% to the lowest 0.4%. There was wide variation in methods and approaches including study designs, size and sampling and whether both pfhrp2 and pfhrp3 double deletions or pfhrp2 single deletion were investigated, with a wide variation in laboratory methods. Conclusion Based on the review, there is evidence of the presence of pfhrp2/3 gene-deleted P. falciparum parasites in Africa. The approaches and methods used for investigation, confirmation and reporting of pfhrp2/3 deleted parasites have varied between studies and across countries. Countries that are considering plans to investigate, confirm and report pfhrp2/3 deletion should use recommended standard and harmonized methods to prevent unnecessary recommendations for costly switch of RDTs in Africa.

Background There are growing concerns regarding the spread of carbapenemase-producing organisms (CPOs) among patients in long-term care facilities (LTCFs) and hospitals in South Korea. We have established a screening protocol for the detection of CPOs in high-risk patients upon admission to intensive care units (ICUs). The diagnostic performance of the Xpert Carba-R assay was compared to that of rectal culture for CPO detection in high-risk patients upon ICU admission. Methods A total of 408 consecutive rectal swabs were obtained from December 2016 to December 2017. CPO screening was performed using the Xpert Carba-R assay (Cepheid, Sunnyvale, CA, USA). When a carbapenemase gene was detected, additional rectal swabs were incubated overnight and inoculated on chromID CARBA medium (bioMérieux, Marcy l’Etoile, France). Bacterial carbapenemase genes, including bla KPC, bla NDM, bla VIM, bla IMP-1, and bla OXA-48, were confirmed by conventional PCR. The diagnostic performance of the Carba-R assay was ascertained based on the culture results. Results The prevalence of CPO carriage was 7.4% according to the Carba-R assay and 3.7% according to rectal culture. The median Ct values of IMP-1 and KPC were significantly different (35.2 vs. 26.6, P = 0.0143). The overall sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of the Carba-R assay were 100.0% (95% confidence interval [CI], 78.2–100.0), 96.7% (94.4–98.2), 53.6% (40.4–66.4) and 100.0% (99.0–100.0), respectively. Conclusions We demonstrated the prevalence of CPO carriage in high-risk patients upon ICU admission and evaluated the diagnostic performance of the Carba-R assay. The combined use of the Xpert Carba-R assay and culture produces rapid and reliable results for the active surveillance of rectal CPO in ICU patients.

BACKGROUND Blood donor screening includes tests using capillary blood, which is usually obtained by finger pricking using a lancet; however, the lancet has some shortcomings, such as skin puncture pain and needle stick injury. Recently, laser lancing devices for finger-prick sampling have been developed. We compared capillary blood Hb (cHb) levels and blood typing results obtained using a laser lancing device with those obtained using a lancet. STUDY DESIGN AND METHODS cHb levels, blood typing results, and skin puncture pain scores were assessed in 191 participants. Finger-prick sampling was performed using LMT-1000 (LaMeditech, Seoul, Korea) and a lancet on the same finger on different hands. Paired venous Hb (vHb) levels were assessed in 103 participants using an automated hematology analyzer and compared with the cHb levels obtained using both lancing devices. RESULTS The paired cHb results obtained with the laser lancing device and lancet showed a strong correlation (r = 0.927, p

Background Genetic diversity and parasite relatedness are essential parameters for assessing impact of interventions and understanding transmission dynamics of malaria parasites, however data on its status in Plasmodium falciparum populations in Uganda is limited. Microsatellite markers and DNA sequencing were used to determine diversity and molecular characterization of P. falciparum parasite populations in Uganda. Methods A total of 147 P. falciparum genomic DNA samples collected from cross-sectional surveys in symptomatic individuals of 2–10 years were characterized by genotyping of seven highly polymorphic neutral microsatellite markers (n = 85) and genetic sequencing of the Histidine Rich Protein 2 (pfhrp2) gene (n = 62). ArcGIS was used to map the geographical distribution of isolates while statistical testing was done using Student's t-test or Wilcoxon's rank-sum test and Fisher’s exact test as appropriate at P ≤ 0.05. Results Overall, 75.5% (95% CI 61.1–85.8) and 24.5% (95% CI14.2–38.9) of parasites examined were of multiclonal (mixed genotype) and single clone infections, respectively. Multiclonal infections occurred more frequently in the Eastern region 73.7% (95% CI 48.8–89.1), P pfhrp2 exon2 revealed a high level of genetic diversity reflected in 96.8% (60/62) unique sequence types. Repeat type AHHAAAHHATD and HRP2 sequence Type C were more frequent in RDT−/PCR + samples (1.9% vs 1.5%) and (13% vs 8%), P Conclusion High level of genetic diversity of P. falciparum parasites reflected in the frequency of multiclonal infections, multiplicity of infection and variability of the pfhrp2 gene observed in this study is consistent with the high malaria transmission intensity in these settings. Parasite genetic analysis suggested spontaneous emergence and clonal expansion of pfhrp2 deleted parasites that require close monitoring to inform national malaria diagnosis and case management policies.

Alere i Influenza AB is an isothermal nucleic acid amplification-based integrated system used for detecting and differentiating between influenza virus A and influenza virus B. We evaluated the clinical performances of Alere i Influenza AB compared to that of real-time PCR, multiplex real-time PCR, and two rapid influenza diagnostic kits.Nasopharyngeal aspiration specimens (n=315) from patients with signs of acute respiratory infection were collected between 2015 and 2016. Samples were tested using real-time PCR, the multiplex RT-PCR Anyplex II RV16 Detection kit, Alere i Influenza AB, BD Veritor™ System Flu A+B, and the Sofia Influenza A+B Fluorescence Immunoassay. Positive influenza specimens detected by the Anyplex II RV16 Detection kit were tested by real-time PCR.Compared to that of multiplex RT-PCR (influenza A, n=88; influenza B, n=82; influenza-negative, n=145), the sensitivities of Alere i, Sofia, and Veritor for influenza A were 97.7%, 72.7%, and 71.6%, respectively, whereas for influenza B, the sensitivities were 96.3%, 80.4%, and 75.6%, respectively. The specificity of Alere i, Sofia, and Veritor was 100.0%.The clinical performance of Alere i Influenza AB is satisfactory, with the advantage of a significantly shorter test time than other molecular assays. It is suitable for point-of-care testing and rapid influenza diagnostic tests because of its high sensitivity and specificity.

Rapid diagnosis and parasitemia measurement is crucial for management of malaria. Microscopic examination of peripheral blood (PB) smears is the gold standard for malaria detection. However, this method is labor-intensive. Here, we aimed to develop a completely automated microscopic system for malaria detection and parasitemia measurement. The automated system comprises a microscope, plastic chip, fluorescent dye, and an image analysis program. Analytical performance was evaluated regarding linearity, precision, and limit of detection and was compared with that of conventional microscopic PB smear examination and flow cytometry. The automated microscopic malaria parasite detection system showed a high degree of linearity for Plasmodium falciparum culture (R2 = 0.958, p = 0.005) and Plasmodium vivax infected samples (R2 = 0.931, p = 0.008). Precision was defined as the %CV of the assay results at each level of parasitemia and the %CV value for our system was lower than that for microscopic examination for all densities of parasitemia. The limit of detection analysis showed 95% probability for parasite detection was 0.00066112%, and a high correlation was observed among all three methods. The sensitivity and specificity of the system was both 100% (n = 21/21) and 100% (n = 50/50), respectively, and the system correctly identified all P. vivax and P. falciparum samples. The automated microscopic malaria parasite detection system offers several advantages over conventional microscopy for rapid diagnosis and parasite density monitoring of malaria.

A newly identified coronavirus, designated as severe acute respiratory syndrome coronavirus 2 (SARS CoV-2), has spread rapidly from its epicenter in China to more than 150 countries across six continents. In this study, we have designed three reverse-transcription loop-mediated isothermal amplification (RT-LAMP) primer sets to detect the RNA-dependent RNA polymerase (RdRP), Envelope (E) and Nucleocapsid protein (N) genes of SARS CoV-2. For one tube reaction, the detection limits for five combination SARS CoV-2 LAMP primer sets (RdRP/E, RdRP/N, E/N, RdRP/E/N and RdRP/N/Internal control (actin beta)) were evaluated with a clinical nasopharyngeal swab sample. Among the five combination, the RdRP/E and RdRP/N/IC multiplex LAMP assays showed low detection limits. The sensitivity and specificity of the RT-LAMP assay were evaluated and compared to that of the widely used Allplex™ 2019-nCoV Assay (Seegene, Inc., Seoul, South Korea) and PowerChek™ 2019-nCoV Real-time PCR kit (Kogenebiotech, Seoul, South Korea) for 130 clinical samples from 91 SARS CoV-2 patients and 162 NP specimens from individuals with (72) and without (90) viral respiratory infections. The multiplex RdRP (FAM)/N (CY5)/IC (Hex) RT-LAMP assay showed comparable sensitivities (RdRP: 93.85%, N: 94.62% and RdRP/N: 96.92%) to that of the Allplex™ 2019-nCoV Assay (100%) and superior to those of PowerChek™ 2019-nCoV Real-time PCR kit (RdRP: 92.31%, E: 93.85% and RdRP/E: 95.38%).

Introduction The rapid and accurate diagnosis of tuberculosis (TB) is important to reduce morbidity and mortality rates and risk of transmission. Therefore, molecular detection methods such as a real-time PCR–based assay for Mycobacterium tuberculosis (MTB) have been commonly used for diagnosis of TB. Loop-mediated isothermal amplification (LAMP) assay was believed to be a simple, quick, and cost-effective isothermal nucleic acid amplification diagnostic test for infectious diseases. In this study, we designed an in-house multiplex LAMP assay for the differential detection of MTB and non-tuberculosis mycobacterium (NTM), and evaluated the assay using clinical samples. Material and methods For the multiplex LAMP assay, two sets of specific primers were designed: the first one was specific for IS6110 genes of MTB, and the second one was universal for rpoB genes of mycobacterium species including NTM. MTB was confirmed with a positive reaction with both primer sets, and NTM was identified with a positive reaction by only the second primer set without a MTB-specific reaction. Total 333 clinical samples were analyzed to evaluate the multiplex LAMP assay. Clinical samples were composed of 195 positive samples (72 MTB and 123NTM) and 138 negative samples. All samples were confirmed positivity or negativity by real-time PCR for MTB and NTM. Analytical sensitivity and specificity were evaluated for the multiplex LAMP assay in comparison with acid fast bacilli staining and the culture method. Results Of 123 NTM samples, 121 were identified as NTM and 72/72 MTB were identified as MTB by the multiplex LAMP assay. False negative reactions were seen only in two NTM positive samples with co-infection of Candida spp. All 138 negative samples were identified as negative for MTB and NTM. Analytical sensitivity of the multiplex LAMP assay was 100% (72/72) for MTB, and 98.4% (121/123) for NTM. And the specificity of assay was 100% (138/138) for all. Conclusions Our newly designed multiplex LAMP assay for MTB and NTM showed relatively good sensitivity in comparison with previously published data to detect isolated MTB. This multiplex LAMP assay is expected to become a useful tool for detecting and differentiating MTB from NTM rapidly at an acceptable sensitivity.

Background: Among major trauma patients in the emergency department (ED), the leading cause of morbidity and mortality is a hemorrhagic shock. The low oxygen flow with hypovolemia in trauma patients is believed to play a significant role. Hence, oxygen supply is essential in severe trauma patients with massive hemorrhage. This study aimed to investigate the effect of different oxygen environments (hypoxia 1%, normoxia 20%, hyperoxia 80% oxygen) and variable treatments such as pentoxifylline (PTX), glycerol, hypertonic saline (HTS), protease inhibitor and dexamethasone (DEXA) in macrophage and T cells. Methods: Nitric oxide synthase (iNOS) and macrophage migration inhibitory factor (MIF) were measured for macrophage. MIF, interleukin (IL)-2 and 8 were measured for T cells. MTT assay was done for measuring T cell viability. Coculture was done on T cells on top of macrophages and the same protocols were carried out. Results: PTX decreased iNOS expression mostly followed by Glycerol under hypoxia. Under the hyperoxia, PTX and other treatments decreased iNOS expressions. MIF expression was lowered with PTX under hypoxia. PTX, glycerol, HTS, and protease inhibitor were effective under hyperoxia. PTX increased T cell survival under hypoxia. Under the hyperoxia, IL-2 expressions were upregulated with PTX, Glycerol, and HTS. PTX and other treatments were effective for IL-8, too. Our results indicate that PTX, HTS, and other medications with oxygen showed an effect on macrophage and T cells under various insults and stimulations. Conclusion: Our study demonstrated potential usefulness in improving immune systems during severe inflammatory conditions similar to septic shock possibly caused by massive hemorrhage.

Background: Immunity is the state of having sufficient biological defenses to avoid infection, trauma, or other unwanted biological invasions. T-cells and macrophages play important role in cell-mediated immunity. Pentoxifylline (PTX) is known to decrease pro-inflammatory cytokine and tumor necrosis factor (TNF-α). However, the effect on the immune system is not known well. This study aims to investigate the effect of PTX on inflammation. Methods: THP-1 derived macrophages were incubated with lipopolysaccharide (LPS) and/or indicated concentration of PTX for 8hr and wash with PBS to eliminate the effect of LPS. In this media, Jurkat cells were plated into trans-well plate and co-culture was done at 12hr. The T cell viability was measured by MTT. Also, the expression of Interleukin-2 (IL-2) was analyzed by RT-PCR and western blots. Results: PTX restored the increased concentration of MIF, TLR4 protein level and mRNA expression of TLR4 in LPS stimulated THP-1 derived macrophages. However, PTX did not restore the decreased T cell proliferation with PGE2 in Jurkat cells. In the co-culture study, The T cell viability was decreased in the THP-1 derived macrophage cells stimulated with LPS. The additional PTX restored the T cell's viability. Besides, PTX restored the decrease in the IL-2 expression of Jurkat cells in the LPS stimulated THP-1 derived macrophages. Conclusion: LPS stimulated THP-1 derived macrophages inhibited the T cell viability in hyper-inflammation conditions. However, PTX restored the T cells viability with increase IL-2. PTX influenced the cell-cell interaction, therefore, had its immunomodulatory effects.

Background: There is insufficient data on the longevity of immunity acquired following severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. We aimed to evaluate the duration of SARS-CoV-2-specific humoral and cellular immunity according to the clinical severity of coronavirus disease 2019 (COVID-19). Methods: Convalescent blood was collected prospectively from 97 patients who had recovered from COVID-19. The level of humoral immunity to SARS-CoV-2 was measured using the anti-SARS-CoV-2 nucleocapsid immunoglobulin G (IgG) assay and the plaque reduction neutralization test with sera collected at three periods: within 8 weeks, at 9-20 weeks, and at 22-27 weeks after diagnosis. IFN-γ ELISpot assays were conducted using overlapping peptides covering spike, nucleocapsid, and membrane proteins of SARS-CoV-2. Findings: The study population comprised asymptomatic (n=14), symptomatic/non-pneumonic (n=42), and pneumonic (n=41) patients. The anti-SARS-CoV-2 IgG and neutralizing antibody (NAb) titers lasted until six months after diagnosis, with positivity rates of 66·7% and 86·9%, respectively. The level of humoral immunity at six months after diagnosis were significantly higher in the pneumonic group than in the symptomatic/non-pneumonic group. Older age, prolonged viral shedding and accompanying pneumonia were more frequently found in patients with sustained humoral immunity. SARS-CoV-2 specific T-cell response was strongly observed in pneumonic patients and prominent in individuals with sustained humoral immunity. Interpretation: Most (> 85%) patients carries NAb until six months after diagnosis of SARS-CoV-2 infection, providing insights for establishing vaccination strategies against COVID-19. Funding Statement: This study was supported by a grant of the Korea National Institute of Health, Korea Disease Control and Prevention Agency (project number: 2020-ER5314-00), Korea University Guro Hospital (Korea research-driven hospital) (No. O2001061), and the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (grant number: HI20C0452). Declaration of Interests: The authors declare no competing interests. Ethics Approval Statement: The study protocol was approved by the Institutional Review Board of the Korea University Guro Hospital (approval number: 2020GR0130). All participants provided written informed consent.

Objective: The AllplexTM Respiratory Panel 1 (ARP) is a new assay based on a real-time polymerase chain reaction (RT-PCR) for the detection of influenza A (Flu A), influenza B virus (Flu B), and respiratory syncytial virus (RSV), including subtyping by multiple detection temperature (MuDT) technology. We evaluated the performance of the Allplex Respiratory Panel compared to the SimplexaTM Flu A/B & RSV assay (SP) and other diagnostic tools. Materials and Methods: A total of 372 samples were collected from patients at the Korea University Guro Hospital in Seoul, Korea. All samples were tested for influenza virus and RSV by ARP, SP, and an in-house RT-PCR. Results: The sensitivity of ARP was 95.56, 100, and 95.24% for Flu A, Flu B, and RSV, respectively. The specificity of ARP was 100, 100, and 100% for Flu A, Flu B, and RSV, respectively. SP had sensitivities and specificities of 98.89 and 100% for Flu A, 100 and 100% for Flu B, and 100 and 100% for RSV. Conclusion: The Allplex panel showed high sensitivity, specificity, positive predictive, and negative predictive values for the detection of Flu A, Flu B, and RSV. This assay is fast and easy to perform because it takes only about 150 min and there is no need for post-PCR electrophoresis. The ARP can be used as a reliable and convenient assay in clinical laboratories.

BACKGROUND: Platelet function analysis is crucial in assessing the hemostatic status to evaluate congenital and acquired platelet function defects. The Anysis-200 analyzer is a new automated lab-on-a-chip-based platelet function analyzer. OBJECTIVE: We aimed to evaluate a new platelet function analyzing system, the Anysis-200 in comparison to the Platelet Function Analyzer (PFA)-200 in cardiac patients. METHODS: Citrated blood was collected from 174 patients who visited the Department of Cardiology. The Anysis-200 consists of two kits, the microchips with collagen and epinephrine-coated membrane (C/EPI) or adenosine diphosphate-coated membrane (C/ADP). Platelet clogging in the Anysis-200 is measured by the blood migration distance obtained by a camera, which is compatible with the closure time in the PFA-200. We performed Anysis-200 and PFA-200 analyzers simultaneously and compared the results. RESULTS: The sensitivity and specificity of the Anysis-200 C/EPI kit in comparison to the PFA-200 C/EPI kit were 63.41% and 91.43%, respectively. Regarding the C/ADP kit, the sensitivity and specificity of the Anysis-200 were 58.97% and 74.29%, respectively. The agreement rate between the Anysis-200 and PFA-200 for C/EPI was 83.35% and 70.14% for C/ADP. CONCLUSIONS: The Anysis-200, which applies a novel method to detect platelet clogging, has shown moderate to fair agreement with the PFA-200. This test is potentially useful for screening cardiac patients with an abnormal platelet function.

Severe fever with thrombocytopenia syndrome (SFTS) and scrub typhus are endemic zoonotic diseases that pose significant public health threats in East Asia. As these two diseases share common clinical features, as well as overlapping disease regions, it is difficult to differentiate between SFTS and scrub typhus. A multiplex reverse-transcription loop‑mediated isothermal amplification (RT-LAMP) assay was developed to detect large segments and GroES genes for SFTS virus (SFTSV) andOrientia tsutsugamushi(OT). The performance of the RT-LAMP assay was compared and evaluated with those of commercial PowerChek™SFTSV real-time PCR and LiliF™TSUTSU nested PCR for 23 SFTS and 12 scrub typhus clinical samples, respectively. The multiplex SFTSV/OT/Internal control (IC) RT-LAMP assay showed comparable sensitivity (91.3%) with that of commercial PowerChek™SFTSV Real-time PCR (95.6%) and higher sensitivity (91.6%) than that of LiliF™TSUTSU nested PCR (75%). In addition, the multiplex SFTSV/OT RT-LAMP assay showed 100% specificity and no cross-reactivity for blood from uninfected healthy patients and samples from patients infected with other fever viruses. Thus, the multiplex SFTSV/OT/IC RT-LAMP assay could serve as a useful point-of-care molecular diagnostic test for SFTS and scrub typhus.

Homogenizing organism distribution in sputum samples is an initial, yet crucial step to diagnose pulmonary diseases with the samples. Although a homogenization platform for a diagnostic system needs to be system-compatible and efficient, it is rarely implemented. Here, we present a pulsatile micromixer for sputum homogenization that is efficient and does not use dynamic external controllers. The device consists of three oscillator and one mixer units. The oscillator unit autonomously converts a constant input of water-head pressure to a pulsatile pressure, and the mixer unit utilizes this pressure to homogeneously mix two solutions. The mixing performance is analyzed with different oscillation frequencies and width of mixer membranes. The device implemented homogeneous micromixing with a 9 Hz pulse for a flow rate up to 350 μL/min and approximately 200 μL/min for 1 cP and 10 cP viscosity solutions, respectively. Importantly, the device demonstrated successful homogenization of sputum samples with cell viabilities similar to those of the standard method for the flow rate up to 100 μL/min, thereby exhibiting biocompatibility with clinical samples. Our device is potentially useful for various lab-on-a-chip applications that require the compatibility of system integration without sacrificing mixing efficiency.

Background Complete blood count (CBC) results play an important role in peripheral blood smear (PBS) examinations. Many descriptions in PBS reports may simply be translated from CBC parameters. We developed a computer program that automatically generates a PBS draft report based on CBC parameters and age- and sex-matched reference ranges. Methods The Java programming language was used to develop a computer program that supports a graphical user interface. Four hematology analyzers from three different laboratories were tested: Sysmex XE-5000 (Sysmex, Kobe, Japan), Sysmex XN-9000 (Sysmex), DxH800 (Beckman Coulter, Brea, CA, USA), and ADVIA 2120i (Siemens Healthcare Diagnostics, Eschborn, Germany). Input data files containing 862 CBC results were generated from hematology analyzers, middlewares, or laboratory information systems. The draft reports were compared with the content of input data files. Results We developed a computer program that reads CBC results from a data file and automatically writes a draft PBS report. Age- and sex-matched reference ranges can be automatically applied. After examining PBS, users can modify the draft report based on microscopic findings. Recommendations such as suggestions for further evaluations are also provided based on morphological findings, and they can be modified by users. The program was compatible with all four hematology analyzers tested. Conclusions Our program is expected to reduce the time required to manually incorporate CBC results into PBS reports. Systematic inclusion of CBC results could help improve the reliability and sensitivity of PBS examinations.

The objective of this study was to comparatively evaluate three commercial whole-blood platelet function analyzer systems: Platelet Function Analyzer-200 (PFA; Siemens Canada, Mississauga, Ontario, Canada), Multiplate analyzer (MP; Roche Diagnostics International Ltd., Rotkreuz, Switzerland), and Plateletworks Combo-25 kit (PLW; Helena Laboratories, Beaumont, TX, USA). Venipuncture was performed on 160 patients who visited a department of cardiology. Pairwise agreement among the three platelet function assays was assessed using Cohen's kappa coefficient and percent agreement within the reference limit. Kappa values with the same agonists were poor between PFA-collagen (COL; agonist)/adenosine diphosphate (ADP) and MP-ADP (-0.147), PFA-COL/ADP and PLW-ADP (0.089), MP-ADP and PLW-ADP (0.039), PFA-COL/ADP and MP-COL (-0.039), and between PFA-COL/ADP and PLW-COL (-0.067). Nonetheless, kappa values for the same assay principle with a different agonist were slightly higher between PFA-COL/ADP and PFA-COL/EPI (0.352), MP-ADP and MP-COL (0.235), and between PLW-ADP and PLW-COL (0.247). The range of percent agreement values was 38.7% to 73.8%. Therefore, various measurements of platelet function by more than one method were needed to obtain a reliable interpretation of platelet function considering low kappa coefficient and modest percent agreement rates among 3 different platelet function tests.

Many patients are admitted to the emergency department due to trauma. Patients with massive hemorrhage and respiratory failure can fall into hypovolemic shock. Thereafter, oxygen is an essential part of the treatment of trauma patients, but the mechanisms of its effects in the management of trauma patients remain unknown. Therefore, we conducted an experiment to apply hypoxia, hyperoxia, and other treatment with the goal of decreasing hypoxic neuronal cells damage, as reflected by cell survival, apoptosis, hydrogen peroxide (H2O2) production, and hypoxia-inducible factor 1α (HIF) expression in SH-SY5Y cells. Under hypoxic insults, cell survival percentages decreased and apoptosis was seen with increased necrotic cell death. High-pressure oxygen (80% O2) had no effect compared with normal-pressure oxygen (20% O2). After exposure to hypoxia, H2O2 production and levels of HIF significantly increased compared with normoxia. However, when pentoxifylline (PTX), steroid, and hypertonic saline (HTS) were added after exposure to hypoxic conditions, the production of H2O2 and HIF levels significantly decreased in the groups treated with PTX and HTS. That is, the neuroprotective effect of PTX and HTS alleviated the impacts of hypoxic insulted on neuronal cells.

We aimed to characterize the lineages of influenza B viruses obtained from clinical specimens during the 2007-2014 seasons in South Korea. RT-PCR for the partial hemagglutinin gene of influenza B virus was performed on laboratory-confirmed influenza B samples from the 2007-2008 season to 2013-2014 season. A phylogenetic tree was generated, and current influenza vaccine strains for the Northern Hemisphere were used as representative strains of Victoria and Yamagata lineages. A total of 571 influenza B virus sequences were analyzed. During the 2009-2010 season, most of the circulating influenza B viruses matched the vaccine strain; 91.0% (91/100) of viruses belonged to the Victoria lineage. In the 2007-2008, 2011-2012, and 2013-2014 seasons, co-circulation of each influenza B lineage was found with a match ratio to the vaccine strain of 53.2% (42/79), 40.9% (63/154), and 58.3% (134/230), respectively. Overall, 41.7% (238/571) of the circulating influenza B viruses belonged to the lineage mismatching the vaccine strain. During the seven influenza seasons, influenza B epidemics were substantial in four seasons in South Korea. Significant mismatches of the vaccine and lineage of the circulating influenza B viruses were found. The current trivalent influenza vaccine may not be fully suitable for effective protection against influenza B.

Blood coagulation tests are a critical part in diagnosis of patients with bleeding disorders and cardiovascular diseases. However, most conventional blood coagulation tests require large and expensive instruments and skilled personnel. Here, we present a simple and low-cost approach to evaluate blood coagulation using Lamb wave-based device. To assess coagulation, we monitored the acoustic streaming of 5 μm fluorescent particles suspended in citrated plasma. Using a Lamb wave, citrated blood plasma and coagulation reagents can be mixed in a few seconds and the viscosity of the droplet increases due to blood coagulation. Then, the acoustic streaming of particles eventually stops, at which the “coagulation time” is defined. As a preliminary test, coagulation time was reduced with increasing calcium concentration from 0 to 25 mM. Prothrombin time results from the Lamb wave-based device were validated by comparing with those of commercial instruments with the correlation coefficient (R2) as 0.9432. Using the Lamb wave for blood coagulation test, the temperature can be controlled as 37 °C simultaneously by modulating experimental conditions. Our device enables lithography-free, simple and low-cost measurement of blood coagulation, which can be applied to clinical settings and developing countries.

Acoustic manipulation of fluids and particles has gained much attention in microfluidics, owing to its advantages of non-invasive manipulation and low power consumption. Microchannel mixing has been limited to diffusion-based mixing, because of low Reynolds number that has a low mixing efficiency. In this study, we firstly introduce a conductive liquid-based focused surface acoustic wave (CL-FSAW) device for mixing. Using the concentrated acoustic force of CL-FSAW, rapid and efficient mixing of deionized water and fluorescent particle suspension was demonstrated in a microfluidic channel. Effects of the applied voltage and the flow rate on the mixing efficiencies were investigated. As the flow rate decreased or the applied voltage increased, the mixing efficiency increased. At 21 V, mixing efficiencies were higher than 90% at a flow rate lower than 120 μL min−1. In addition, our device was applied to silver nanoparticle synthesis at the optimal mixing condition (100 μL min−1 and 21 V).

Efficient mixing in a microfluidic system is difficult to achieve, since it is dependent solely on molecular diffusion due to low Reynolds number. In this paper, we newly propose a novel mixing technique using three-dimensional dual surface acoustic waves (3D-dSAWs) generated from two interdigitated transducers of top and bottom piezoelectric substrates. By using the 3D-dSAW, internal swirling in a single direction is induced, which can facilitate more efficient mixing of a fluorescent particle suspension and deionized water. Therefore, by using the 3D-dSAW mixer, higher efficiency mixing performance can be achieved compared to the single SAW mixer at the same flow rate and applied voltage. With the applied voltage of 14 V, 3D-dSAW mixing device could achieve 100% mixing efficiency at the flow rate of 50 μl/min, while the mixing efficiency of the traditional single SAW mixer was approximately 38%. Moreover, the throughput of 3D-dSAW mixing device could be enhanced up to 120 μl/min with the applied voltage of 18 V, at which the measured temperature was lower than 40 °C and the efficiency could reach approximately ∼95.6%.

To reduce the morbidity and mortality of candidemia patients through rapid treatment, the development of a simple, rapid molecular diagnostic method that is based on nucleic acid extraction and is superior to conventional methods for detecting Candida in the blood is necessary. We developed a multiplex Candida Pan/internal control (IC) loop-mediated isothermal amplification (LAMP) assay and a simple DNA extraction boiling protocol using Chelex-100 that could extract yeast DNA in blood within 20 min. The Chelex-100/boiling method for DNA extraction showed comparable efficiency to that of the commercial QIAamp UCP Pathogen Mini Kit using Candida albicans qPCR. In addition, the Candida Pan/IC LAMP assay showed superior sensitivity to that of general Candida Pan and species qPCRs against clinical DNA samples extracted with the QIAamp UCP Pathogen Mini Kit and Chelex-100/boiling method. The Candida Pan/IC LAMP assay followed by Chelex-100/boiling-mediated DNA extraction showed high sensitivity (100%) and specificity (100%) against clinical samples infected with Candida. These results suggest that the Candida Pan/IC LAMP assay could be used as a rapid molecular diagnostic test for candidemia.

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 (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 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.