Analysis of the Vaginal Microbiome by Next-Generation Sequencing and Evaluation of its Performance as a Clinical Diagnostic Tool in Vaginitis

BACKGROUND: Next-generation sequencing (NGS) can detect many more microorganisms of a microbiome than traditional methods. This study aimed to analyze the vaginal microbiomes of Korean women by using NGS that included bacteria and other microorganisms. The NGS results were compared with the results of other assays, and NGS was evaluated for its feasibility for predicting vaginitis. METHODS: In total, 89 vaginal swab specimens were collected. Microscopic examinations of Gram staining and microbiological cultures were conducted on 67 specimens. NGS was performed with GS junior system on all of the vaginal specimens for the 16S rRNA, internal transcribed spacer (ITS), and Tvk genes to detect bacteria, fungi, and Trichomonas vaginalis. In addition, DNA probe assays of the Candida spp., Gardnerella vaginalis, and Trichomonas vaginalis were performed. Various predictors of diversity that were obtained from the NGS data were analyzed to predict vaginitis. RESULTS: ITS sequences were obtained in most of the specimens (56.2%). The compositions of the intermediate and vaginitis Nugent score groups were similar to each other but differed from the composition of the normal score group. The fraction of the Lactobacillus spp. showed the highest area under the curve value (0.8559) in ROC curve analysis. The NGS and DNA probe assay results showed good agreement (range, 86.2-89.7%). CONCLUSIONS: Fungi as well as bacteria should be considered for the investigation of vaginal microbiome. The intermediate and vaginitis Nugent score groups were indistinguishable in NGS. NGS is a promising diagnostic tool of the vaginal microbiome and vaginitis, although some problems need to be resolved.

Hematological Significance of RT-PCR Test for bcr-abl Rearrangement and the Breakpoint Distribution within the Major bcr in Chronic Myelogenous Leukemia Patients / 대한혈액학회지

BACKGROUND: Bcr-abl rearrangement is the molecular hallmark of chronic myelogenous leukemia (CML). The test for bcr-abl rearrangement, especially using RT-PCR, is the standard test for the diagnosis of CML. We analyzed hematological significances of bcr-abl rearrangement by RT-PCR and the breakpoint distribution within the major bcr in CML patients. METHODS: From 1994 October to 1997 September, we performed the bcr-abl rearrangement using RT-PCR, in 268 untreated patients with various hematologic diseases, and classified the breakpoints within BCR gene as three types (b2a2, b3a2, e1a2) according to PCR product sizes. We compared hematologic parameters between two groups of b2a2 and b3a2 breakpoints in CML. RESULTS: Among the patients with clinically diagnosed CML, 96.8% (61/63) were bcr-abl positive. In ALL, 52.8% (19/36) were bcr- abl positive. All patients with hematologic diseases other than CML or ALL were bcr- abl negative. Among 61 CML patients with positive bcr-abl rearrangement, 19 patients (31.1%) showed b2a2 type and 42 patients (68.9%) b3a2 type. Patients with b3a2 breakpoints showed more frequent peripheral basophilia (P<0.01) than those with b2a2 type. However, other hematologic parameters were not statistically significant. CONCLUSION: RT-PCR test for bcr-abl rearrangement is a specific and efficient test for the diagnosis of CML. However, the hematological significance of b2a2 and b3a2 types is uncertain in CML.

Abnormalities of erythrocyte membrane proteins in Korean patients with hereditary spherocytosis

Hereditary spherocytosis (HS) is a common inherited erythrocyte membrane disorder characterized by chronic hemolytic anemia. Clinical manifestations and biochemical abnormalities of HS are heterogeneous. In this study, we investigated erythrocyte membrane protein defects in 27 Korean HS cases. Utilizing both the Fairbanks system and the Laemmli system, sodium dodecyl sulfate polyacrylamide gel electrophoresis of erythrocyte membrane proteins was performed. Proteins were stained with Coomassie brilliant blue and gels were scanned using a densitometer. We detected spectrin deficiency in 7.4% of cases (2/27), ankyrin deficiency in 29.6% (8/27), combined spectrin and ankyrin deficiency in 3.7% (1/27), band 3 deficiency in 11.1% (3/27) and protein 4.2 deficiency in 14.8% (4/27). Membrane protein deficiencies were not observed in nine cases (33.3%, 9/27). Members of two of seven families tested showed the same protein defects as the proband. Ankyrin deficiency alone and combined with spectrin deficiency accounted for 33.3% of cases (9/27), and they were the most common biochemical defects in Korean HS cases. Protein 4.2 deficiency caused HS more frequently in Koreans than in Caucasians.

Abnormalities of erythrocyte membrane proteins in Korean patients with hereditary spherocytosis

Hereditary spherocytosis (HS) is a common inherited erythrocyte membrane disorder characterized by chronic hemolytic anemia. Clinical manifestations and biochemical abnormalities of HS are heterogeneous. In this study, we investigated erythrocyte membrane protein defects in 27 Korean HS cases. Utilizing both the Fairbanks system and the Laemmli system, sodium dodecyl sulfate polyacrylamide gel electrophoresis of erythrocyte membrane proteins was performed. Proteins were stained with Coomassie brilliant blue and gels were scanned using a densitometer. We detected spectrin deficiency in 7.4% of cases (2/27), ankyrin deficiency in 29.6% (8/27), combined spectrin and ankyrin deficiency in 3.7% (1/27), band 3 deficiency in 11.1% (3/27) and protein 4.2 deficiency in 14.8% (4/27). Membrane protein deficiencies were not observed in nine cases (33.3%, 9/27). Members of two of seven families tested showed the same protein defects as the proband. Ankyrin deficiency alone and combined with spectrin deficiency accounted for 33.3% of cases (9/27), and they were the most common biochemical defects in Korean HS cases. Protein 4.2 deficiency caused HS more frequently in Koreans than in Caucasians.

SDS-PAGE Analysis of Red Cell Membrane Proteins in Hereditary Hemolytic Anemia / 대한혈액학회지

BACKGROUND: Red cell membrane is a lipid bilayer laminated by the membrane cytoskeleton at the surface of inner monolayer. A class of hemolytic anemia, such as hereditary spherocytosis (HS) or hereditary elliptocytosis (HE) is mainly caused by the abnormalities of the protein components in the cytoskeleton, which is useful to diagnosis each disorder. We investigated red cell membrane protein defects in HS and HE using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). METHODS: We studied 10 normal healthy volunteers, 27 HS cases and 5 HE cases. Erythrocyte membrane proteins were prepared by hypotonic lysis, and fractionated by SDS-PAGE using both the Fairbanks system (3.5~17% exponential gradient gel), and the Laemmli system (4~17% linear gradient gel). Fractionated proteins were stained with Coomassie brilliant blue and scanned to quantitate each protein using a densitometer. RESULTS: We detected nine peaks in Fairbanks' gel and eight peaks in Laemmli's. We identified red cell membrane abnormalities in 18 of 27 HS patients (66.7%) : Spectrin deficiency alone was in 7.4% of HS cases (2/27), ankyrin deficiency alone in 29.6% (8/27), combined spectrin and ankyrin deficiency in 3.7% (1/27), band 3 deficiency in 11.1% (3/27) and protein 4.2 deficiency in 14.8% (4/27). In HE, three of five cases showed protein 4.1 deficiency. RBC membrane protein deficiencies were not observed in nine HS cases and two HE case. CONCLUSION: In HS, Ankyrin deficiency is the most common RBC membrane protein abnor mality, and protein 4.2 deficiency is more frequently found in Korean HS patients than in Caucasians. In HE patients, protein 4.1 deficiencies is the main red cell membrane protein defect, which is rarely reported in Caucasians.