Outbreak investigation of Serratia marcescens bloodstream infection in an obstetric ward for high-risk pregnant women.

BACKGROUND: Serratia marcescens is a gram-negative bacterium that is widespread in the environment. S. marcescens bacteremia can be fatal during pregnancy and cause persistent chorioamnionitis. This study reports an outbreak of Serratia marcescens bloodstream infection (BSI) among high-risk pregnant women in an obstetric ward. The purpose of this study is to report our experience with the usefulness of the ATP test in hospital environmental management and to confirm that bloodstream infections of patients with the same strain were correlated by WGS testing. METHODS: This retrospective study collected the data of inpatients with S. marcescens bacteremia in obstetric ward for high-risk pregnant women from August 22, 2021, to October 14, 2021. We performed: an adenosine triphosphate (ATP) bioluminescence test in the environment with a high-contact area; environmental culture; on-site monitoring and staff education; and whole-genome sequencing (WGS) to evaluate genetic relationships among S. marcescens isolates. RESULTS: S. marcescens BSI occurred in four consecutive patients. None of the patients had central venous catheters. An ATP bioluminescence test revealed that high-contact areas and areas for injection preparation were not clean (≥ 1000 relative light units). However, S. marcescens was not identified in the environmental cultures, likely due to intensive environmental cleaning and discarding of potentially contaminated specimens before the culture test. On-site monitoring and education were conducted for 1 month. There were no further reports of BSI until 6 months after the last patient was discharged. WGS performed on three isolates from three patients indicated that the isolated S. marcescens was likely from the same strain. CONCLUSIONS: We controlled an S. marcescens outbreak by improving environmental cleaning as well as education of and behavior changes in healthcare workers. Using the ATP bioluminescence test can provide feedback on environmental cleaning and education. WGS played a role in determining the spread of BSI caused by the same strain.

A DNA-derived phage nose using machine learning and artificial neural processing for diagnosing lung cancer.

There is a growing interest in electronic nose-based diagnostic systems that are fast and portable. However, existing technologies are suitable only for operation in the laboratory, making them difficult to apply in a rapid, non-face-to-face, and field-suitable manner. Here, we demonstrate a DNA-derived phage nose (D2pNose) as a portable respiratory disease diagnosis system requiring no pretreatment. D2pNose was produced based on phage colour films implanted with DNA sequences from mammalian olfactory receptor cells, and as a result, it possesses the comprehensive reactivity of these cells. The manipulated surface chemistry of the genetically engineered phages was verified through a correlation analysis between the calculated and the experimentally measured reactivity. Breaths from 31 healthy subjects and 31 lung cancer patients were collected and exposed to D2pNose without pretreatment. With the help of deep learning and neural pattern separation, D2pNose has achieved a diagnostic success rate of over 75% and a classification success rate of over 86% for lung cancer based on raw human breath. Based on these results, D2pNose can be expected to be directly applicable to other respiratory diseases.

Novel PKD1 Mutations in Patients with Autosomal Dominant Polycystic Kidney Disease.

OBJECTIVE: Autosomal dominant polycystic kidney disease (ADPKD) is the most common genetic kidney disease. Identifying mutated causative genes can provide diagnostic and prognostic information. In this study, we describe the clinical application of a next generation sequencing (NGS)-based, targeted multi-gene panel test for the genetic diagnosis of patients with ADPKD. METHODS: We applied genetic analysis on 26 unrelated known or suspected patients with ADPKD. A total of 10 genes related to cystic change of kidney were targeted. Detected variants were classified according to standard guidelines. RESULTS: We identified 19 variants (detection rate: 73.1%), including PKD1 (n = 18) and PKD2 (n = 1). Of the 18 PKD1 variants, 8 were novel. CONCLUSION: Multigene panel test can be a comprehensive tool in a clinical setting for genetic diagnosis of ADPKD. It allows us to identify clinically significant novel variants and confirm the diagnosis, and these objectives are difficult to achieve using conventional diagnostic tools.

Rufomycin Targets ClpC1 Proteolysis in Mycobacterium tuberculosis and M. abscessus.

ClpC1 is an emerging new target for the treatment of Mycobacterium tuberculosis infections, and several cyclic peptides (ecumicin, cyclomarin A, and lassomycin) are known to act on this target. This study identified another group of peptides, the rufomycins (RUFs), as bactericidal to M. tuberculosis through the inhibition of ClpC1 and subsequent modulation of protein degradation of intracellular proteins. Rufomycin I (RUFI) was found to be a potent and selective lead compound for both M. tuberculosis (MIC, 0.02 µM) and Mycobacterium abscessus (MIC, 0.4 µM). Spontaneously generated mutants resistant to RUFI involved seven unique single nucleotide polymorphism (SNP) mutations at three distinct codons within the N-terminal domain of clpC1 (V13, H77, and F80). RUFI also significantly decreased the proteolytic capabilities of the ClpC1/P1/P2 complex to degrade casein, while having no significant effect on the ATPase activity of ClpC1. This represents a marked difference from ecumicin, which inhibits ClpC1 proteolysis but stimulates the ATPase activity, thereby providing evidence that although these peptides share ClpC1 as a macromolecular target, their downstream effects are distinct, likely due to differences in binding.

T-Cell Receptor Rearrangements Determined Using Fragment Analysis in Patients With T-Acute Lymphoblastic Leukemia.

BACKGROUND: Chromosomal abnormalities and common genetic rearrangements related to T-acute lymphoblastic leukemia (T-ALL) are not clear. We investigated T-cell receptor (TCR) rearrangement in Korean T-ALL patients by fragment analysis, examining frequency, association between clinicopathologic characteristics and TCR clonality, and feasibility for detecting minimal residual disease (MRD). METHODS: In 51 Korean patients diagnosed as having T-ALL, TCR rearrangement was analyzed using the IdentiClone TCR gene clonality assay (InVivoScribe Technologies, San Diego, CA, USA) from archived bone marrow specimens. Limit of detection (LOD) and clonal stability at relapse were evaluated. The association between clinical prognosis and TCR clonality was examind by age and immunophenotypic classification. RESULTS: Thirty-eight patients (74.5%) had 62 clonal products of TCRß, TCRγ, and/or TCRδ rearrangements at diagnosis. Children with T-ALL (<12 years) showed a higher frequency of clonality (93.8%) than adolescents/adults (65.7%; ≥12 years). Patients with a mature immunophenotype (84.4%) showed a relatively higher frequency of clonality than those with the immature immunophenotype (57.9%). Survival and event-free survival were not influenced by immunophenotype or TCR clonality. The LOD was 1%. Clonal evolution at the relapse period was noted. CONCLUSIONS: The overall detection rate of TCR clonality was 74.5%. Survival did not differ by TCR clonality or immunophenotype and age group. Fragment analysis of TCR rearrangement cannot be used to assess MRD due to low sensitivity. Further research on the relationship between prognosis and frequency of TCR rearrangements is needed, using more sensitive methods to detect clonality and monitor MRD.

Increasing use of therapeutic apheresis as a liver-saving modality.

INTRODUCTION: Therapeutic plasma exchange (TPE) is used for temporary support of liver function in patients presenting with early graft dysfunction after liver transplantation (LT) or liver surgery. We analyzed the effect of therapeutic apheresis on patients with liver disease. METHODS: Between January 2011 and August 2016, 93 apheresis procedures were performed for 26 patients at our institution. Anti-ABO isoagglutination immunoglobulin (Ig) M titer was checked using a type A and type B 3% red blood cell (RBC) suspension in saline with two-fold serial dilutions of patient serum. Anti-ABO isoagglutination IgG titer was checked by a type A and B 0.8% RBC suspension using a low-ionic strength/Coombs card. RESULTS: ABO-incompatible (ABOi) LT was the most common (n=10, 38.5%) indication for apheresis; early graft dysfunction after LT (n=8, 30.7%) was the second most common. Median initial IgM and IgG anti-ABO titers for ABOi LT recipients were 1:16 (range, 1:8-1:128) and 1:48 (range, 1:8-1:2048). We performed preoperative TPE in 10 recipients (median number of sessions, 1.5; range, 1-11). Among patients with early graft dysfunction, those who underwent living donor LT had better survival (4/4; 100%) than those who underwent nonliving donor LT (0/3; 0%). Patients who underwent living donor LT first and then additional LT also survived after three TPE sessions. CONCLUSION: Therapeutic apheresis is associated with a good survival rate and is essential for liver support in patients with early graft dysfunction after LT or posthepatectomy liver failure and during preparation for ABOi LT.

Effective High-Throughput Blood Pooling Strategy before DNA Extraction for Detection of Malaria in Low-Transmission Settings.

In the era of (pre) elimination setting, the prevalence of malaria has been decreasing in most of the previously endemic areas. Therefore, effective cost- and time-saving validated pooling strategy is needed for detection of malaria in low transmission settings. In this study, optimal pooling numbers and lowest detection limit were assessed using known density samples prepared systematically, followed by genomic DNA extraction and nested PCR. Pooling strategy that composed of 10 samples in 1 pool, 20 µl in 1 sample, was optimal, and the parasite density as low as 2 p/µl for both falciparum and vivax infection was enough for detection of malaria. This pooling method showed effectiveness for handling of a huge number of samples in low transmission settings (<9% positive rate). The results indicated that pooling of the blood samples before DNA extraction followed by usual nested PCR is useful and effective for detection of malaria in screening of hidden cases in low-transmission settings.

Correlation of NPM1 Type A Mutation Burden With Clinical Status and Outcomes in Acute Myeloid Leukemia Patients With Mutated NPM1 Type A.

BACKGROUND: Nucleophosmin gene (NPM1) mutation may be a good molecular marker for assessing the clinical status and predicting the outcomes in AML patients. We evaluated the applicability of NPM1 type A mutation (NPM1-mutA) quantitation for this purpose. METHODS: Twenty-seven AML patients with normal karyotype but bearing the mutated NPM1 were enrolled in the study, and real-time quantitative PCR of NPM1-mutA was performed on 93 bone marrow (BM) samples (27 samples at diagnosis and 56 at follow-up). The NPM1-mutA allele burdens (represented as the NPM1-mutA/Abelson gene (ABL) ratio) at diagnosis and at follow-up were compared. RESULTS: The median NPM1-mutA/ABL ratio was 1.3287 at diagnosis and 0.092 at 28 days after chemotherapy, corresponding to a median log10 reduction of 1.7061. Significant correlations were observed between BM blast counts and NPM1-mutA quantitation results measured at diagnosis (γ=0.5885, P=0.0012) and after chemotherapy (γ=0.5106, P=0.0065). Total 16 patients achieved morphologic complete remission at 28 days after chemotherapy, and 14 (87.5%) patients showed a >3 log10 reduction of the NPM1-mutA/ABL ratio. The NPM1-mutA allele was detected in each of five patients who had relapsed, giving a median increase of 0.91-fold of the NPM1-mutA/ABL ratio at relapse over that at diagnosis. CONCLUSIONS: The NPM1-mutA quantitation results corresponded to BM assessment results with high stability at relapse, and could predict patient outcomes. Quantitation of the NPM1-mutA burden at follow-up would be useful in the management of AML patients harboring this gene mutation.

Risk-Reducing Genetic Variant of Wilms Tumor 1 Gene rs16754 in Korean Patients With BCR-ABL1-Negative Myeloproliferative Neoplasm.

The genetic variant rs16754 of Wilms tumor gene 1 (WT1) has recently been described as an independent prognostic factor in AML patients. It is of great interest to test whether WT1 single nucleotide polymorphism can be used as a molecular marker in other types of cancer, to improve risk and treatment stratification. We performed sequencing analysis of exons 7 and 9 of WT1, which are known mutational hotspots, in a total of 73 patients with BCR-ABL1-negative myeloproliferative neoplasm (MPN) and 93 healthy controls. No previously reported WT1 mutations were identified in the present study. In Korean patients with BCR-ABL1-negative MPN, WT1 genetic variant rs16754 had no significant impact on clinical outcomes. We observed a significant difference in the allelic frequencies of WT1 rs16754 in Koreans between BCR-ABL1-negative MPN cases and healthy controls. Individuals carrying variant G alleles of WT1 rs16754 showed a relatively low prevalence of BCR-ABL1-negative MPN, compared with those carrying wild A alleles of WT1 rs16754 (Hazard ratio 0.10-0.65, P<0.05). Therefore, possession of the variant G allele of WT1 rs16754 may reduce the risk of developing BCR-ABL1-negative MPN.

The first Korean case of Sphingobacterium spiritivorum bacteremia in a patient with acute myeloid leukemia.

Sphingobacterium spiritivorum has been rarely isolated from clinical specimens of immunocompromised patients, and there have been no case reports of S. spiritivorum infection in Korea to our knowledge. We report a case of S. spiritivorum bacteremia in a 68-yr-old woman, who was diagnosed with acute myeloid leukemia and subsequently received chemotherapy. One day after chemotherapy ended, her body temperature increased to 38.3℃. A gram-negative bacillus was isolated in aerobic blood cultures and identified as S. spiritivorum by an automated biochemical system. A 16S rRNA sequencing analysis confirmed that the isolate was S. spiritivorum. The patient received antibiotic therapy for 11 days but died of septic shock. This is the first reported case of human S. spiritivorum infection in Korea. Although human infection is rare, S. spiritivorum can be a fatal opportunistic pathogen in immunocompromised patients.

Clinical relevance of elevated levels of serum soluble interleukin-2 receptor alpha (sIL-2Rα) in patients with non-Hodgkin's lymphoma.

Levels of soluble interleukin-2 receptor alpha (sIL-2Rα) are known to increase in the sera of patients with certain malignancies, including malignant lymphoma. This study aimed to assess the clinical significance of the sIL-2Rα level in non-Hodgkin's lymphoma (NHL). We used ELISA to measure the sIL-2Rα levels in 48 newly diagnosed and untreated patients with NHL and evaluated the correlation between the sIL-2Rα levels and clinical characteristics and the International Prognostic Index (IPI). We monitored serum sIL-2Rα in 7 patients to compare the changes in their clinical progress with these levels. High levels of serum sIL-2Rα (≥ 2,000 U/mL) correlated well with parameters defining the high risk group according to the IPI, i.e., high tumor burden at diagnosis (stage III+IV) and lactate dehydrogenase ≥ 472 U/L. The levels were also associated with B symptoms, bone marrow involvement, and poor response to therapy. The sIL-2Rα level decreased during complete remission and was elevated during disease progression or relapse. A high level of sIL-2Rα was significantly associated with a low survival rate. These results suggest that serum sIL-2Rα might be useful as a biomarker for evaluating the prognosis of patients with NHL at the time of diagnosis and during therapy. A well-controlled, large-scale study is needed to clarify the clinical significance of sIL-2Rα in specific groups of NHL.

Impact of amino acid substitution at residue 9 of HLA-A2 on the development of acute GVHD in Korean pediatric patients receiving unrelated hematopoietic stem cell transplantation.

Incompatibility of human leukocyte antigen (HLA) alleles between donors and recipients of unrelated hematopoietic stem cell transplantation (UHSCT) increases the risk of acute graft-versus-host disease (GVHD). We evaluated the positional effect of amino acid substitutions in HLA molecules on severe acute GVHD in Korean pediatric recipients of UHSCT. All of 64 donor-recipient pairs were serologically matched for HLA-A, -B, and -DR loci. Only substitution at residue 9 resulting from an HLA-A*02 polymorphism was significantly associated with the risk of severe acute GVHD in patients (OR=7.0, P=0.033) on multivariate analysis. Recipients of this mismatched HLA also showed shortened overall survival (HR=9.7, P<0.001) and increased risk for transplant-related mortality (HR=9.1, P=0.027). Structural modeling showed that the amino acid substitution could alter the peptide preference of the ligand-binding pocket. A single amino acid substitution at position 9 was a major predictor of severe acute GVHD in Korean pediatric patients.

[Comparative quantification of plasma hnRNP B1 mRNA in non-small cell lung cancer patients by real-time PCR].

BACKGROUND: Circulating cell-free nucleic acids are known to be a noninvasive diagnostic tool for cancer detection. Heterogeneous nuclear ribonucleoprotein (hnRNP) B1, a nuclear core complex, is overexpressed in early stage lung cancer. We intended to evaluate the usefulness of plasma hnRNP B1 mRNA in differentiating non-small cell lung cancer (NSCLC) from other benign lung diseases, especially pulmonary tuberculosis, which is highly prevalent in Korea and often difficult to distinguish from lung cancer. METHODS: Plasma RNA was extracted from 30 patients with NSCLC, 30 patients with benign lung diseases including pulmonary tuberculosis, and 10 healthy controls. Plasma hnRNP B1 mRNA was measured by TaqMan Gene Expression Assay (Applied Biosystems, USA), and pre-developed beta-actin (ACTB) mRNA was used for normalization. We analyzed the relative gene expression data using the delta-delta Ct method. RESULTS: Plasma hnRPN B1 mRNA was measurable in 93.3% (28/30) of NSCLC patients. Normalized 2-DeltaDeltaCt of plasma hnRPN B1 mRNA was 62.2 (95%Cl, 6.4-210.1) in NSCLC patients and 2.7 (95%Cl, 0.5-13.6) in benign lung disease patients (P<0.001, Mann-Whitney U test). CONCLUSIONS: Plasma hnRNP B1 mRNA was significantly increased in patients with lung cancer compared with that in patients with other benign lung diseases. Plasma hnRNP B1 mRNA may be useful as a potential marker for the detection of NSCLC.

Cancer-derived lysophosphatidic acid stimulates differentiation of human mesenchymal stem cells to myofibroblast-like cells.

Lysophosphatidic acid (LPA) is enriched in ascites of ovarian cancer patients and is involved in growth and invasion of ovarian cancer cells. Accumulating evidence suggests cancer-associated myofibroblasts play a pivotal role in tumorigenesis through secreting stromal cell-derived factor-1 (SDF-1). In the present study, we demonstrate that LPA induces expression of alpha-smooth muscle actin (alpha-SMA), a marker for myofibroblasts, in human adipose tissue-derived mesenchymal stem cells (hADSCs). The LPA-induced expression of alpha-SMA was completely abrogated by pretreatment of the cells with Ki16425, an antagonist of LPA receptors, or by silencing LPA(1) or LPA(2) isoform expression with small interference RNA (siRNA). LPA elicited phosphorylation of Smad2/3, and siRNA-mediated depletion of endogenous Smad2/3 or adenoviral expression of Smad7, an inhibitory Smad, abrogated the LPA induced expression of alpha-SMA and phosphorylation of Smad2/3. LPA-induced secretion of transforming growth factor (TGF)-beta1 in hADSCs, and pretreatment of the cells with SB431542, a TGF-beta type I receptor kinase inhibitor, or anti-TGF-beta1 neutralizing antibody inhibited the LPA-induced expression of alpha-SMA and phosphorylation of Smad2. Furthermore, ascites from ovarian cancer patients or conditioned medium from ovarian cancer cells induced expression of alpha-SMA and phosphorylation of Smad2, and pretreatment of the cells with Ki16425 or SB431542 abrogated the expression of alpha-SMA and phosphorylation of Smad2. In addition, LPA increased the expression of SDF-1 in hADSCs, and pretreatment of the cells with Ki16425 or SB431562 attenuated the LPA-stimulated expression of SDF-1. These results suggest that cancer-derived LPA stimulates differentiation of hADSCs to myofibroblast-like cells and increases SDF-1 expression through activating autocrine TGF-beta1-Smad signaling pathway.

Lysophosphatidic acid in malignant ascites stimulates migration of human mesenchymal stem cells.

Lysophosphatidic acid (LPA) is elevated in ascites of ovarian cancer patients and is involved in growth and invasion of ovarian cancer cells. Accumulating evidence suggests a pivotal role of mesenchymal stem cells (MSCs) or stromal cells in tumorigenesis. In the present study, we demonstrated that ascites from ovarian cancer patients and LPA increased migration of human MSCs. The migration of MSCs induced by LPA and malignant ascites was completely abrogated by pretreatment with Ki16425, an antagonist of LPA receptors, and by silencing of endogenous LPA(1), but not LPA(2), with small interference RNA, suggesting a key role of LPA played in the malignant ascites-induced migration. LPA induced activation of ERK through pertussis toxin-sensitive manner, and pretreatment of MSCs with U0126, a MEK inhibitor, or pertussis toxin attenuated the LPA-induced migration. Moreover, LPA induced activation of RhoA in MSCs, and pretreatment of the cells with Y27632, a Rho kinase inhibitor, markedly inhibited the LPA-induced migration. In addition, LPA and malignant ascites increased intracellular concentration of calcium in MSCs, and Ki16425 completely inhibited the elevation of intracellular calcium. These results suggest that LPA is a crucial component of the malignant ascites which induce the migration of MSCs and elevation of intracellular calcium.

Successful treatment of localized cutaneous infection caused by Mycobacterium scrofulaceum with clarithromycin.

Cutaneous infection arising from Mycobacterium scrofulaceum, a nontuberculous mycobacteria, has rarely been reported, and most of the reported infections were disseminated forms in patients with AIDS or other immunocompromising illness. We describe an occurrence of localized mycobacterial skin infection caused by M. scrofulaceum in a previously healthy child that manifested as a red nodule on the cheek. A biopsy specimen of the lesion demonstrated granulomatous infiltration in the dermis. M. scrofulaceum was isolated from culture of a tissue specimen. Polymerase chain reaction amplified specific fragments for M. scrofulaceum. The patient was treated successfully with clarithromycin as monotherapy for 6 months, leading to complete healing without recurrence during a follow-up period of 2 years.

Acute hemolytic transfusion reactions due to multiple alloantibodies including anti-E, anti-c and anti-Jkb.

We report a case of two consecutive episodes of acute hemolytic transfusion reactions (HTRs) due to multiple alloantibodies in a 34-yr-old man who suffered from avascular necrosis of left femoral head. He received five units of packed red blood cells (RBCs) during surgery. Then the transfusion of packed RBCs was required nine days after the surgery because of the unexplained drop in hemoglobin level. The transfusion of the first two units resulted in fever and brown-colored urine, but he received the transfusion of another packed RBCs the next day. He experienced even more severe symptoms during the transfusion of the first unit. We performed antibody screening test, and it showed positive results. Multiple alloantibodies including anti-E, anti-c and anti-Jkb were detected by antibody identification study. Acute HTRs due to multiple alloantibodies were diagnosed, and the supportive cares were done for 6 days. We suggest the antibody screening test should be included in the panel of pretransfusion tests for safer transfusion, and it is particularly mandatory for the patients with multiple transfusions, pregnant women, and preoperative patients.