•SARS-CoV-2 RNA detection is performed for clinical diagnosis of COVID-19.•The detection of two different targets on the SARS-CoV-2 genome is recommended.•An internal control is required for ...evaluating the assay qualities.•A multiplex rRT-PCR methodology was developed for SARS-CoV-2 RNA detection.•A multiplex rRT-PCR will enable reducing reagent use and cost, and handling time.
The detection of SARS-CoV-2 RNA by real-time reverse transcription–polymerase chain reaction (rRT-PCR) is used to confirm the clinical diagnosis of COVID-19 by molecular diagnostic laboratories. We developed a multiplex rRT-PCR methodology for the detection of SARS-CoV-2 RNA.
Three genes were used for multiplex rRT-PCR: the Sarbecovirus specific E gene, the SARS-CoV-2 specific N gene, and the human ABL1 gene as an internal control.
Good correlation of Cq values was observed between the simplex and multiplex rRT-PCR methodologies. Low copies (<25 copies/reaction) of SARS-CoV-2 RNA were detected by the novel multiplex rRT-PCR method.
The proposed multiplex rRT-PCR methodology will enable highly sensitive detection of SARS-CoV-2 RNA, reducing reagent use and cost, and time required by clinical laboratory technicians.
Invasive fungal and bacterial infections are becoming increasingly important concerns in infants hospitalized in neonatal intensive care units (NICUs). Obtaining information on airborne pathogens in ...the NICU is important for evaluating the risk of hospital-acquired infections (HAIs) and for implementing measures to prevent HAIs in the NICU. This study aimed to evaluate the distribution of culturable airborne fungal and bacterial species in indoor air from six different hospital NICUs in Chiba Prefecture, Japan. Bacterial and fungal isolation from the air was performed using a low-volume air sampler.Fungi isolated from the samples were identified using molecular methods. The bacteria isolated from the samples were identified using matrix-assisted laser desorption time-of-flight mass spectrometry. A few pathogenic fungi, including Candida parapsilosis, were detected in the air samples. Staphylococcus spp., including methicillin-resistant Staphylococcus aureus, were mainly detected in the air samples. Based on the results of this study, the analysis of air samples in the NICU can be considered a good method for identifying the source of infection and preventing infection during outbreaks of fungi and resistant bacteria in the NICU.
Aspergillus spp. belong to filamentous fungi and sometimes cause invasive aspergillosis which has high mortality. Filamentous fungi are generally identified morphologically. However, morphologic ...identification is time consuming and requires advanced skills. It is difficult to train technicians and ensure a high level of quality. Therefore, an identification technique that is both accurate and relatively easy to learn is needed. In the present study, we focused on the effects of Yatalase and silica beads, which enable the efficient extraction of proteins via cell wall disruption of Aspergillus spp., and aimed to establish a novel sample preparation method using Yatalase and silica beads to enhance the efficiency of Aspergillus spp. identification with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The sample preparation method using the combination of Yatalase and silica beads showed higher accuracy for the identification of Aspergillus spp. compared with Yatalase or silica beads alone. The Yatalase/silica beads method also resulted in significantly higher identification scores compared with the conventional method for the identification of Aspergillus fumigatus (n = 33). These findings indicate that our novel Yatalase/silica beads method provides more reliable identification of A. fumigatus than does the conventional method.
Aspergillus spp., which belong to filamentous fungi, occasionally cause invasive aspergillosis, the mortality rate of has been high. Filamentous fungi are generally identified morphologically. However, morphologic identification is time consuming and requires advanced skills. It is difficult to train technicians and ensure quality morphologic identification. Therefore, an identification technique that is both accurate and relatively easy to learn is needed. We aimed to establish a novel sample preparation method using Yatalase and silica beads to enhance the efficiency of Aspergillus spp. identification with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The obtained results indicated that our novel Yatalase/silica bead protocol more reliably identified Aspergillus fumigatus compared to the conventional method. Display omitted
•It is difficult to identify Aspergillus spp. including Aspergillus fumigatus using MALDI-TOF MS.•We evaluated a novel sample preparation method for MALDI-TOF MS identification that combines Yatalase and silica beads.•The novel Yatalase/silica beads method provides more reliable identification of A. fumigatus than the conventional method.
Although polymerase chain reaction (PCR) amplification and sequencing of the bacterial 16S rDNA region has numerous scientific applications, it does not provide DNA methylation information. Herein, ...we propose a simple extension for bisulfite sequencing to investigate 5-methylcytosine residues in the bacterial 16S rDNA region from clinical isolates or flora. Multiple displacement amplification without DNA denaturation was used to preferentially pre-amplify single-stranded bacterial DNA after bisulfite conversion. Following the pre-amplification, the 16S rDNA region was analyzed using nested bisulfite PCR and sequencing, enabling the simultaneous identification of DNA methylation status and sequence data. We used this approach (termed sm16S rDNA PCR/sequencing) to identify novel methylation sites and a methyltransferase (M. MmnI) in Morganella morganii and different methylation motifs among Enterococcus faecalis strains from small volumes of clinical specimens. Further, our analysis suggested that M. MmnI may be correlated to erythromycin resistance. Thus, sm16S rDNA PCR/sequencing is a useful extension method for analyzing the DNA methylation of 16S rDNA regions in a microflora, providing additional information not provided by conventional PCR. Given the relationship between DNA methylation status and drug resistance in bacteria, we believe this technique can be effectively applied in clinical sample testing.
The prevalence of nonencapsulated Streptococcus pneumoniae (NESp) has increased with the introduction of pneumococcal conjugate vaccines in children; however, the bacteriological characteristics of ...NESp have not been sufficiently clarified. In this study, NESp strains isolated from the nasopharyngeal carriage of children from four nursery schools in Japan were analyzed for molecular type, antibiotic susceptibility, and biofilm productivity. A total of 152 putative S. pneumoniae strains were identified by optochin-susceptibility analysis, of which 21 were not serotypeable by slide agglutination, quellung reaction, or multiplex PCR. Among these 21 strains, three were lytA-negative and, therefore, not S. pneumoniae. The remaining 18 strains were positive for lytA, ply, pspK, and bile solubility and were confirmed as NESp. Therefore, the isolation rate of NESp in the S. pneumoniae strains in this study was 12.0% (18/149). Molecular-typing analyses classified five strains as two existing sequence types (STs; ST7502 and ST7786), and 13 strains formed four novel STs. Horizontal spread was suspected, because strains with the same ST were often isolated from the same nursery school. The NESp isolates were generally susceptible to most antimicrobials, with the exception of macrolides; however, all isolates possessed more than one abnormal penicillin-binding protein gene. Furthermore, NESp strains were more effective than encapsulated counterparts at forming biofilms, which showed obvious differences in morphology. These data indicated that NESp strains should be continuously monitored as emerging respiratory pathogens.
Matrix-assisted laser desorption ionization–time-of-flight mass spectrometry (MALDI-TOF MS) is one of the most promising technologies for the identification of microbial pathogens directly from ...positive blood culture bottles. As blood culture bottle medium contains various nonbacterial proteins, including those derived from blood cells, pretreatment to effectively remove host cells is key for successful proteome-based identification of microorganisms. Although the Sepsityper® kit is the most widely used pretreatment protocol, its performance is not satisfactory, particularly for gram-positive isolates. We developed a new in-house protocol, the centrifugation and membrane filtration technique (CMFT), in which vacuum-filtration is coupled with differential centrifugation. We prospectively evaluated the performance of this novel method compared with that of the Sepsityper®. For gram-negative bacterial isolates, the species-level identification rates obtained with the CMFT and the Sepsityper® were comparable (98.8% vs 92.9%). By contrast, for gram-positive isolates, the performance of the CMFT was significantly better than that of the Sepsityper® (P < 0.05). Using our new protocol, 81 (95.3%) isolates were identified with a score >2.0, and 85 (100%) isolates were identified with a score >1.7, versus 46 (54.1%) and 69 (81.2%), respectively, for the Sepsityper®. These results are preliminary, but considering that this novel protocol provides notably high species-level identification rates for gram-positive isolates, it deserves assessment in a larger-scale study with a variety of platforms for MS-based identification of microorganisms.
Methicillin-resistant Staphylococcus aureus (MRSA) is one of the major pathogens responsible for nosocomial infections. The presence of MRSA in a hospital is detrimental to patients and to hospital ...management. Thus, rapid identification of MRSA is needed. Here, we report on a prospective method to rapidly discriminate of MSSA from MRSA using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and support vector machine (SVM) analysis in 160 clinical isolates of S. aureus. The predictive model was tested using 100 S. aureus isolates (50 MSSA and 50 MRSA). The identification rates were 90.0% for MSSA and 87.5% for MRSA in a 10-fold cross-validation SVM. In blind test sets, 60 S. aureus isolates (30 MSSA and 30 MRSA) were correctly classified, with identification rates of 93.3% for MSSA and 86.7% for MRSA. The method proposed in this study using the predictive model enables detection of one colony in 5 minutes, and thus is useful at clinical sites at which rapid discrimination of MRSA from MSSA is required.
Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) is increasingly used for identification of microorganisms from positive blood cultures. Pretreatments to ...effectively remove non-bacterial components and selectively collect microorganisms are a prerequisite for successful identification, and a variety of home-brew and commercial protocols have been reported. Although commercially available kits, mainly the Sepsityper Kit, are increasingly used, the identification rates reported often are not satisfactory, particularly for Gram-positive isolates. We recently developed a method to collect bacteria from positive blood culture bottles using a polyallylamine-polystyrene copolymer that has been used in wastewater processing. This pretreatment protocol is now commercially available as the rapid BACpro® II kit (Nittobo Medical Co., Tokyo, Japan). The operation time required for processing using this novel kit is approximately 10 min, and the entire procedure can be completed within a biosafety cabinet.
Since the performance of the rapid BACpro® II kit has not been tested using the MALDI Biotyper system, we prospectively evaluated the performance of the rapid BACpro® II kit as compared with the Sepsityper® kit.
Performance of the rapid BACpro® II kit was evaluated using a total of 193 monomicrobial cases of positive blood culture. Medium from blood culture bottles was pretreated by the rapid BACpro® II kit or the Sepsityper® Kit, and isolated cells were subjected to direct identification by MS fingerprinting in parallel with conventional subculturing for reference identification. The overall MALDI-TOF MS-based identification rates with >1.7 score and >2.0 score obtained using the rapid BACpro® II kit were 99.5% and 80.8%, respectively, whereas those obtained using the Sepsityper® Kit were 89.1% and 68.4%, respectively (P < 0.05 for >1.7 and P < 0.05 for >2.0 by Pearsons's chi-square). In Gram-positive cases, the rapid BACpro® II kit gave identification rate of 100% with >1.7 score and 69.4% with >2.0 score, whereas there were 84.7% and 56.8%, respectively by the Sepsityper® Kit (P < 0.05 for >1.7).
These results are preliminary, but considering that this new kit is easy to perform and the identification rates are promising, the rapid BACpro® II kit deserves assessment in a larger-scale study with a variety of platforms for MS-based bacterial identification.
•We prospectively evaluated the performance of the rapid BACpro® II kit.•The kit consists of four simple steps that can be done within a biosafety cabinet.•The kit protocol is a rapid method for collecting bacteria within about 10 min.
•Mass spectrometry is a highly efficient tool for clinical bacterial identification.•Sample preparation at 40% humidity provides optimal serum peptidomic results.•Sample preparation at 30–40% ...humidity is optimal for bacterial identification.•These results could lead to improved accuracy of bacterial identification.
Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a highly reliable and efficient technology for the identification of microbial pathogens. We previously found that 40% humidity was the optimal condition for the preparation of samples (co-crystallization of the sample and matrix) for serum peptidomic analysis via MALDI-TOF MS profiling. This optimum temperature was applied to obtain the highest reproducibility and throughput and greatest number of peaks. We therefore hypothesized that humidity control was also essential for MALDI-TOF MS bacterial identification. In this study, we constructed a simple sample preparation device that enables humidity control and used it for co-crystallization of the sample and matrix. Identification scores for five Gram-negative bacteria and six Gram-positive bacteria were determined using the MALDI BioTyper® system at three humidity ranges (10–20%, 30–40%, and 50–60%). As a result, higher identification scores were obtained at 30–40% humidity than at 10–20% or 50–60% humidity. At 30–40% humidity, 517/550 (94.0%) isolates scored greater than 2.0, indicating the success of species-level identification. Similarly, 537/550 (97.6%) isolates scored greater than 1.7, indicating the success of genus-level identification. Thus, 30–40% humidity generated optimal MALDI-TOF MS identification scores and the highest percentage of correct identifications. These results could lead to further improvements in the accuracy of MALDI-TOF MS bacterial identification.
In a clinical diagnosis microbiology laboratory, the current method of identifying bacterial isolates is based mainly on phenotypic characteristics, for example growth pattern on different media, ...colony morphology, Gram stain, and various biochemical reactions. These techniques collectively enable great accuracy in identifying most bacterial isolates, but are costly and time-consuming. In our clinical microbiology laboratory, we prospectively assessed the ability of matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI–TOF MS) to identify bacterial strains that were routinely isolated from clinical samples. Bacterial colonies obtained from a total of 468 strains of 92 bacterial species isolated at the Department of Clinical Laboratory at Chiba University were directly placed on target MALDI plates followed by addition of CHCA matrix solution. The plates were then subjected to MALDI–TOF MS measurement and the microorganisms were identified by pattern matching with the libraries in the BioTyper 2.0 software. Identification success at the species and genus levels was 91.7% (429/468) and 97.0% (454/468), respectively. MALDI–TOF MS is a rapid, simple, and high-throughput proteomic technique for identification of a variety of bacterial species. Because colony-to-colony differences and effects of culture duration on the results are minimal, it can be implemented in a conventional laboratory setting. Although for some pathogens, preanalytical processes should be refined, and the current database should be improved to obtain more accurate results, the MALDI–TOF MS based method performs, in general, as well as conventional methods and is a promising technology in clinical laboratories.
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