Detection of Plasmodium vivax using Automated Hematology Analyzer in the Korean Army.

BACKGROUND: This study aimed to evaluate whether our equation model developed from the Sysmex hematology analyzer can discriminate patients with Plasmodium vivax (P. vivax) infection from those with acute febrile illness (AFI) and healthy controls. Besides, we compared our model with the previously studied models. METHODS: A total of 312 blood samples were collected from the P. vivax, AFI, and healthy control groups. All samples were tested for routine complete blood count conducted by using a Sysmex XE-2100 or XE-5000 analyzer. We compared the reportable and research parameters generated from the Sysmex analyzer among the three groups. The selected parameters that showed a significant difference between the P. vivax and the other group were included in the logistic regression analysis to develop our model (N-OIpv model). Moreover, we analyzed the CBC data according to the previous models, such as the presence of abnormal blue coded events in the WBC/BASO scattergram called the observer-interpretation (OIpv) model, and the previous equation model (N-OD1pv model) developed by Campuzano-Zuluaga et al. Results: The N-OIpv model, which consists of three parameters, such as mean cell volume, plateletcrit, and Lymph-X, showed the best performance for detection of malaria (97.4% accuracy). Also, this model can increase the sensitivity by about 11.9% to 18.1% compared with the OIpv and N-OD1pv models, respectively. CONCLUSIONS: We concluded that the N-OIpv model using the Sysmex hematology analyzer is a useful diagnostic tool in the routine laboratory workup for malaria.

Comparison of three molecular diagnostic assays for SARS-CoV-2 detection: Evaluation of analytical sensitivity and clinical performance.

BACKGROUND: 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. METHODS: 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. RESULTS: Limits of detection ranged from 650 to 1300 copies/ml for rRT-PCR assays, and the mean differences in cycle threshold (Ct ) values of the two RNA controls were within 1.0 for each target in the rRT-PCR assays (0.05-0.73), without any prominent Ct value shift or dropouts in the SARS-COV-2 B.1.351 RNA control. Using the consensus criterion as the reference standard, 89 samples were positive, whereas 160 were negative. The overall clinical performance of rRT-PCR assays was comparable (sensitivity 98.88%-100%; specificity 99.38%-100%), whereas the sensitivities of each target gene were more variable. CONCLUSIONS: The 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.

Longitudinal Assessment of Antisevere Acute Respiratory Syndrome Coronavirus 2 Immune Responses for Six Months Based on the Clinical Severity of Coronavirus Disease 2019.

BACKGROUND: There is insufficient data on the longevity of immunity acquired after severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. METHODS: 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). The study population comprised asymptomatic (n = 14), symptomatic/nonpneumonic (n = 42), and pneumonic (n = 41) patients. RESULTS: The anti-SARS-CoV-2 immunoglobulin class G and neutralizing antibody (NAb) titers lasted until 6 months after diagnosis, with positivity rates of 66.7% and 86.9%, respectively. Older age, prolonged viral shedding, and accompanying pneumonia were more frequently found in patients with sustained humoral immunity. Severe acute respiratory syndrome coronavirus 2-specific T-cell response was strongly observed in pneumonic patients and prominent in individuals with sustained humoral immunity. CONCLUSIONS: In conclusion, most (>85%) patients carry NAb until 6 months after diagnosis of SARS-CoV-2 infection, providing insights for establishing vaccination strategies against COVID-19.

Application of a multiplex immunochromatographic assay for rapid identification of carbapenemases in a clinical microbiology laboratory: performance and turn-around-time evaluation of NG-test Carba 5.

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.

Comparison between a laser lancing device and lancet for capillary blood sampling, capillary blood hemoglobin measurement, and blood typing.

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 < .001) without any significant difference (p = .113) and a substantial agreement (κ = 0.654) for the identification of participants with a low Hb level (<12.5 g/dl). cHb levels were significantly higher than vHb levels with both lancing devices (mean differences: 0.27-0.43 g/dl). The results of blood typing using the laser lancing device showed 100% accuracy. Use of the laser lancing device showed significantly lower skin puncture pain scores (p < .001). CONCLUSION: Use of a laser lancing device for capillary Hb measurement and blood typing showed accurate results, with significantly reduced skin puncture pain. Laser lancing devices could be feasible for donor screening tests.

Genetic diversity and genetic relatedness in Plasmodium falciparum parasite population in individuals with uncomplicated malaria based on microsatellite typing in Eastern and Western regions of Uganda, 2019-2020.

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 < 0.05. Overall, multiplicity of infection (MOI) was 1.9 (95% CI 1.7-2.1), P = 0.01 that was similar between age groups (1.8 vs 1.9), P = 0.60 and regions (1.9 vs 1.8), P = 0.43 for the < 5 and ≥ 5 years and Eastern and Western regions, respectively. Genomic sequencing of the 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 < 0.05 respectively. Genetic relatedness analysis revealed small clusters of gene deleted parasites in Uganda, but no clustering with Eritrean parasites. 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.

An in vitro study on the differentiated metabolic mechanism of chloroquine-resistant Plasmodium falciparum using high-resolution metabolomics.

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.

Clinical Usefulness of LabChip Real-time PCR using Lab-On-a-Chip Technology for Diagnosing Malaria.

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.

Performance Evaluation of Biozentech Malaria Scanner in Plasmodium knowlesi and P. falciparum as a New Diagnostic Tool.

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<0.05) was observed between each method. The parasitemia analysis using this new diagnostic tool needs technical improvement, particularly in the differentiation of malaria species.

Molecular surveillance reveals the presence of pfhrp2 and pfhrp3 gene deletions in Plasmodium falciparum parasite populations in Uganda, 2017-2019.

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.

Clinical Significance of a High SARS-CoV-2 Viral Load in the Saliva.

BACKGROUND: Patients with coronavirus disease 2019 (COVID-19) can unknowingly spread the virus to several people during the early subclinical period. METHODS: We evaluated the viral dynamics in various body fluid specimens, such as nasopharyngeal swab, oropharyngeal swab, saliva, sputum, and urine specimens, of two patients with COVID-19 from hospital day 1 to 9. Additional samples of the saliva were taken at 1 hour, 2 hours, and 4 hours after using a chlorhexidine mouthwash. The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral load was determined by real-time reverse transcriptase polymerase chain reaction (rRT-PCR). RESULTS: SARS-CoV-2 was detected from all the five specimens of both patients by rRT-PCR. The viral load was the highest in the nasopharynx (patient 1 = 8.41 log10 copies/mL; patient 2 = 7.49 log10 copies/mL), but it was also remarkably high in the saliva (patient 1 = 6.63 log10 copies/mL; patient 2 = 7.10 log10 copies/mL). SARS-CoV-2 was detected up to hospital day 6 (illness day 9 for patient 2) from the saliva of both patients. The viral load in the saliva decreased transiently for 2 hours after using the chlorhexidine mouthwash. CONCLUSION: SARS-CoV-2 viral load was consistently high in the saliva; it was relatively higher than that in the oropharynx during the early stage of COVID-19. Chlorhexidine mouthwash was effective in reducing the SARS-CoV-2 viral load in the saliva for a short-term period.

Seroprevalence of Anti-SARS-CoV-2 Antibodies among Outpatients in Southwestern Seoul, Korea.

Serosurveillance studies reveal the actual disease burden and herd immunity level in the population. In Seoul, Korea, a cross-sectional investigation showed 0.07% anti-severe acute respiratory syndrome coronavirus-2 antibody seropositivity among 1,500 outpatients of the university hospitals. Low seroprevalence reflects well-implemented social distancing. Serosurveillance should be repeated as the pandemic progresses.

Discovery of metabolic alterations in the serum of patients infected with Plasmodium spp. by high-resolution metabolomics.

INTRODUCTION: Despite the advances in diagnosis and treatment, malaria has still not been eradicated. Metabolic interactions between the host and Plasmodium may present novel targets for malaria control, but such interactions are yet to be deciphered. An exploration of metabolic interactions between humans and two Plasmodium species by high-resolution metabolomics may provide fundamental insights that can aid the development of a new strategy for the control of malaria. OBJECTIVES: This study aimed at exploring the metabolic changes in the sera of patients infected with Plasmodium falciparum and Plasmodium vivax. METHODS: Uni- and multivariate metabolomic analyses were performed on the sera of four groups of patients, namely normal control (N, n = 100), P. falciparum-infected patients (PF, n = 21), P. vivax-infected patients (PV, n = 74), and non-malarial pyretic patients (Pyr, n = 25). RESULTS: Univariate and multivariate analyses of N, PF, and PV groups showed differential metabolic phenotypes and subsequent comparisons in pairs revealed significant features. Pathway enrichment test with significant features showed the affected pathways, namely glycolysis/gluconeogenesis for PF and retinol metabolism for PV. The metabolites belonging to the affected pathways included significantly low 2,3-diphosphoglycerate and glyceraldehyde-3-phosphate in the sera of PF. The sera of PV had significantly low levels of retinol but high levels of retinoic acid. CONCLUSION: Our study reveals metabolic alterations induced by Plasmodium spp. in human serum and would serve as a milestone in the development of novel anti-malarial strategies.

Systematic review of the status of pfhrp2 and pfhrp3 gene deletion, approaches and methods used for its estimation and reporting in Plasmodium falciparum populations in Africa: review of published studies 2010-2019.

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.

Clinical Performance of the AllplexTM Respiratory Panel 1 Test Compared to SimplexaTM Flu A/B and RSV for Detection of Influenza Virus and Respiratory Syncytial Virus Infection Including Their Subtyping.

OBJECTIVE: TheAllplexTM 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 panelshowed 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.

Comparison of three multiplex PCR assays for detection of respiratory viruses: Anyplex II RV16, AdvanSure RV, and Real-Q RV.

BACKGROUND: Due to its great sensitivity, the nucleic acid amplification test (NAAT) is widely used for detection of respiratory viruses (RV). However, few reports have described a direct comparison between multiplex RT-PCR assays for RV. The objective of this study was to perform a direct comparison of three multiplex RT-PCR assays for the detection of respiratory viruses. METHODS: A total of 201 respiratory samples (161 nasopharyngeal swab samples and 40 sputum samples) were tested with three commercial RV assays: Seegene Anyplex II RV16 (AP), LG AdvanSure RV (AD), and Biosewoom Real-Q RV (RQ). The additional tests for the discrepant results were conducted by repeat RV assay or monoplex PCR coupled direct sequencing. Data analysis using percent agreement, kappa, and prevalence-adjusted and bias-adjusted kappa (PABAK) values was performed for comparisons among the three RV assays. RESULTS: Of the 201 samples, AP, AD, and RQ detected 105 (52.2%), 99 (49.3%), and 95 (47.3%) positive cases respectively. The overall agreement, kappa, and PABAK values for the three assays ranged between 97%-98%, 0.76-0.86, and 0.93-0.96 respectively. The performance of the three assays was very similar, with 94%-100% agreement for all comparisons, each virus types. The additional testing of samples showed discrepant results demonstrating that AD assay had the highest rate of concordance with original results. CONCLUSIONS: We suggest that all multiplex assay would be suitable for the detection of for respiratory viruses in clinical setting.

Comparison between Saliva and Nasopharyngeal Swab Specimens for Detection of Respiratory Viruses by Multiplex Reverse Transcription-PCR.

Nasopharyngeal swabs (NPSs) are being widely used as specimens for multiplex real-time reverse transcription (RT)-PCR for respiratory virus detection. However, it remains unclear whether NPS specimens are optimal for all viruses targeted by multiplex RT-PCR. In addition, the procedure to obtain NPS specimens causes coughing in most patients, which possibly increases the risk of nosocomial spread of viruses. In this study, paired NPS and saliva specimens were collected from 236 adult male patients with suspected acute respiratory illnesses. Specimens were tested for 16 respiratory viruses by multiplex real-time RT-PCR. Among the specimens collected from the 236 patients, at least 1 respiratory virus was detected in 183 NPS specimens (77.5%) and 180 saliva specimens (76.3%). The rates of detection of respiratory viruses were comparable for NPS and saliva specimens (P = 0.766). Nine virus species and 349 viruses were isolated, 256 from NPS specimens and 273 from saliva specimens (P = 0.1574). Adenovirus was detected more frequently in saliva samples (P < 0.0001), whereas influenza virus type A and human rhinovirus were detected more frequently in NPS specimens (P = 0.0001 and P = 0.0289, respectively). The possibility of false-positive adenovirus detection from saliva samples was excluded by direct sequencing. In conclusion, neither of the sampling methods was consistently more sensitive than the other. We suggest that these cost-effective methods for detecting respiratory viruses in mixed NPS-saliva specimens might be valuable for future studies.

Naturally acquired humoral and cellular immune responses to Plasmodium vivax merozoite surface protein 8 in patients with P. vivax infection.

BACKGROUND: Thirty-one glycosylphosphatidylinositol (GPI)-anchored proteins of Plasmodium vivax, merozoite surface protein 1 (MSP1), MSP1 paralogue, MSP4, MSP5, MSP8, and MSP10 have been reported from homologs of Plasmodium falciparum by gene annotation with bioinformatics tools. These GPI-anchored proteins contain two epidermal growth factor (EGF)-like domains at its C-terminus. Here, P. vivax merozoite surface protein 8 (PvMSP8) are considered as potential targets of protective immunity. METHODS: Recombinant PvMSP8 (rPvMSP8) was expressed, purified, and used for the assessment of humoral and cellular immune responses in P. vivax-infected patients and immune mice. Moreover, the target epitope of ant-PvMSP8 antibodies and subcellular localization of PvMSP8 was also determined. RESULTS: The rPvMSP8 was successfully expressed and purified as soluble form as ~55 kDa. PvMSP8 was localized to the outer circle of pigments associated with the food vacuole. The rPvMSP8 protein had a high antigenicity (73.2% in sensitivity and 96.2% in specificity) in patients infected with P. vivax. IgG2 antibody subtype was the predominantly responses to this antigen. Antibody response to PvMSP8 increased up to day 7 and after that slightly decreased within a month. The longevity of anti-PvMSP8 antibody was stably sustained up to 12-year recovery patient samples. Most anti-PvMSP8 antibodies recognized two epitopes that were located outside the C-terminal EGF-like domain. The cellular immune response in P. vivax-exposed individuals produced high levels of IFN-γ and IL-10 upon PvMSP8 antigen stimulation in vitro. CONCLUSIONS: All data in this study suggest that PvMSP8 antigen has a potential to induce both humoral and cellular immune responses in patients with P. vivax infection. The subcellular localization of PvMSP8 confirmed that it was associated with the parasite food vacuole in blood-stage parasites. A further characterization of this protein will be useful for blood stage P. vivax vaccine development.

Performance evaluation of four rapid antigen tests for the detection of Respiratory syncytial virus.

Rapid identification of Respiratory syncytial virus (RSV) is important in the management of infected patients. Rapid diagnostic tests (RDT) are widely used for this purpose. This study aimed to evaluate the clinical performance of four RSV antigen tests including the BinaxNow RSV Card test, SD Bioline RSV test, BD Veritor RSV test, and Humasis RSV antigen test in comparison with real-time RT-PCR as the reference method. Nasopharyngeal swabs were collected from 280 patients with symptoms of lower respiratory tract infection and stored at -80°C. All swabs were tested for RSV using four rapid antigen tests and real time RT-PCR. The sensitivity of the BinaxNow RSV Card test, SD Bioline RSV test, BD Veritor RSV test, and Humasis RSV Antigen tests were 62.5%, 61.3%, 65.0%, and 67.5% for RSV A, and 61.3%, 65.0%, 61.3%, and 67.5% for RSV B compared to real time RT-PCR, respectively. The specificity of BD Veritor RSV test was 95.8% and those of the other three RDTs was 100%. Commercial RSV antigen detection assays are useful tools for the rapid diagnosis of RSV infection. However, confirmatory testing is always recommended. J. Med. Virol. 88:1720-1724, 2016. © 2016 Wiley Periodicals, Inc.

Combined Use of Malaria Antigen and Antibody Enzyme-Linked Immunosorbent Assay for Blood Screening of Plasmodium vivax in the Republic of Korea.

OBJECTIVE: To evaluate the clinical sensitivity and specificity of the newly developed Genedia malaria antigen enzyme-linked immunosorbent assay (ELISA) test and to evaluate the diagnostic efficiency of the combined use of the Genedia malaria antigen and antibody ELISA tests to detect Plasmodium vivax in blood samples. MATERIALS AND METHODS: In all, 1,070 samples were analyzed: 300 P. vivax-infected patients, 41 samples from posttreatment patients upon follow-up and 729 healthy volunteers. The Genedia malaria antigen ELISA test and the Genedia malaria antibody ELISA 2.0 test were evaluated and compared to polymerase chain reaction and microscopy. RESULTS: The Genedia malaria antigen ELISA test had a clinical sensitivity of 94.7% (284/300) and a clinical specificity of 99.3% (724/729). The Genedia malaria antibody ELISA 2.0 test had a clinical sensitivity of 94.0% (282/300) and a clinical specificity of 98.4% (717/729). The Genedia malaria antigen ELISA test was able to detect 13 confirmed P. vivax cases without antibodies against P. vivax, whereas the Genedia malaria antibody ELISA 2.0 test detected 11 confirmed P. vivax cases nonreactive to the Genedia malaria antigen ELISA test, and 25 cases from 41 follow-up samples nonreactive in the Genedia malaria antigen ELISA test. The combined Genedia malaria antigen and antibody ELISA 2.0 tests had a clinical sensitivity of 98.3% (295/300) and a clinical specificity of 97.9% (714/729). CONCLUSION: The combination of antigen and antibody ELISAs improved the diagnostic sensitivity in P. vivax-confirmed cases in the Republic of Korea.