Rapid identification of pathogens directly from blood culture bottles by Bruker matrix-assisted laser desorption laser ionization-time of flight mass spectrometry versus routine methods

The use of matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) for identification of microorganisms directly from blood culture is an exciting dimension to the microbiologists. We evaluated the performance of Bruker SepsiTyper kit™ (STK) for direct identification of bacteria from positive blood culture. This was done in parallel with conventional methods. Nonrepetitive positive blood cultures from 160 consecutive patients were prospectively evaluated by both methods. Of 160 positive blood cultures, the STK identified 114 (75.6%) isolates and routine conventional method 150 (93%). Thirty-six isolates were misidentified or not identified by the kit. Of these, 5 had score of >2.000 and 31 had an unreliable low score of <1.7. Four of 8 yeasts were identified correctly. The average turnaround time using the STK was 35 min, including extraction steps and 30:12 to 36:12 h with routine method. The STK holds promise for timely management of bacteremic patients.     

Mycobacterium heckeshornense lung infection diagnosed by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS)

Identification of microorganisms by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has been widely accepted. However, the significance of MALDI-TOF MS for identifying mycobacteria, particularly rare nontuberculous mycobacteria, has not been established. M. heckeshornense is one such bacteria, and distinguishing it from M. xenopi is difficult. The patient was a 40-year-old man with Beh?et's disease who had started treatment with prednisolone and azathioprine. A lung nodule in the right lower lobe was pointed out, and it increased in size 6 months later. Bronchoscopy was performed, and was culture positive for mycobacteria. It was identified as M. heckeshornense by MALDI-TOF MS with a score value of 1.928. Analysis of the 16S rRNA, rpoB, and hsp65 genes confirmed the result of MALDI-TOF MS. MALDI-TOF MS seems reliable for the diagnosis of M. heckeshornense infection.     

应用基质辅助激光解析电离飞行时间质谱鉴定常见细菌和酵母菌

目的 应用MALDI-TOF MS鉴定临床常见病原菌.方法 复苏560株临床分离株,其中革兰阳性细菌(G+ )260株、革兰阴性细菌(G-)180株、酵母菌60株、肠道致病菌60株.MALDI-TOF MS鉴定结果与Vitek2 Compact全自动微生物鉴定系统比对,结果差异菌株采用16S rDNA测序验证.结果 MALDI-TOF MS与Vitek2 Compact鉴定结果比较,G+细菌鉴定符合率为94.6％( 246/260),G-细菌鉴定符合率为96.7％ (174/180),酵母菌鉴定符合率为95.0％ (57/60),肠道致病菌鉴定符合率为93.3％( 56/60).15株鉴定结果不符的菌株经16S rDNA测序确认,结果与Vitek2Compact和MALDI-TOF MS鉴定符合率分别为26.7％ (4/15)和66.7％( 10/15).结论 MALDI-TOF MS可用于常见病原微生物鉴定,其快速、低成本的特点具有推广价值.     

Multiplex detection of KRAS mutations by a matrix-assisted laser desorption/ionization-time of flight mass spectrometry assay

Objectives

Mutations of the Kirsten rat sarcoma viral oncogene (KRAS) gene are known to be important in the pathogenesis of a variety of cancers. Patients with mutant KRAS tumors do not respond to epidermal growth factor receptor (EGFR) inhibitors and fail to benefit from adjuvant chemotherapy. Testing for KRAS mutations is now being recommended as a tailored therapeutic strategy prior to anti-EGFR treatment; however, the low sensitivity of direct sequencing frequently leads to failure of detection of KRAS mutations in clinical samples.

Design and methods

We developed restriction fragment mass polymorphism (RFMP) assays, to detect KRAS mutations in codons 12, 13, and 61. We performed RFMP analysis for KRAS on DNA isolated from eight different KRAS mutant cell lines and 34 formalin-fixed paraffin-embedded (FFPE) lung cancer tissues.

Results

Overall, the RFMP assay was in good concordance with direct sequencing analysis in the detection of KRAS mutations. By using dilutions of KRAS mutant DNA in wild type DNA from mutation cell lines with a known KRAS status, we confirmed that the RFMP assay has a higher analytical sensitivity, requiring only 3% of cells in a sample to have mutant alleles, compared to direct sequencing, which had a detection threshold of ~ 25%. In the FFPE samples, RFMP successfully detected KRAS genotypes in codons 12, 13, and 61.

Conclusion

The RFMP might be efficiently applicable for the detection of KRAS mutations in a clinical setting, particularly for tumor samples containing abundant non-neoplastic cells.     

基质辅助激光解吸电离飞行时间质谱在临床病原菌鉴定中的应用

目的探讨基质辅助激光解吸电离飞行时间质谱(matrix-assisted laser desorption ionization-time of flight mass spectrometry,MALDI-TOF MS)系统用于快速鉴定临床分离菌的可靠性和实用性。方法收集南京军区南京总医院2013年7~10月自临床标本分离的非重复细菌1 061株,分别使用MALDI-TOF MS和Vitek 2 Compact全自动细菌鉴定仪系统进行鉴定,结果不一致的菌株采用16S r DNA测序验证。结果 1 061株临床分离菌中1 058株(99.7%)经MALDI-TOF MS系统正确鉴定到属水平,1 016株(95.8%)正确鉴定到种,其余3株(0.3%)未给出鉴定结果。5株鉴定结果不一致的菌株经16S r DNA测序确认,结果与MALDI-TOF MS和Vitek 2 Compact鉴定符合率分别为40.0%(2/5)和0(0/5)。结论 MALDI-TOF MS可以作为一个快速、准确和价廉的工具应用于临床分离菌的鉴定。     

Differentiation of Raoultella ornithinolytica/planticola and Klebsiella oxytoca clinical isolates by matrix-assisted laser desorption/ionization-time of flight mass spectrometry

Ninety-nine clinical isolates previously identified as Klebsiella oxytoca were evaluated using matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS). Eight isolates were identified as Raoultella spp., being 5 Raoultella spp. and 3 K. oxytoca, by 16S rRNA sequencing. These isolates were correctly identified by applying the 10% differential rule for the MALDI-TOF MS score values. This approach might be useful to discriminate Raoultella species from K. oxytoca.     

Lactococcus lactis cholangitis and bacteremia identified by MALDI-TOF mass spectrometry: A case report and review of the literature on Lactococcus lactis infection

Lactococcus lactis is a rare causative organism in humans. Cases of L. lactis infection have only rarely been reported. However, because it is often difficult to identify by conventional commercially available methods, its incidence may be underestimated. We herein report the case of a 70-year-old man with cholangiocarcinoma who developed L. lactis cholangitis and review previously reported cases of L. lactis infection. Our case was confirmed by matrix-assisted desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). This case shows L. lactis is a potential causative pathogen of cholangitis and that MALDI-TOF MS can be useful for the rapid and accurate identification of L. lactis infection. We searched the literature for published case reports on cholangitis and any other infections caused by L. lactis, and thereby identified 36 cases, including our case. At least 66.7% (n = 24) of the cases had significant underlying conditions; 15 of the cases involved patients with an immunocompromised status. At least 41.7% (n = 15) had a significant food consumption history, such as the consumption of unpasteurized dairy products. The clinical sources of L. lactis were diverse and endocarditis was the most common diagnosis (n = 8), followed by hepatobiliary infection (n = 6), central nervous system infection (n = 5), and peritonitis (n = 4). The prognosis was favorable in most cases.     

Liver abscess caused by Burkholderia pseudomallei in a young man: A case report and review of literature

Pyogenic liver abscess is a common entity in Indian subcontinent and is mostly caused by gram negative bacteria. Melioidosis is not commonly seen in India and only a few cases are reported. It can give rise to multiple abscesses at different sites including liver. We report a case of isolated liver abscess caused by Burkholderia pseudomallei (B. pseudomallei) in a 29-year-old recently diagnosed diabetic, immunocompetent male. Diagnosis was made by imaging and culture of pus aspirated from the abscess and he was treated with percutaneous pigtail catheter drainage followed by antibiotics (meropenem and trimethoprim-sulphmethoxazole). Melioidosis is an emerging infection in India and has high mortality rate, so early diagnosis and prompt management is warranted which requires clinical vigilance and an intensive microbiological workup. Clinicians should be aware of isolated liver abscess caused by B. pseudomallei in appropriate clinical settings.     

MALDI-TOF 质谱仪在临床常见细菌鉴定中的应用

目的：探讨两种质谱仪 Biotyper MS 和 Vitek-MS 系统在临床常规分离细菌鉴定中的应用价值。方法收集沈阳军区总医院2012年3月至2013年1月分离自血液、尿液、脑脊液、分泌物和痰液等标本的临床常见菌细菌149株（包括14个菌属和30个菌种）。同时采用两种 MALDI-TOF-MS 系统对上述菌株进行鉴定,结果与 Vitek2 compact 常规生化鉴定进行比较,对3种方法检测结果不一致菌株用16S rRNA 测序进行确认。结果在149株常见细菌中,Bruker Biotype 属和种的正确鉴定率分别为98％和96％,Vitek-MS 属和种的正确鉴定率分别为97％和95％。两者均无错误鉴定菌株。结论对该组常见细菌,两种质谱仪鉴定结果比较差异无统计学意义（P ＞0．05）,均呈现出优异的鉴定水平,适合用于临床微生物的常规鉴定。     

侵肺戴阿李斯特菌致菌血症1例

戴阿李斯特菌属(Dialister sp.)是厌氧或微需氧革兰阴性细小球杆菌,含有5个人源性菌种:侵肺戴阿李斯特菌(D. pneumosintes)[1]、浑浊戴阿李斯特菌(D.invisus)[2]、微嗜氧戴阿李斯特菌(D. micraerophilus)、产丙酸戴阿李斯特菌(D.propionicifaciens)...     

Clinical utility of direct application of matrix-assisted laser desorption ionization time-of-flight mass spectrometry and rapid disk diffusion test in presumptive antimicrobial therapy for bacteremia

Objective

To study how and to what degree the rapid pathogen identification by MALDI-TOF MS coupled with rapid disk diffusion test improve the current clinical practice of patients with bacteremia in a tertiary teaching hospital with full-time ID consultation service.

Development and validation of a whole-cell inhibition assay for bacterial methionine aminopeptidase by surface-enhanced laser desorption ionization-time of flight mass spectrometry

Bacterial methionine aminopeptidase (MAP) is a protease that removes methionine from the N termini of newly synthesized bacterial proteins after the peptide deformylase enzyme cleaves the formyl group from the initiator formylmethionine. MAP is an essential bacterial gene product and thus represents a potential target for therapeutic intervention. A fundamental challenge in the antibacterial drug discovery field is demonstrating conclusively that compounds with in vitro enzyme inhibition activity produce the desired antibacterial effect by interfering with the same target in whole bacterial cells. One way to address the activity of inhibitor compounds is by profiling cellular biomarkers in whole bacterial cells using compounds that are known inhibitors of a particular target. However, in the case of MAP, no specific inhibitors were available for such studies. Instead, a genetically attenuated MAP strain was generated in which MAP expression was placed under the control of an inducible arabinose promoter. Thus, MAP inhibition in whole cells could be mimicked by growth in the absence of arabinose. This genetically attenuated strain was used as a benchmark for MAP inhibition by profiling whole-cell lysates for unprocessed proteins using surface-enhanced laser desorption ionization-time of flight mass spectrometry (MS). Eight proteins between 4 and 14 kDa were confirmed as being unprocessed and containing the initiator methionine by adding back purified MAP to the preparations prior to MS analysis. Upon establishing these unprocessed proteins as biomarkers for MAP inhibition, the assay was used to screen small-molecule chemical inhibitors of purified MAP for whole-cell activity. Fifteen compound classes yielded three classes of compound with whole-cell activity for further optimization by chemical expansion. This report presents the development, validation, and implementation of a whole-cell inhibition assay for MAP.     

基质辅助激光解吸电离飞行时间质谱快速鉴定革兰阴性杆菌的研究

目的评价基质辅助激光解吸电离飞行时间质谱（matrix assisted laser desorption ionisation time of flight mass spectrometry, MALDI-TOF-MS）技术检测临床常见革兰阴性杆菌的临床应用价值。方法1665株革兰阴性杆菌经VITEK2Compact鉴定到属和种,采用MALDI—TOF—MS对1665株革兰阴性杆菌进行种属检测,并计算鉴定正确率。结果1665株菌包括27个菌属,73个菌种。主要革兰阴性杆菌菌属鉴定整体正确率为95．7％,菌种整体正确率为85．5％。其中菌属鉴定正确率最高的是布鲁菌属和沙门菌属,正确率均为100．0％;菌种鉴定正确率最高的为危险罗尔斯顿菌和摩氏摩根菌,正确率均为100．0％。结论MALDI—TOF—MS技术鉴定细菌快速、准确、方便,且成本低,适用于临床实验室的快速鉴定诊断。     

The expanding use of matrix-assisted laser desorption/ionization-time of flight mass spectroscopy in the diagnosis of patients with mycotic diseases

Introduction: Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has emerged as a powerful new tool to identify human fungal pathogens and has radically altered the diagnostic mycology workflow at many medical centers around the world.

Areas covered: While most experience is with the identification of yeasts, including species of Candida and Cryptococcus, there is ongoing work investigating the role of MALDI-TOF MS to detect molds, including species of Aspergillus, Fusarium, Scedosporium, and Mucormyctes as well as thermally dimorphic fungi.

Expert commentary: In this paper, we review the current knowledge about this important new platform and examine how its expanding use may impact molecular diagnostics and patient care in the years ahead.     

Rapid detection of twenty-nine common Chinese glucose-6-phosphate dehydrogenase variants using a matrix-assisted laser desorption/ionization-time of flight mass spectrometry assay on dried blood spots

BackgroundGlucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common inherited disease. Current neonatal screening methods for G6PD deficiency primarily rely on the use of biochemical tests. However, only 15%–20% of female carriers were estimated to have been detected using these tests. As a better alternative, DNA-based tests could be used for G6PD deficiency screening. We aimed to develop a matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) assay for G6PD variant detection.MethodsA MALDI-TOF MS assay with multiprimer extension (multi-PEX) was developed to rapidly and accurately detect the 29 common G6PD variants in the Chinese population using a dried blood spot as a template. A parallel study screening 571 unrelated neonatal samples using the MALDI-TOF MS and fluorescence quantitative enzymatic assays was performed. All results were confirmed by Sanger sequencing in a blind study.ResultsIn 571 unrelated neonatal samples, 34 positive samples, including 26 samples from hemizygous males and eight samples from heterozygous females, were correctly identified, yielding a clinical sensitivity of 100%. The results were validated using Sanger sequencing with 100% concordance. In contrast, the fluorescence quantitative enzymatic assay had a 75% false negative and 88.8% false positive rate for the detection of heterozygous G6PD deficient females.ConclusionsWe established a reliable MALDI-TOF MS assay for G6PD deficiency screening in the Chinese population maximizing the chance of detection of heterozygous G6PD deficient females and reducing the false negative and false positive rates associated with routinely used newborn screening procedures.     

Improvement of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry identification of difficult-to-identify bacteria and its impact in the workflow of a clinical microbiology laboratory

This study evaluates matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) capability for the identification of difficult-to-identify microorganisms. A total of 150 bacterial isolates inconclusively identified with conventional phenotypic tests were further assessed by 16S rRNA sequencing and by MALDI-TOF MS following 2 methods: a) a simplified formic acid-based, on-plate extraction and b) performing a tube-based extraction step. Using the simplified method, 29 isolates could not be identified. For the remaining 121 isolates (80.7%), we obtained a reliable identification by MALDI-TOF: in 103 isolates, the identification by 16S rRNA sequencing and MALDI TOF coincided at the species level (68.7% from the total 150 analyzed isolates and 85.1% from the samples with MALDI-TOF result), and in 18 isolates, the identification by both methods coincided at the genus level (12% from the total and 14.9% from the samples with MALDI-TOF results). No discordant results were observed. The performance of the tube-based extraction step allowed the identification at the species level of 6 of the 29 unidentified isolates by the simplified method. In summary, MALDI-TOF can be used for the rapid identification of many bacterial isolates inconclusively identified by conventional methods.     

Muscular abscess caused by Cupriavidus gilardii in a renal transplant recipient

Cupriavidus gilardii is a rare cause of human infection. We report a muscular abscess on the right thigh caused by this organism in a renal transplant recipient, who had suffered a septic shock associated with an extensive cellulitis caused by Streptococcus pyogenes. The patient was successfully treated with surgical drainage and intravenous ciprofloxacin for 13 days. This is the first time that C. gilardii is isolated from a human abscess. C. gilardii should be considered as a cause of human infection, especially in immunocompromised patients. Infection caused by this organism may be underdiagnosed because the identification is very difficult.     

A rare case of a prostatic abscess,bacteremia and chronic granulomatous disease associated with Klebsiella pneumoniae

Chronic granulomatous disease (CGD) is a primary immunodeficiency disease characterized by severe recurrent infections such as pneumonia, liver and skin infections. However, prostatic abscesses are rare as only two cases have been reported thus far. We present the case of a 41-year-old patient with CGD who was admitted to the hospital with fever and subsequently, Klebsiella pneumoniae was identified on blood culture. Abdominal computed tomography revealed a prostatic abscess. He improved with intravenous antibiotics and drainage of the abscess. After he was taken off the intravenous antibiotics and started on an oral agent, he was discharged from the hospital. We confirmed a reduction in the prostatic abscess size and continued the antibiotic therapy for 52 days. A prostatic abscess is an uncommon disease being diagnosed at a median age of 49 years. Sometimes it is discovered in patients with fever of unknown origin and might be considered as an infection site of CGD patients.