Clinical performance of the STANDARD M10 SARS-CoV-2 rapid RT-PCR assay in patients visiting an emergency department.

In emergency departments, rapid screening of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was important for arranging limited isolation resources and patient care during the coronavirus disease 2019 (COVID-19) pandemic. STANDARD M10 SARS-CoV-2 (SD Biosensor) is a recently developed cartridge-based RT-PCR that provides a turnaround time of 1 h, which is shorter than that for conventional RT-PCR. This study evaluated the clinical performance of STANDARD M10 in patients visiting an emergency department. From March to June 2022, two specimens were collected from patients visiting an emergency department. Each specimen comprised one nasopharyngeal and one oropharyngeal swab. Respective specimens underwent rapid RT-PCR using STANDARD M10 and conventional RT-PCR using Allplex SARS-CoV-2 (Seegene). When discordant results occurred, specimens undergoing the STANDARD M10 were retested with the Allplex to exclude specimen variations. Retest results replaced initial results of the Allplex. Clinical performance of STANDARD M10 was compared with Allplex. The study enrolled 1971 patients. COVID-19 prevalence was 6.2% based on the Allplex. Compared with the Allplex, overall agreement, positive percent agreement, and negative percent agreement of STANDARD M10 were 99.5% (95% CI: 99.1%-99.8%), 95.9% (95% CI: 90.8%-98.3%), and 99.8% (95% CI: 99.4%-99.9%), respectively. Nine discordant results were all positive on droplet digital PCR, except for one specimen that was positive with STANDARD M10. The STANDARD M10 showed reliable diagnostic performance for detecting SARS-CoV-2 from patients visiting in emergency departments and is a useful tool in emergency healthcare systems because of its easy-to-use cartridge-based assay and short resulting time for detecting SARS-CoV-2.

Pre-Pandemic Distribution of Bacterial Species in Nasopharyngeal Swab Specimens from Pediatric and Adult Patients Detected via RT-PCR Using the Allplex Respiratory Panel.

Background: Recently, panel-based molecular diagnostics for the simultaneous detection of respiratory viruses and bacteria in nasopharyngeal swab (NPS) specimens have been highlighted. We identified the distribution of bacterial species in NPS specimens collected from pediatric and adult patients by employing RT-PCR (Allplex respiratory panel 4, RP4, Seegene) to estimate its applicability in a panel-based assay for detecting respiratory viruses. Methods: We used 271 and 173 NPS specimens from pediatric and adult patients, respectively. The results of the Allplex RP4 panel using NPS (NPS-RP4) from adult patients were compared with those of the Seeplex PneumoBacter ACE Detection assay (Seegene), which used sputum for testing (sputum-Seeplex). Results: A total of 147 specimens (54.2%) were positive for the NPS-RP4 panel in pediatric patients. There were 94, 77, 10, 3, 3, and 2 specimens that were positive for Haemophilus influenzae (HI), Streptococcus pneumoniae (SP), Mycoplasma pneumoniae (MP), Chlamydia pneumoniae (CP), Bordetella pertussis (BP), and B. parapertussis (BPP), respectively. Among 173 adult patients, 39 specimens (22.5%) were positive in the NPS-RP4. Thirty specimens were positive for HI, and 13 were positive for SP. One specimen tested positive for both MP and Legionella pneumophila (LP). CP, BP, and BPP results were all negative. However, 126 specimens (72.8%) had positive results with sputum-Seeplex (99 SP, 59 HI, three LP, and two MP), and the overall percentage of agreement between the two assays was 39.3% in the adult patients. Conclusions: Bacterial species in NPS from more than half of pediatric patients were detected. Performing the Allplex RP4 assay with NPS revealed additional respiratory bacteria that are not detected in current clinical practices, which do not include bacterial testing, demanding the use of sputum specimens. However, the use of NPS showed low agreement with standard assays using sputum in adult patients. Thus, more research is needed to develop a reliable RT-PCR method using NPS specimens in adult patients.

A Case of Marrow Fibrosis with Megakaryocytic Hyperplasia after Short-Term use of Granulocyte Colony-Stimulating Factor.

BACKGROUND: Administration of granulocyte colony-stimulating factors G-SCFs can cause diagnostic challenges because of morphologic alteration of hematopoietic cells. METHODS: We experienced a patient who showed distinctive clinical and morphologic findings after short time use of G-CSF. The clinical information and examination results of the morphology of bone marrow (BM) specimen and karyotype were analyzed by reviewing relevant literature. RESULTS: White blood cell (WBC) counts of the patient were unresponsive to G-CSF and marrow fibrosis and megakaryocytic hyperplasia were accompanied with increase of blasts in BM. Presence of malignant clones was confirmed by cytogenetic aberrations of monosomy 7. CONCLUSIONS: We concluded, BM study including cytogenetic analysis should be performed when such clinical findings are encountered and the possibility of hematologic malignancy should be considered.

Comparison of Throat and Nasopharyngeal Swabs for the Molecular Detection of Enterovirus in Pediatric Patients.

BACKGROUND: Enterovirus infections frequently occur in children worldwide. Molecular assays are widely used to detect enterovirus. Nasopharyngeal swabs (NPS) and throat swabs (TS) are common specimen types used in clinical practice. Here, the reliability of TS for detecting enterovirus in pediatric patients was compared with that of NPS using real-time reverse transcription polymerase chain reaction (RT-rPCR). METHODS: Results obtained using the Allplex Respiratory Panel 2 (Seegene, Korea) for NPS (NPS-RP) and Accu-Power EV Real-time RT-PCR (Bioneer, Korea) for TS (TS-EV), which were performed simultaneously between September 2017 to March 2020, were initially compared. Cross examination (Allplex Respiratory Panel 2 assay using TS and AccuPower EV assay with NPS) was performed for specimens collected between July 2019 to March 2020 to evaluate the performance of the enterovirus assays based on each specimen type. RESULTS: Among the 742 case results of initial tests, 597 cases (80.5%) tested negative in both assays, and 91 cases (12.6%) tested positive in both assays. Fifty-four discrepant results were observed: 39 cases (5.3%) tested positive in TS-EV and negative in NPS-RP, and 15 cases (2.0%) tested positive in NPS-RP and negative in TS-EV. The overall percent agreement was 92.7%. In the 99 cases cross examined, overall percent agreements were 98.0%, 94.9%, 92.9%, and 89.9% for TS-EV vs. TS-RP, NPS-RP vs. NPS-EV, TS-EV vs. NPS-EV, and NPS-RP vs. TS-RP, respectively. CONCLUSIONS: TS yields a high agreement rate with NPS in detecting enterovirus, regardless of single-plex or multiplex RT-rPCR assays. Thus, TS could be a good alternative specimen in pediatric patients who are reluctant to NPS sampling.

Changing Epidemiology of Pathogenic Bacteria Over the Past 20 Years in Korea.

BACKGROUND: The epidemiology of pathogenic bacteria varies according to the socioeconomic status and antimicrobial resistance status. However, longitudinal epidemiological studies to evaluate the changes in species distribution and antimicrobial susceptibility of pathogenic bacteria nationwide are lacking. We retrospectively investigated the nationwide trends in species distribution and antimicrobial susceptibility of pathogenic bacteria over the last 20 years in Korea. METHODS: From 1997 to 2016, annual cumulative antimicrobial susceptibility and species distribution data were collected from 12 university hospitals in five provinces and four metropolitan cities in South Korea. RESULTS: The prevalence of Staphylococcus aureus was the highest (13.1%) until 2012 but decreased to 10.3% in 2016, consistent with the decrease in oxacillin resistance from 76.1% in 2008 to 62.5% in 2016. While the cefotaxime resistance of Escherichia coli increased from 9.0% in 1997 to 34.2% in 2016, E. coli became the most common species since 2013, accounting for 14.5% of all isolates in 2016. Pseudomonas aeruginosa and Acinetobacter baumannii rose to third and fifth places in 2008 and 2010, respectively, while imipenem resistance increased from 13.9% to 30.8% and 0.7% to 73.5% during the study period, respectively. Streptococcus agalactiae became the most common pathogenic streptococcal species in 2016, as the prevalence of Streptococcus pneumoniae decreased since 2010. During the same period, pneumococcal penicillin susceptibility decreased to 79.0%, and levofloxacin susceptibility of S. agalactiae decreased to 77.1% in 2016. CONCLUSION: The epidemiology of pathogenic bacteria has changed significantly over the past 20 years according to trends in antimicrobial resistance in Korea. Efforts to confine antimicrobial resistance would change the epidemiology of pathogenic bacteria and, consequently, the diagnosis and treatment of infectious diseases.

Effect of changes in lymphocyte subsets at diagnosis in acute myeloid leukemia on prognosis: association with complete remission rates and relapse free survivals.

We prospectively investigated whether the characteristics of lymphocyte subsets at diagnosis in acute myeloid leukemia (AML) patients are different from healthy controls and affect treatment outcomes. A total of 91 AML patients classified into 3 genetic risk subgroups (favorable/intermediate/poor) according to 2022 NCCN guidelines were enrolled. We measured lymphocyte subsets by flow cytometry with peripheral blood samples at diagnosis and compared results with healthy controls. Influences of lymphocyte subsets on complete remission (CR) rates and survivals were also evaluated. AML patients had significantly lower numbers and proportions of CD56dimCD16+ natural killer (NK) cells, central memory T cells, and regulatory T cells than healthy controls. Higher proportion of helper/inducer T cells, CD4+CD31+ naïve T cells, and decreased proportion of NK cells significantly increased CR rates in 65 non-promyelocytic leukemia patients (P = 0.034, 0.027, and 0.019, respectively), and it was also significant in multivariable analysis with age/risk adjusted (P = 0.014, 0.016, and 0.045, respectively). NK cells < 4.8% of lymphocytes demonstrated significantly shorter relapse free survivals (RFS) in both univariate and multivariate analyses with risk adjusted (P = 0.006 and 0.037, respectively). AML patients showed significant lower numbers of CD56dimCD16+ NK cells, central memory T cells, and regulatory T cells than healthy controls at diagnosis. Higher proportion of helper/inducer T cells and CD4+CD31+ naïve T cells and decreased proportion of NK cells at diagnosis were independent factor of increasing probability of CR, and proportion of NK cells < 4.8% at diagnosis had adverse impact in RFS.

Relationship between the appropriateness of antibiotic treatment and clinical outcomes/medical costs of patients with community-acquired acute pyelonephritis: a multicenter prospective cohort study.

BACKGROUND: Inappropriate use of antibiotics not only increases antibiotic resistance as collateral damage but also increases clinical failure rates and medical costs. The purpose of this study was to determine the relationship between the appropriateness of antibiotic prescription and outcomes of community-acquired acute pyelonephritis (CA-APN). METHODS: A multicenter prospective cohort study was conducted at eight hospitals in Korea between September 2017 and August 2018. All hospitalized patients aged ≥ 19 years who were diagnosed with CA-APN on admission were recruited. The appropriateness of empirical and definitive antibiotics, as well as the appropriateness of antibiotic treatment duration and route of administration, was evaluated in accordance with the guideline and expert opinions. Clinical outcomes and medical costs were compared between patients who were administered antibiotics 'appropriately' and 'inappropriately.' RESULTS: A total of 397 and 318 patients were eligible for the analysis of the appropriateness of empirical and definitive antibiotics, respectively. Of them, 10 (2.5%) and 18 (5.7%) were administered 'inappropriately' empirical and definitive antibiotics, respectively. Of the 119 patients whose use of both empirical and definitive antibiotics was classified as 'optimal,' 57 (47.9%) received antibiotics over a longer duration than that recommended; 67 (56.3%) did not change to oral antibiotics on day 7 of hospitalization, even after stabilization of the clinical symptoms. Patients who were administered empirical antibiotics 'appropriately' had shorter hospitalization days (8 vs. 10 days, P = 0.001) and lower medical costs (2381.9 vs. 3235.9 USD, P = 0.002) than those who were administered them 'inappropriately.' Similar findings were observed for patients administered both empirical and definitive antibiotics 'appropriately' and those administered either empirical or definitive antibiotics 'inappropriately'. CONCLUSIONS: Appropriate use of antibiotics leads to better outcomes, including reduced hospitalization duration and medical costs.

Changes in the characteristics of community-onset fluoroquinolone-resistant Escherichia coli isolates causing community-acquired acute pyelonephritis in South Korea.

PURPOSE: This study aimed to examine the changes in the characteristics of community-onset fluoroquinolone-resistant (FQ-R) Escherichia coli isolates causing community-acquired acute pyelonephritis (APN) in South Korea. METHODS: Blood or urine samples were prospectively collected from patients aged ≥15 years with community-acquired APN who were admitted to one of the eight Korean hospitals included in this study between September 2017 and August 2018. Phylogenetic typing, multilocus sequence typing, and molecular characterization of ß-lactamase resistance and plasmid-mediated quinolone resistance (PMQR) determinants were performed. The data were compared with those from a previous study with the same design conducted in 2010-2011. RESULTS: A total of 300 and 346 isolates were identified in 2010-2011 and 2017-2018, respectively. Among them, 76 (22.0%) and 77 (25.7%) FQ-R isolates were identified in 2010-2011 and 2017-2018, respectively. A significantly higher antimicrobial resistance against third-to fourth-generation cephalosporins, including cefotaxime (23.9% vs. 77.9%, P < 0.001), were observed among FQ-R isolates in 2017-2018 than among those in 2010-2011. A higher proportion of ST131 isolates (27.6% vs. 66.2%, P < 0.001), as well as isolates that had extended-spectrum ß-lactamase (ESBL)/plasmid-mediated AmpC ß-lactamase (PABL) (23.7% vs. 79.2%, P < 0.001), was observed in 2017-2018 than in 2010-2011. Further, more PMQR determinants (11.8% vs. 40.8%, P < 0.001) were observed in 2017-2018 than in 2010-2011. CONCLUSIONS: Among uropathogenic FQ-R E. coli isolates in South Korea, the prevalence of ST131 and the proportion of isolates containing ESBL and/or PMQR determinants have increased.

Genetic Relatedness of 5-Year Isolates of Clostridioides difficile Polymerase Chain Reaction Ribotype 017 Strains in a Hospital.

The objective of this study was to analyse the genetic relatedness of Clostridioides difficile polymerase chain reaction ribotype 017 (RT017) strains from patients with hospital-acquired C. difficile infection (HA-CDI) in a hospital with a high RT017 prevalence. From 2009 to 2013, 200 RT017 strains (26.8%) were collected from 745 HA-CDI patient isolates. They comprised 64 MLVA types, and 197 (98.5%) strains were genetically related to 5 clonal complexes (CCs). The largest cluster, CC-A, included 163 isolates of 40 MLVA types. CC-A accounted for 20% of RT017 strains in 2009 and sharply increased to 94.9% in 2010, 94% in 2011, 86.2% in 2012, and 73.5% in 2013. The other 4 CCs included 20 isolates with 7 MLVA types. The resistance rates of antimicrobials were as follows: clindamycin 100%, moxifloxacin 99%, rifaximin 88.5%, and vancomycin 1%. All isolates were susceptible to metronidazole and piperacillin/tazobactam. Comparing antibiotic resistance among CCs, the geometric mean of the minimum inhibitory concentrations of moxifloxacin, vancomycin, and piperacillin/tazobactam were significantly higher for CC-A isolates than for the other CCs. RT017 clones constantly evolved over the 5 years studied with regard to genetic relatedness. The levels of antibiotic resistance may contribute to the persistence of organisms in the institution.

Increased Monocytic Myeloid-Derived Suppressor Cells in Whole Blood Predict Poor Prognosis in Patients with Plasma Cell Myeloma.

Myeloid-derived suppressor cells (MDSCs) are heterogeneous populations of immature myeloid cells with immunosuppressive effects that have prognostic potential in patients with malignancies; however, survival analysis studies are sparse. In this study, the prognostic implication of MDSCs was investigated in peripheral blood (PB) and bone marrow (BM) samples from 81 patients with plasma cell myeloma at diagnosis. MDSCs were quantified as monocytic MDSCs (mMDSCs) (CD11b+HLA-DR-/lowCD14+) and granulocytic MDSCs with neutrophils (gMDSCs-N) (CD11b+HLA-DR-/lowCD14-CD33+CD15+). Serum creatinine and lactate dehydrogenase levels showed a moderate correlation with all MDSC types, except BM-gMDSCs-N; mMDSCs correlated with serum ß2-microglobulin level, and PB-mMDSCs showed an inverse correlation with hemoglobin. PB-mMDSC levels were significantly higher in patients with progressive disease than those in patients at diagnosis and complete response. BM-mMDSC levels in patients with progressive disease were also higher than those in patients at diagnosis. Patients with high mMDSCs showed significantly poorer prognosis than patients with low mMDSCs. Multivariate analysis showed high PB-mMDSCs (≥0.3%) as a significant adverse prognostic marker for overall survival. This study demonstrated the independent adverse prognostic impact of PB-mMDSCs in patients with myeloma. PB-mMDSC measurement using whole blood is readily accessible in clinical laboratories, and may be used as a prognostic marker in clinical practice.

Granulocytic and Monocytic Myeloid-Derived Suppressor Cells are Functionally and Prognostically Different in Patients with Chronic Myeloid Leukemia.

Myeloid-derived suppressor cells (MDSCs) represent phenotypically heterogeneous populations that suppress tumor-specific T-cell responses. MDSCs are produced from myeloid precursors in emergent states and are increased in several hematologic malignancies. We evaluated the differences in the levels and prognostic significance of MDSCs according to the clinical status of chronic myeloid leukemia (CML). The percentages and numbers of granulocytic (g)MDSCs and monocytic (m)MDSCs in peripheral blood (PB) and bone marrow (BM) aspirates were determined by five-color flow cytometry (HLA-DR/CD11b/CD15/CD33/CD14). The median BM-gMDSC% and PB-gMDSC% of the CML group were lower than those of the complete hematologic response (CHR) and control groups (P<0.001). In the CHR group, patients with major molecular response (MMR) showed higher median BM-gMDSC% than those without MMR (P=0.039). Conversely, the PB-mMDSC number of the CML group was higher than those of the CHR and control groups (P<0.001). Patients with high PB-gMDSC number exhibited superior survival to those with low PB-gMDSC number (P=0.021), and patients with high PB-mMDSC% showed inferior survival to those with low PB-mMDSC%, but there was no statistical significance (P=0.182). Increased gMDSCs at CHR may reflect non-leukemic granulopoiesis, and a high number of PB-gMDSCs suggests better prognosis in CML. However, mMDSCs may be associated with malignant conditions and poor prognosis.

Comparison of the clinical characteristics of community-acquired acute pyelonephritis between male and female patients.

BACKGROUND: Community-acquired acute pyelonephritis (CA-APN) is relatively rare in men. This study aimed to compare the clinical characteristics of CA-APN between male and female patients. METHODS: We prospectively collected the clinical and microbiological data of hospitalized CA-APN patients aged ≥19 years in South Korea from March 2010 to February 2011 in 11 hospitals and from September 2017 to August 2018 in 8 hospitals. Only the first episodes of APN of each patient during the study period were included. RESULTS: From 2010 to 2011, 573 patients from 11 hospitals were recruited, and from 2017 to 2018, 340 patients were recruited from 8 hospitals. Among them, 5.9% (54/913) were male. Male patients were older (66.0 ± 15.2 vs. 55.3 ± 19.0 years, P < 0.001), had a higher Charlson comorbidity index (1.3 ± 1.5 vs. 0.7 ± 1.2, P = 0.027), and had a higher proportion of structural problems in the urinary tract (40.7% vs. 6.1%, P < 0.001) than female patients. Moreover, the total duration of antibiotic treatment was longer (21.8 ± 17.8 d vs. 17.3 ± 9.4 d, P = 0.001) and the proportion of carbapenem usage was higher (24.1% vs. 9.5%, P = 0.001) in men than in women. Male patients were hospitalized for longer durations than female patients (median, 10 d vs. 7 d, P < 0.001). CONCLUSIONS: Male CA-APN patients were older and had more comorbidities than female CA-APN patients. In addition, male patients received antibiotic treatment for a longer duration than female patients.

Quantitative characterization of Clostridioides difficile population in the gut microbiome of patients with C. difficile infection and their association with clinical factors.

Objective was to analyse bacterial composition and abundance of Clostridioides difficile in gut microbiome of patients with C. difficile infection (CDI) in association with clinical characteristics. Whole metagenome sequencing of gut microbiome of 26 CDI patients was performed, and the relative abundance of C. difficile and its toxin genes was measured. Clinical characteristics of the patients were obtained through medical records. A strong correlation between the abundance of C. difficile and tcdB genes in CDI patients was found. The relative abundance of C. difficile in the gut microbiome ranged from undetectable to 2.8% (median 0.089). Patients with fever exhibited low abundance of C. difficile in their gut, and patients with fewer C. difficile organisms required long-term anti-CDI treatment. Abundance of Bifidobacterium and Bacteroides negatively correlated with that of C. difficile at the genus level. CDI patients were clustered using the bacterial composition of the gut: one with high population of Enterococcus (cluster 1, n = 12) and another of Bacteroides or Lactobacillus (cluster 2, n = 14). Cluster1 showed significantly lower bacterial diversity and clinical cure at the end of treatment. Additionally, patients with CDI exhibited increased ARGs; notably, blaTEM, blaSHV and blaCTX-M were enriched. C. difficile existed in variable proportion of the gut microbiome in CDI patients. CDI patients with Enterococcus-rich microbiome in the gut had lower bacterial diversity and poorer clinical cure.

Changes in Clinical Characteristics of Community-Acquired Acute Pyelonephritis and Antimicrobial Resistance of Uropathogenic Escherichia coli in South Korea in the Past Decade.

This study examined changes in the clinical characteristics of community-acquired acute pyelonephritis (CA-APN) in South Korea between the period 2010-2011 and 2017-2018. We recruited all CA-APN patients aged ≥19 years who visited eight hospitals in South Korea from September 2017 to August 2018, prospectively. Data collected were compared with those from the previous study in 2010-2012, with the same design and participation from 11 hospitals. A total of 617 patients were enrolled and compared to 818 patients' data collected in 2010-2011. Escherichia coli was the most common causative pathogen of CA-APN in both periods (87.3% vs. 86.5%, p = 0.680). E. coli isolates showed significantly higher antimicrobial resistance against fluoroquinolone (32.0% vs. 21.6%, p < 0.001), cefotaxime (33.6% vs. 8.3%, p < 0.001), and trimethoprim/sulfamethoxazole (37.5% vs. 29.2%, p = 0.013) in 2017-2018 than in 2010-2011. Total duration of antibiotic treatment increased from 16.55 ± 9.68 days in 2010-2011 to 19.12 ± 9.90 days in 2017-2018 (p < 0.001); the duration of carbapenem usage increased from 0.59 ± 2.87 days in 2010-2011 to 1.79 ± 4.89 days in 2010-2011 (p < 0.001). The median hospitalization was higher for patients in 2017-2018 than in 2010-2011 (9 vs. 7 days, p < 0.001). In conclusion, antimicrobial resistance of E. coli to almost all antibiotic classes, especially third generation cephalosporin, increased significantly in CA-APN in South Korea. Consequently, total duration of antibiotic treatment, including carbapenem usage, increased.

Evaluation of the performance of two real-time PCR assays for detecting Mycobacterium species.

BACKGROUNDS: Rapid discrimination between Mycobacterium tuberculosis (MTB) and nontuberculous mycobacteria (NTM) is critical for patient treatment and to avoid unnecessary expenditure on infection control. Because real-time PCR assays distinguish MTB from NTM, we evaluated the performance of two real-time PCR assays (AdvanSure and PowerChek). METHODS: This study used 143 DNA samples from respiratory specimens which were collected based on routine PCR results using Anyplex kit. A total of 87 positive samples (65 MTB and 22 NTM) and 56 negative samples were collected consecutively during 6 months and 1 month, respectively. The diagnostic performance of PCR assays (AdvanSure and PowerChek) was retrospectively analyzed based on the results of conventional mycobacterial tests and routine PCR assay. RESULTS: Based on culture results, the sensitivities/specificities of AdvanSure and PowerChek were 90.7%/87.6% and 92.6%/85.4%, respectively, for MTB detection. For PCR-positive specimens, the quantification cycle (Cq) values of smear-negative specimens were higher than those of the smear-positive specimens (P < 0.001). As expected, the two PCR assays had the same sensitivities for NTM detection, viz. 90.0%, and their specificities were 99.2% and 98.4%, respectively. The overall agreement rate between the three PCR assays was 96.5% for MTB and 97.9% for NTM. CONCLUSION: The sensitivities of PCR assays in our study might be overestimated, because this study enrolled relatively lower number of PCR-negative samples which potentially missed PCR-negative but culture-positive specimens. However, the two real-time PCR assays for detecting MTB and NTM perform equally well in relative performance evaluation and their Cq values can be considered suitable for predicting smear-positive specimens.

Prognostic Impact of Lymphocyte Subpopulations in Peripheral Blood after Hematopoietic Stem Cell Transplantation for Hematologic Malignancies.

BACKGROUND: We prospectively evaluated prognostic value of lymphocyte subpopulations in peripheral blood of allogeneic hematopoietic stem cell transplant (HSCT) recipients. METHODS: 113 allogeneic HSCT (47 sibling matched, 37 unrelated matched, 29 haploidentical)-performed patients diagnosed as AML (n = 66), ALL (n = 28), and MDS (n = 19) were prospectively enrolled. 14 lymphocyte subpopulations were quantified by flow cytometry of PB at specific time-points after HSCT, and their prognostic impacts were analyzed. RESULTS: At 1, 2, and 3 months post-HSCT, significant adverse impact on overall survival (OS) and/or event free survival (EFS) was exhibited by low levels of natural killer (NK) cells (≤32 and ≤90/µL at 1 and 2 months on OS and EFS); regulatory T cells (≤1/µL) on EFS at 2 months; and B cells (≤19 and ≤92/µL for OS and EFS at 3 months). At 12 months, low levels of T cells (≤1180/µL), helper/inducer (H/I) T cells (≤250/µL), cytotoxic/suppressor (C/S) T cells (≤541/µL), and NK cells (≤138/µL) were associated with significantly higher risk of relapse. Low levels of T cells (≤879/µL) and C/S T cells (≤541/µL), and high level of naïve thymic T cells (>115/µL) showed a significant association with poor OS; low levels of C/S T cells (≤541/µL) and NK cells (≤138/µL) showed a significant adverse impact on EFS. CONCLUSIONS: Low levels of NK cells, regulatory T cells, and B cells at early stage post-HSCT are adverse prognostic indicators. At late stage, low levels of T cells and their subpopulations, NK cells, and high level of naïve thymic T cells are adverse prognostic indicators. © 2017 International Clinical Cytometry Society.

Increased circulating plasma cells detected by flow cytometry predicts poor prognosis in patients with plasma cell myeloma.

BACKGROUND: Flow cytometry (FC) is a reliable tool for diagnosing and monitoring of plasma cell myeloma (PCM). Recent studies used FC for quantifying plasma cells (PCs) in peripheral blood (PB) using various panels, and an adverse prognostic effect of circulating PCs (cPCs) has been reported. We investigated the prognostic implication of cPCs quantified using a simple panel in patients with PCM. METHODS: Bone marrow (BM) and PB of 85 patients with PCM were analyzed by five-color FC at time of diagnosis. A serial gating strategy for quantification used CD38/CD138 to gate PCs in 100,000-200,000 acquired events, with subsequent gating for CD19, CD56, and CD45, to identify aberrant immunophenotypes. RESULTS: cPCs were observed in 74.1% patients (63/85, median 0.067% leukocytes). Patients were grouped based on a cPC cut-off level of 0.02% derived using the receiver operating characteristic curves. Compared with patients with cPCs < 0.02% (n = 28), those with cPCs ≥ 0.02% (n = 57) showed lower hemoglobin (P = 0.003) and platelets (P = 0.014), but higher calcium, M-protein and BM PCs (P = 0.013, 0.029, and P < 0.001, respectively). Survival analysis of 74 patients showed that cPCs ≥ 0.02% predicted shorter progression-free and overall survival (P = 0.001 and 0.013, respectively), and this negative prognostic impact was retained in multivariate analysis (P = 0.023). CONCLUSIONS: Flow cytometric quantification of cPCs using five surface antigens (CD138, CD38, CD56, CD19, and CD45) is a sensitive and simple method that can be used for assessing PCM prognosis; it would allow clinical laboratories to readily adopt a risk stratification strategy based on cPC levels in PCM patients. © 2017 International Clinical Cytometry Society.

Flow cytometric measurement of respiratory burst activity and surface expression of neutrophils for septic patient prognosis.

BACKGROUND: The hyperinflammation that begins with sepsis is essential for eradicating infection but also causes hypoperfusion and organ failure. To understand the innate immune status of septic patients, the functional and phenotypic changes of neutrophils during sepsis and their clinical implication were studied. METHODS: Seventy-four patients who were admitted to intensive care unit due to severe sepsis or septic shock were enrolled. Surface antigens of neutrophils (CD64, CD10, and CD16) were detected by flow cytometry. Respiratory burst activity (RBA) was measured by flow cytometry using 2',7'-dichlorofluorescein diacetate and phorbol-12-myristate-13-acetate. The parameters were serially examined at Days 1 and 8 in septic shock patients. RESULTS: High CD64 and low CD10 and CD16 on Day 1 was associated with sepsis severity (P = 0.003, 0.017, and 0.007, respectively). On Day 1, RBA and CD64 were higher in survivors than in nonsurvivors of septic shock patients (P = 0.012 and 0.027, respectively), and on Day 8, CD10 and CD16 were higher in survivors than in nonsurvivors (P = 0.019 and 0.036, respectively). High RBA and high CD64 on Day 1 showed low 28-day mortality in univariate analysis (P = 0.018 and 0.034, respectively). In multivariate analysis, RBA maintained statistical significance (P = 0.042) but CD64 revealed only a tendency (P = 0.064). CONCLUSIONS: Neutrophil surface antigen (CD64, CD10, and CD16) could reflect sepsis severity. High CD64 expression and high RBA at early phase of sepsis might be associated with better prognosis, whereas high expression of CD10 and CD16 at late phase of sepsis might be associated with better prognosis. © 2015 International Clinical Cytometry Society.

JAK2 V617F, MPL, and CALR Mutations in Korean Patients with Essential Thrombocythemia and Primary Myelofibrosis.

Mutations in the calreticulin gene, CALR, have recently been discovered in subsets of patients with essential thrombocythemia (ET) or primary myelofibrosis (PMF). We investigated Korean patients with ET and PMF to determine the prevalence, and clinical and laboratory correlations of CALR/JAK2/MPL mutations. Among 84 ET patients, CALR mutations were detected in 23 (27.4%) and were associated with higher platelet counts (P=0.006) and lower leukocyte counts (P=0.035) than the JAK2 V617F mutation. Among 50 PMF patients, CALR mutations were detected in 11 (22.0%) and were also associated with higher platelet counts (P=0.035) and trended to a lower rate of cytogenetic abnormalities (P=0.059) than the JAK2 V617F mutation. By multivariate analysis, triple-negative status was associated with shorter overall survival (HR, 7.0; 95% CI, 1.6-31.1, P=0.01) and leukemia-free survival (HR, 6.3; 95% CI, 1.8-22.0, P=0.004) in patients with PMF. The type 1 mutation was the most common (61.1%) type among all patients with CALR mutations, and tended toward statistical predominance in PMF patients. All 3 mutations were mutually exclusive and were never detected in patients with other myeloid neoplasms showing thrombocytosis. CALR mutations characterize a distinct group of Korean ET and PMF patients. Triple-negative PMF patients in particular have an unfavorable prognosis, which supports the idea that triple-negative PMF is a molecularly high-risk disease.