Ho Eun Chang

Rapid, Sensitive, and Specific Detection of Mycobacterium tuberculosis Complex by Real-Time PCR on Paraffin-Embedded Human Tissues

The detection of Mycobacterium tuberculosis complex (MTB) in clinical specimens is important for diagnosing and caring for patients in whom tuberculosis is clinically suspected. We collected 129 FFPE specimens, including 56 nontuberculosis cases, 63 MTB cases, and 10 nontuberculous mycobacteria (NTM) cases determined by acid-fast bacilli (AFB) culture. We performed AFB staining; nested MTB PCR, targeting the IS6110 gene; and real-time MTB PCR, targeting the senX3-regX3 intergenic region in the 129 FFPE specimens. The sensitivity and specificity of AFB staining were 37.0% and 98.2%, respectively, using AFB culture results as the reference standard. The sensitivity and specificity of detecting MTB were 68.3% and 98.5%, respectively, by nested PCR; and 74.6% and 98.5% by real-time PCR, respectively. Among the 129 specimens, four were positive by AFB staining but negative by nested or real-time PCR. NTM grew in all four of these cases by AFB culture. AFB density in FFPE tissue sections significantly correlated with MTB DNA load. Thus, real-time PCR is a useful diagnostic tool for rapid and sensitive MTB detection in FFPE specimens, whereas NTM should be included in differential diagnoses of cases positive by AFB staining but negative by PCR.

Human bloodstream infection caused by Staphylococcus pettenkoferi

Staphylococcus pettenkoferi is a recently isolated human pathogen with only a few reported cases of infection. We report a case of bloodstream infection caused by S. pettenkoferi in a patient with pulmonary tuberculosis.

[Detection of mycobacterium tuberculosis complex using real-time polymerase chain reaction].

BACKGROUND For the detection of Mycobacterium tuberculosis complex (MTB), PCR is known to be sensitive, specific, and rapid compared to the conventional methods of acid-fast-bacilli (AFB) smear and culture. We evaluated a new approach for MTB detection using real-time PCR. METHODS The specificity of real-time PCR was evaluated using 20 MTB isolates and 37 nontuberculous mycobacteria (NTM) isolates identified by AccuProbe Mycobacterium tuberculosis complex colony identification test (Gen-Probe Inc., USA) and Myco-ID (M&D, Korea). One hundred sputum specimens (50 AFB smear-positive and 50 negative specimens) were analyzed using real-time PCR and Amplicor Mycobacterium tuberculosis test (Roche, Germany). The results of real-time PCR positives (55 samples) and negatives (598 samples) were analyzed by AFB smear and culture. RESULTS The real-time PCR assay accurately discriminated between MTB and NTM species. Realtime PCR and Amplicor test yielded the same results in 96.0% (96/100) of the sputum specimens tested. The sensitivity and specificity of real-time PCR based on AFB culture were 97.4% and 88.5%, respectively. Of the 55 real-time PCR positive specimens, 83.6% (46/55) were culture-positive, 30.9% (17/55) were smear-positive, 52.7% (29/55) were smear-negative and culture-positive, and 14.5% (8/55) were both smear and culture-negative. Among the 598 real-time PCR negative specimens, 60 were not tested for AFB smear or culture and 10 were contaminated. Of the remaining 528 specimens, 478 (90.5%) were both smear and culture-negative and 39 (7.4%) were culture-positive. CONCLUSIONS For the detection of MTB, real-time PCR was sensitive and specific and comparable to conventional methods. It can be used for rapid identification of M. tuberculosis in clinical laboratories.

Novel Multiplex PCR Using Dual-Priming Oligonucleotides for Detection and Discrimination of the Mycobacterium tuberculosis Complex and M. bovis BCG

ABSTRACT We developed a novel multiplex PCR assay using dual-priming oligonucleotide primers targeting the RD1 gene for simultaneous identification of the Mycobacterium tuberculosis complex and M. bovis bacillus Calmette-Guérin (BCG). This assay would be useful both for detection of the M. tuberculosis complex and for differentiation of M. bovis BCG from pathogenic M. tuberculosis complex species.

Performance Characteristics of Nested Polymerase Chain Reaction vs Real-Time Polymerase Chain Reaction Methods for Detecting Mycobacterium tuberculosis Complex in Paraffin-Embedded Human Tissues

OBJECTIVES Nucleic acid amplification tests on formalin-fixed, paraffin-embedded (FFPE) tissue specimens enable Mycobacterium tuberculosis complex (MTB) detection and rapid tuberculosis diagnosis in the absence of microbiologic culture tests. We aimed to evaluate the efficacy of different polymerase chain reaction (PCR) methods for detecting Mycobacterium species in FFPE tissues. METHODS We examined 110 FFPE specimens (56 nonmycobacterial cases, 32 MTB, and 22 nontuberculous mycobacteria [NTM] determined by acid-fast bacilli [AFB] culture) to assess five PCR methods: nested PCR (N-PCR) (Seeplex MTB Nested ACE Detection; Seegene, Seoul, South Korea), an in-house real-time PCR (RT-PCR) method, and three commercial RT-PCR methods (AccuPower MTB RT-PCR [Bioneer, Seoul, Korea], artus M tuberculosis TM PCR [Qiagen, Hilden, Germany], and AdvanSure tuberculosis/NTM RT-PCR [LG Life Sciences, Seoul, Korea]). RESULTS The results of N-PCR, in-house RT-PCR, and AdvanSure RT-PCR correlated well with AFB culture results (concordance rates, 94.3%, 87.5%, and 89.5%, respectively). The sensitivity of N-PCR (87.5%) was higher than that of the RT-PCR methods, although these differences were not statistically significant between N-PCR and the in-house and AdvanSure RT-PCR methods (68.8% and 80.0%, respectively). All the PCR methods had high specificities, ranging from 98.2% to 100%. Only two NTM cases were detected by AdvanSure RT-PCR, implying a very low sensitivity. CONCLUSIONS Well-designed RT-PCR and N-PCR can effectively identify MTB in FFPE specimens.

Human platelet antigen genotyping and expression of CD109 (human platelet antigen 15) mRNA in various human cell types.

CD109 gene encodes a glycosylphosphatidylinositol-linked glycoprotein found in a subset of platelets and endothelial cell, and human platelet antigen (HPA) 15 is found on CD109. We evaluated the HPA genotype and/or the CD109 mRNA expression on two peripheral blood stem cells (PBSC), two peripheral bloods (PB), 12 granulocyte products, natural killer (NK)-92, B-lymphocyte (CO88BV59-1), K-562 leukemia cell line, human embryonic stem cell (hESC), and human fibroblasts (HF). HPA genotyping was performed by SNaPshot assay and CD109 mRNA expression was evaluated by real-time PCR with SYBR green and melting curve analysis. Genotype HPA-15a/-15a was found in PBSC#1 and two granulocyte products, and HPA-15a/-15b was found in PBSC#2, eight granulocyte products, NK-92, K-562, hESC, and HF, and HPA-15b/-15b was found in two granulocyte products. CD109 mRNA expression was highly increased in HF and increased in CD34+ and CD34− PBSCs and some granulocyte products, compared to the PB. However, the increase of expression level varied among the PBSC and granulocyte products. The CD109 mRNA expression of NK-92, K-562, hESC, and CO 88BV59-1 was not detected. HPA genotype was evaluated in various cells and the expression of CD109, which contains HPA 15, was different among cell lines and high in HF and PBSCs.

Molecular and biochemical characterization of the GALT gene in Korean patients with galactose-1-phosphate uridyltransferase deficiency.

BACKGROUND Three different types of galactosemia have been described, and the most common form occurs due to a deficiency in the galactose-1-phosphate uridyltransferase (GALT) enzyme activity. METHODS To investigate the molecular defects of the GALT gene, PCR-direct sequencing was performed with genomic DNA from 18 Korean patients with reduced GALT activity. RESULTS Of the 18 patients tested, 13 (72.2%) had previously reported variants: Duarte variant (12 patients), p.R201H (1 patient), and g.A1962G. In addition, we identified six novel sequence variations by PCR-direct sequencing: five sequence variations in coding regions (p.H31R, p.L116I, p.Q169H, p.H186P and p.R333R), and one in an intron (g.2621A>G). Of 100 normal individuals tested, 4 were heterozygous for the Duarte variant, which indicates a Duarte allele frequency of 2%. Biochemical characteristics of the novel genetic alterations were determined: enzyme activity for exonic alterations and splicing for intron. CONCLUSION The genetic constitution of the GALT gene is responsible for galactosemia in the Korean population.

Real-time multiplex PCR assay for genotyping of three apolipoprotein E alleles and two choline acetyltransferase alleles with three hybridization probes.

Abstract Background: Apolipoprotein E (APOE) and choline acetyltransferase (ChAT) have been suggested as candidate genes for determining the risk of late-onset Alzheimers disease. The aim of this study was to simultaneously detect polymorphisms in codons 112 and 158 of APOE and codon 2 of ChAT by hybridization probe multiplexing. Methods: The decrease in fluorescence emitted by LC-Red 610, LC-Red 640, and LC-Red 705 channels was quantified during a gradual temperature increase after amplification. The melting curves were converted to melting peaks by plotting the negative derivative of the fluorescence with respect to temperature (–dF/dT) as a function of temperature. A single pair of hybridization probes and PCR restriction fragment length polymorphism (RFLP) were used to confirm the genotyping of APOE and ChAT, respectively, in 183 subjects. Results: When a homozygous sample with the CGC/CGC sequence in codon 112 of APOE was analyzed, the mean sequence-specific melting point (Tm) was 62.8°C, whereas a sample with the TGC/TGC sequence had a Tm of 54.7°C. A similar fluorescence pattern appeared with a different Tm at 66.9°C (CGC/CGC) and 59.7°C (TGC/TGC) for codon 158 of APOE. For the ChAT polymorphism, the melting temperature (61.4°C) of the G allele was higher than that of the A allele (54.7°C). Conclusions: This real-time multiplex PCR technique can be carried out in a single tube and can differentiate between the three polymorphic sites of the two genes associated with Alzheimers disease. Clin Chem Lab Med 2007;45:346–50.

A Review of Haptoglobin Typing Methods for Disease Association Study and Preventing Anaphylactic Transfusion Reaction

Haptoglobin, the product of the Hp gene, is a glycoprotein involved in the scavenging of free hemoglobin. Haptoglobin levels increase or decrease in response to various acquired conditions, and they are also influenced by genetic predisposition. There were 2 major alleles, Hp 1 and Hp 2, and 1 minor allele, Hp del. Many researchers have attempted to study the haptoglobin types and their association with disease; however, no definitive conclusions have been reached yet. It is reported that patients who are genetically deficient in haptoglobin are at risk of anaphylaxis against blood components containing haptoglobin. Haptoglobin genotypes also affect the reference intervals of haptoglobin levels. Many studies have attempted to establish simple and accurate typing methods. In this paper, we have broadly reviewed several methods for haptoglobin typing—phenotyping, Southern blotting, conventional PCR, real-time PCR, and loop-mediated isothermal amplification. We discuss their characteristics, clinical applications, and limitations. The phenotyping methods are time consuming and labor intensive and not designed to detect patients harboring Hp del. The rapid and robust haptoglobin genotyping may help in preventing fatal anaphylactic reactions and in establishing the relationships between the haptoglobin phenotypes and diseases.

Pulmonary infection caused by Mycobacterium conceptionense.

To the Editor: Mycobacterium conceptionense was first identified in 2006 from a patient with posttraumatic osteitis (1). Since then, 3 more isolates have been recovered from a subcutaneous abscess (2), a wound after breast surgery (3), and an abscess after a fat injection (4). During November 2009 through April 2010, M. conceptionense was isolated from sputum from 4 patients in 2 tertiary hospitals in South Korea.