Multiplex Real-Time PCR Assay Targeting Eight Parasites Customized to the Korean Population: Potential Use for Detection in Diarrheal Stool Samples from Gastroenteritis Patients.

Intestinal parasitic diseases occur worldwide and can cause diarrhea or gastroenteritis; however, their diagnosis is quite difficult, especially in low-endemism countries. We developed a multiplex real-time PCR assay for detection of eight intestinal parasites and prospectively evaluated it for patients with gastroenteritis. The assay targeted Cryptosporidium parvum, Giardia lamblia, Entamoeba histolytica, Blastocystis hominis, Dientamoeba fragilis, Clonorchis sinensis, Metagonimus yokogawai, and Gymnophalloides seoi. Performance characteristics were evaluated based on recovery after DNA extraction, analytical sensitivity, specificity, reproducibility, cross-reactivity, and interference characteristics. Clinical performance was validated against microscopy on 123 diarrheal samples. The assay demonstrated strong correlations between DNA concentrations and Ct values (R2, 0.9924-0.9998), and had a high PCR efficiency (83.3%-109.5%). Polymerase chain reactions detected as few as 10-30 copies of genomic DNA, and coefficient of variance was 0-7%. There was no cross-reactivity to the other 54 microorganisms tested. Interference occurred only in presence of high concentrations of erythrocytes or leukocytes. This assay had a higher correct identification rate (100.0% vs. 90.2%) and lower incorrect ID rate (0.0% vs. 9.8%) when compared to microscopy. Overall, this assay showed a higher sensitivity (100.0%; 95% confidence interval [CI] of 80.5-100.0) than microscopy (29.4%; 95% CI 10.31-55.96), and the specificity levels were comparable for both methods (100.0%; 95% CI 96.58-100.0). This newly developed multiplex real-time PCR assay offers a potential use for detecting intestinal parasitic pathogens customized to the Korean population.

Resistance Mechanisms and Clinical Features of Fluconazole-Nonsusceptible Candida tropicalis Isolates Compared with Fluconazole-Less-Susceptible Isolates.

We investigated the azole resistance mechanisms and clinical features of fluconazole-nonsusceptible (FNS) isolates of Candida tropicalis recovered from Korean surveillance cultures in comparison with fluconazole-less-susceptible (FLS) isolates. Thirty-five clinical isolates of C. tropicalis, comprising 9 FNS (fluconazole MIC, 4 to 64 µg/ml), 12 FLS (MIC, 1 to 2 µg/ml), and 14 control (MIC, 0.125 to 0.5 µg/ml) isolates, were assessed. CDR1, MDR1, and ERG11 expression was quantified, and the ERG11 and UPC2 genes were sequenced. Clinical features of 16 patients with FNS or FLS bloodstream isolates were analyzed. Both FNS and FLS isolates had >10-fold higher mean expression levels of CDR1, MDR1, and ERG11 genes than control isolates (P values of <0.02 for all). When FNS and FLS isolates were compared, FNS isolates had 3.4-fold higher mean ERG11 expression levels than FLS isolates (P = 0.004), but there were no differences in those of CDR1 or MDR1 Of all 35 isolates, 4 (2 FNS and 2 FLS) and 28 (8 FNS, 11 FLS, and 9 control) isolates exhibited amino acid substitutions in Erg11p and Upc2p, respectively. Both FNS and FLS bloodstream isolates were associated with azole therapeutic failure (3/4 versus 4/7) or uncleared fungemia (4/6 versus 4/10), but FNS isolates were identified more frequently from patients with previous azole exposure (6/6 versus 3/10; P = 0.011) and immunosuppression (6/6 versus 3/10; P = 0.011). These results reveal that the majority of FNS C. tropicalis isolates show overexpression of CDR1, MDR1, and ERG11 genes, and fungemia develops after azole exposure in patients with immunosuppression.

Molecular Description of Macroorchis spinulosus (Digenea: Nanophyetidae) Based on ITS1 Sequences.

We performed a molecular genetic study on the sequences of 18S ribosomal RNA (ITS1 region) gene in 4-day-old adult worms of Macroorchis spinulosus recovered in mice experimentally infected with metacercariae from crayfish in Jeollanam-do Province, Korea. The metacercariae were round, 180 µm in average diameter, encysted with 2 layers of thick walls, but the stylet on the oral sucker was not clearly seen. The adult flukes were oval shape, and 760-820 µm long and 320-450 µm wide, with anterolateral location of 2 large testes. The phylogenetic tree based on ITS1 sequences of 6 M. spinulosus samples showed their distinguished position from other trematode species in GenBank. The most closely resembled group was Paragonimus spp. which also take crayfish or crabs as the second intermediate host. The present study is the first molecular characterization of M. spinulosus and provided a basis for further phylogenetic studies to compare with other trematode fauna in Korea.

Molecular identification and amphotericin B susceptibility testing of clinical isolates of Aspergillus from 11 hospitals in Korea.

BACKGROUND: We investigated the species distribution and amphotericin B (AMB) susceptibility of Korean clinical Aspergillus isolates by using two Etests and the CLSI broth microdilution method. METHODS: A total of 136 Aspergillus isolates obtained from 11 university hospitals were identified by sequencing the internal transcribed spacer (ITS) and ß-tubulin genomic regions. Minimal inhibitory concentrations (MICs) of AMB were determined in Etests using Mueller-Hinton agar (Etest-MH) and RPMI agar (Etest-RPG), and categorical agreement with the CLSI method was assessed by using epidemiological cutoff values. RESULTS: ITS sequencing identified the following six Aspergillus species complexes: Aspergillus fumigatus (42.6% of the isolates), A. niger (23.5%), A. flavus (17.6%), A. terreus (11.0%), A. versicolor (4.4%), and A. ustus (0.7%). Cryptic species identifiable by ß-tubulin sequencing accounted for 25.7% (35/136) of the isolates. Of all 136 isolates, 36 (26.5%) had AMB MICs of ≥2 µg/mL by the CLSI method. The categorical agreement of Etest-RPG with the CLSI method was 98% for the A. fumigatus, A. niger, and A. versicolor complexes, 87% for the A. terreus complex, and 37.5% for the A. flavus complex. That of Etest-MH was ≤75% for the A. niger, A. flavus, A. terreus, and A. versicolor complexes but was higher for the A. fumigatus complex (98.3%). CONCLUSIONS: Aspergillus species other than A. fumigatus constitute about 60% of clinical Aspergillus isolates, and reduced AMB susceptibility is common among clinical isolates of Aspergillus in Korea. Molecular identification and AMB susceptibility testing by Etest-RPG may be useful for characterizing Aspergillus isolates of clinical relevance.

Comparison of FcRγ-Deficient and CD57+ Natural Killer Cells Between Cord Blood and Adult Blood in the Cytomegalovirus-Endemic Korean Population.

BACKGROUND: FcRγ-deficient natural killer (NK) cells (g(-)NK cells) have been associated with cytomegalovirus (CMV) infection. However, the frequency of g(-)NK cells in a CMV-endemic area (i.e., Korea) has not yet been studied. We examined the frequency of g(-)NK cells and expression of CD57 on NK cells in cord blood (CB) and adult blood (AB). METHODS: Of the 24 AB samples collected, 95.8% (23/24) were CMV IgG(+)/IgM(-), while 100% of the 13 healthy CB samples were CMV IgG(+)/IgM(-). We performed whole-blood flow cytometry assays to analyze intracellular FcRγ and CD3ζ expression of CD3(-)/CD56(dim) NK cells from 13 CB and 24 AB samples, and surface CD57 expression on CD3(-)/CD56(dim)/CD16(+) NK cells from 13 CB and 19 AB samples. RESULTS: All CMV seropositive AB samples contained g(-)NK cells (23/23), and the median proportion of g(-)NK cells in the CD3(-)/CD56(dim) NK cell pool was 35.0% (range: 11-77%). CD57(+) NK cells in the CD3(-)/CD56(dim)/CD16(+) NK cell population were detected in all 19 AB samples tested, but not in any CB samples. CONCLUSIONS: Our data suggest that g(-)NK cells and CD57(+) NK cells are present at a very high frequency in CMV-seropositive AB, but rare in CMV-naïve CB.

Evaluation of Modified Formalin-Ether Concentration Method Using Para Tube in Clinical Settings.

Conventional formalin-ether concentration method is a gold standard for the diagnosis of parasite infection. However, it may be time-consuming and laborious. We aimed to reveal the clinical usefulness of a modified formalin-ether concentration method using the Para Tube (KS Corporation, Korea) compared with the conventional method. A total of 117 fresh, unpreserved fecal samples composed to 90 negative controls and 27 positive controls with ova of Diphyllobothrium latum/D. nihonkaiense, ova of Clonorchis sinensis and cysts of Giardia lamblia were used in this study. Both methods showed comparable correct identification rate (87.2% for the Para Tube vs. 86.3% for the conventional method).When five samples were examined at once, the Para Tube method reduced the procedure time compared with the conventional method (19 min 58 sec vs. 23 min 18 sec, P=0.0286). We concluded that the modified formalin-ether concentration method using the Para Tube is a rapid, simple, and reliable fecal concentration method for clinical use.

Seroepidemiology of Toxocariasis and Its Clinical Implications in Gwangju and Jeonnam-province, Korea.

We investigated the seroepidemiological, clinical, and laboratory characteristics of patients suspected to have toxocariasis in Gwangju and Jeonnam-province, Korea. In total, 228 specimens were analyzed for anti-Toxocara canis IgG at two university hospitals from 2010 to 2012. The overall seropositive rate was 67.1%, and the seropositive rates among the eosinophilic and non-eosinophilic groups were 76.1% (105/138) and 53.3% (48/90), respectively. Risk factors for eosinophilia and toxocariasis were male sex (odds ratios [OR]=2.632 and 3.477, respectively) and a history of ingesting raw meat (OR=2.884 and 3.274, respectively), especially raw cow liver (OR=2.089 and 10.038, respectively). T. canis seropositivity (OR=5.807, P=0.004) and a history of consuming raw cow liver (OR=2.766, P=0.052) were risk factors for organ involvement. The anti-T. canis IgG level showed weakly positive correlations with eosinophil counts (r=0.234, P<0.001) and the duration of eosinophilia (r=0.155, P=0.019). Although limited to the regions of Gwangju and Jeonnam-province, this study supports the opinion that toxocariasis is a reasonable focus as a cause of eosinophilia and that it is also associated with organ involvement.

Comparison of AdvanSure TB/NTM PCR and COBAS TaqMan MTB PCR for Detection of Mycobacterium tuberculosis Complex in Routine Clinical Practice.

The AdvanSure tuberculosis/non-tuberculous mycobacterium (TB/NTM) PCR (LG Life Science, Korea) and COBAS TaqMan Mycobacterium tuberculosis (MTB) PCR (Roche Diagnostics, USA) are commonly used in clinical microbiology laboratories. We aimed to evaluate these two commercial real-time PCR assays for detection of MTB in a large set of clinical samples over a two-year period. AdvanSure TB/NTM PCR and COBAS TaqMan MTB PCR were performed on 9,119 (75.2%) and 3,010 (24.8%) of 12,129 (9,728 respiratory and 2,401 non-respiratory) MTB specimens, with 361 (4.0%) and 102 (3.4%) acid-fast bacilli (AFB)-positive results, respectively. In MTB culture, 788 (6.5%) MTB and 514 (4.2%) NTM were identified. The total sensitivity and specificity of the AdvanSure assay were 67.8% (95% confidence interval [CI], 63.9-71.6) and 98.3% (95% CI, 98.0-98.6), while those of the COBAS TaqMan assay were 67.2% (95% CI, 60.0-73.8) and 98.4% (95% CI, 97.9-98.9), respectively. The sensitivities and specificities of the AdvanSure and COBAS TaqMan assays for AFB-positive and AFB-negative samples were comparable. Furthermore, the AdvanSure assay showed fewer invalid results compared with the COBAS TaqMan assay (5.0 vs. 20.4 invalid results/1,000 tests, P<0.001). AdvanSure assay represents a comparable yet more reliable method than COBAS TaqMan for the identification of mycobacteria in routine clinical microbiology.

Strongyloidiasis in a diabetic patient accompanied by gastrointestinal stromal tumor: cause of eosinophilia unresponsive to steroid therapy.

We report here a case of strongyloidiasis in a 72-year-old diabetic patient (woman) accompanied by gastrointestinal stromal tumor receiving imatinib therapy, first diagnosed as hypereosinophilic syndrome and treated with steroids for uncontrolled eosinophilia. She suffered from lower back pain and intermittent abdominal discomfort with nausea and diagnosed with gastrointestinal stromal tumor. After post-operative imatinib treatment eosinophilia persisted, so that steroid therapy was started under an impression of hypereosinophilic syndrome. In spite of 6 months steroid therapy, eosinophilia persisted. Stool examination was performed to rule out intestinal helminth infections. Rhabditoid larvae of Strongyloides stercoralis were detected and the patient was diagnosed as strongyloidiasis. This diagnosis was confirmed again by PCR. The patient was treated with albendazole for 14 days and her abdominal pain and diarrhea improved. This case highlights the need for thorough investigation, including molecular approaches, to test for strongyloidiasis before and during steroid therapies.

Feasible quantitative detection of cytomegalovirus from urine sediment in stem cell transplant patients.

BACKGROUND: Urine is an important source for the detection of infections caused by CMV in stem cell transplant patients. Currently, there is no agreement about the type of urine specimen. In order to investigate which is the better specimen type for quantitative detection of CMV, we compared the results from urine supernatant and sediment from the same patients. METHODS: Seventy urine specimens were collected from patients with hematological disorders or solid tumors. After performing shell vial culture, residual urine specimens were centrifuged. Then, 10 mL of each urine supernatant and sediment were taken and immediately frozen at -70 degrees C. Afterwards, archived urine specimens were thawed at room temperature and CMV-quantitative PCR was performed on both the supernatant and sediment fraction of urine. The results from each patient were reviewed for CMV antigenemia, blood shell vial culture, CMV-IgM or IgG, and clinical symptoms. RESULTS: CMV-qPCR results for the urine sediment fraction revealed a significant difference (p = 0.012) between the active CMV infection group and the latent CMV infection group. In addition, receiver operating characteristic curves for active CMV infection revealed that CMV-qPCR using urine sediment produced more accurate results than urine supernatant. CONCLUSIONS: These findings suggest that the sediment fraction of urine is a more suitable specimen in CMV-qPCR testing.

Direct confirmation of quiescence of CD34+CD38- leukemia stem cell populations using single cell culture, their molecular signature and clinicopathological implications.

BACKGROUND: The proliferating activity of a single leukemia stem cell and the molecular mechanisms for their quiescent property remain unknown, and also their prognostic value remains a matter of debate. Therefore, this study aimed to demonstrate the quiescence property and molecular signature of leukemia stem cell and their clinicopathological implications. METHODS: Single cell sorting and culture were performed in the various sets of hematopoietic stem cells including CD34+CD38- acute myeloid leukemia (AML) cell population (ASCs) from a total of 60 patients with AML, and 11 healthy controls. Their quiescence related-molecular signatures and clinicopathological parameters were evaluated in AML patients. RESULTS: Single cell plating efficiency of ASCs was significantly lower (8.6%) than those of normal hematopoietic stem cells i.e.: cord blood, 79.0%; peripheral blood, 45.3%; and bone marrow stem cell, 31.1%. Members of the TGFß super-family signaling pathway were most significantly decreased; as well as members of the Wnt, Notch, pluripotency maintenance and hedgehog pathways, compared with non ASC populations. mtDNA copy number of ASCs was significantly lower than that of corresponding other cell populations. However, our data couldn't support the prognostic value of the ASCs in AML. CONCLUSIONS: ASCs showed remarkable lower plating efficiency and slower dividing properties at the single cell level. This quiescence is represented as a marked decrease in the mtDNA copy number and also linked with down-regulation of genes in various molecular pathways.

Antifungal susceptibilities of bloodstream isolates of Candida species from nine hospitals in Korea: application of new antifungal breakpoints and relationship to antifungal usage.

We applied the new clinical breakpoints (CBPs) of the Clinical and Laboratory Standards Institute (CLSI) to a multicenter study to determine the antifungal susceptibility of bloodstream infection (BSI) isolates of Candida species in Korea, and determined the relationship between the frequency of antifungal-resistant Candida BSI isolates and antifungal use at hospitals. Four hundred and fifty BSI isolates of Candida species were collected over a 1-year period in 2011 from nine hospitals. The susceptibilities of the isolates to four antifungal agents were determined using the CLSI M27 broth microdilution method. By applying the species-specific CBPs, non-susceptibility to fluconazole was found in 16.4% (70/428) of isolates, comprising 2.6% resistant and 13.8% susceptible-dose dependent isolates. However, non-susceptibility to voriconazole, caspofungin, or micafungin was found in 0% (0/370), 0% (0/437), or 0.5% (2/437) of the Candida BSI isolates, respectively. Of the 450 isolates, 72 (16.0%) showed decreased susceptibility to fluconazole [minimum inhibitory concentration (MIC) ≥4 µg/ml]. The total usage of systemic antifungals varied considerably among the hospitals, ranging from 190.0 to 7.7 defined daily dose per 1,000 patient days, and fluconazole was the most commonly prescribed agent (46.3%). By Spearman's correlation analysis, fluconazole usage did not show a significant correlation with the percentage of fluconazole resistant isolates at hospitals. However, fluconazole usage was significantly correlated with the percentage of fluconazole non-susceptible isolates (r = 0.733; P = 0.025) or the percentage of isolates with decreased susceptibility to fluconazole (MIC ≥4 µg/ml) (r = 0.700; P = 0.036) at hospitals. Our work represents the first South Korean multicenter study demonstrating an association between antifungal use and antifungal resistance among BSI isolates of Candida at hospitals using the new CBPs of the CLSI.

Evaluation of matrix-assisted laser desorption ionization-time of flight mass spectrometry-based VITEK MS system for the identification of Acinetobacter species from blood cultures: comparison with VITEK 2 and MicroScan systems.

BACKGROUND: Acinetobacter species are the leading cause of bloodstream infection (BSI), but their correct identification is challenging. We evaluated the matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS)-based VITEK MS (bioMérieux, France), and two automated systems, VITEK 2 (bioMérieux) and MicroScan (Siemens, USA) for identification of Acinetobacter BSI isolates. METHODS: A total of 187 BSI isolates recovered at a university hospital in Korea between 2010 and 2012 were analyzed. The identification results obtained using VITEK MS and two automated systems were compared with those of rpoB sequencing. RESULTS: Of 187 isolates analyzed, 176 were identified to the species level by rpoB sequencing: the Acinetobacter baumannii group (ABG; 101 A. baumannii, 43 A. nosocomialis, 10 A. pittii isolates) was most commonly identified (82.4%), followed by Acinetobacter genomic species 13BJ/14TU (5.3%), A. ursingii (2.1%), A. soli (2.1%), A. bereziniae (1.1%), and A. junii (1.1%). Correct identification rates to the species group (ABG) level or the species level was comparable among the three systems (VITEK MS, 90.3%; VITEK 2, 89.2%; MicroScan, 86.9%). However, VITEK MS generated fewer misidentifications (0.6%) than VITEK 2 (10.8%) and MicroScan (13.1%) (P<0.001). In addition, VITEK MS demonstrated higher specificity (100%) for discrimination between ABG and non-ABG isolates than the other systems (both, 31.8%) (P<0.001). CONCLUSIONS: The VITEK MS system is superior to the VITEK 2 and MicroScan systems for identification of Acinetobacter BSI isolates, with fewer misidentifications and better discrimination between the ABG and non-ABG isolates.

Alteration of the SETBP1 gene and splicing pathway genes SF3B1, U2AF1, and SRSF2 in childhood acute myeloid leukemia.

BACKGROUND: Recurrent somatic SET-binding protein 1 (SETBP1) and splicing pathway gene mutations have recently been found in atypical chronic myeloid leukemia and other hematologic malignancies. These mutations have been comprehensively analyzed in adult AML, but not in childhood AML. We investigated possible alteration of the SETBP1, splicing factor 3B subunit 1 (SF3B1), U2 small nuclear RNA auxiliary factor 1 (U2AF1), and serine/arginine-rich splicing factor 2 (SRSF2) genes in childhood AML. METHODS: Cytogenetic and molecular analyses were performed to reveal chromosomal and genetic alterations. Sequence alterations in the SETBP1, SF3B1, U2AF1, and SRSF2 genes were examined by using direct sequencing in a cohort of 53 childhood AML patients. RESULTS: Childhood AML patients did not harbor any recurrent SETBP1 gene mutations, although our study did identify a synonymous mutation in one patient. None of the previously reported aberrations in the mutational hotspot of SF3B1, U2AF1, and SRSF2 were identified in any of the 53 patients. CONCLUSIONS: Alterations of the SETBP1 gene or SF3B1, U2AF1, and SRSF2 genes are not common genetic events in childhood AML, implying that the mutations are unlikely to exert a driver effect in myeloid leukemogenesis during childhood.

Profiling of biomarkers for the exposure of polycyclic aromatic hydrocarbons: lamin-A/C isoform 3, poly[ADP-ribose] polymerase 1, and mitochondria copy number are identified as universal biomarkers.

This study investigated the profiling of polycyclic aromatic hydrocarbon- (PAH-) induced genotoxicity in cell lines and zebrafish. Each type of cells displayed different proportionality of apoptosis. Mitochondrial DNA (mtDNA) copy number was dramatically elevated after 5-day treatment of fluoranthene and pyrene. The notable deregulated proteins for PAHs exposure were displayed as follows: lamin-A/C isoform 3 and annexin A1 for benzopyrene; lamin-A/C isoform 3 and DNA topoisomerase 2-alpha for pentacene; poly[ADP-ribose] polymerase 1 (PARP-1) for fluoranthene; and talin-1 and DNA topoisomerase 2-alpha for pyrene. Among them, lamin-A/C isoform 3 and PARP-1 were further confirmed using mRNA and protein expression study. Obvious morphological abnormalities including curved backbone and cardiomegaly in zebrafish were observed in the 54 hpf with more than 400 nM of benzopyrene. In conclusion, the change of mitochondrial genome (increased mtDNA copy number) was closely associated with PAH exposure in cell lines and mesenchymal stem cells. Lamin-A/C isoform 3, talin-1, and annexin A1 were identified as universal biomarkers for PAHs exposure. Zebrafish, specifically at embryo stage, showed suitable in vivo model for monitoring PAHs exposure to hematopoietic tissue and other organs.

The p.R168Q mutation is associated with the Bw phenotype and a predicted decrease in the stability of the resulting ABO glycosyltransferase.

BACKGROUND: Mutation of ABO glycosyltransferase (GT) can cause protein stability changes that can result in a weak ABO phenotype. To explain the Bw phenotype of a novel ABO*Bw allele, a protein stability of the mutant GT, which enhances the information of the three-dimensional (3D) structural analysis, was calculated. STUDY DESIGN AND METHODS: ABO serology and genotyping were performed on a neonate and her five family members. A 3D structural analysis of the wild-type GTB and enzymes with a variety of mutations at Residue 168, along with predicted protein stability changes (ΔΔG) and flow cytometric analysis of ABO antigen expression on HeLa cells transfected with plasmids containing R168Q, R168L, and R168P mutants was also performed. RESULTS: A novel ABO*Bw allele (c.503G>A, p.R168Q) was discovered. The structural analysis of 3D homology modeling predicted reduced protein stability of the mutant GTB, and the ΔΔG values, which inversely correlated with the mean relative fluorescence intensity of ABO antigen expression, quantitatively explained the reduced ABO antigen expression. CONCLUSIONS: The predicted protein stability change of a mutant GT enzyme might be a useful and convenient approach to objectively and quantitatively explain the reduced ABO antigen expression.

Serum parathyroid hormone is a new potential risk factor in multiple myeloma.

We hypothesized that serum PTH might be associated with various clinicopathological parameters in multiple myeloma (MM). So we investigated the implications of serum PTH in MM patients and the relationship with other risk factors of MM. A total of 115 patients who were newly diagnosed with MM were enrolled. Serum PTH level was 24.7 ± 34.9 (ranged 0.0-284.1) pg/mL. Serum levels of IgG, IgM, FLC-lambda, albumin, and LDH were in positive correlation with serum PTH. Compared to non-high PTH (<68.3 pg/mL) group, the hazard ratio (HR) for overall survival was higher for group with high PTH level (≥ 68.3 pg/mL) (HR, 1.710). Furthermore, the patient group with high PTH level showed inferior progression-free survival than non-high PTH group (P = 0.056). Interestingly, subgroup analysis showed that serum PTH level at diagnosis was associated with risk factors and clinical outcome in MM patients, especially in complete remission group, transplantation cases, ISS stage II cases, and cases without chromosome abnormality. In conclusion, this study showed that blood PTH level in MM at diagnosis was associated with risk factors and clinical outcome in MM patients.