The impact analysis of a multiplex PCR respiratory panel for hospitalized pediatric respiratory infections in Japan.

BACKGROUND: Rapid molecular diagnosis of infections has contributed to timely treatments and antimicrobial stewardship. However, the benefit and cost-effectiveness vary in each country or community because they have different standard practices and health care systems. In Japan, rapid antigen tests (RATs) have been frequently used for pediatric respiratory infections. We investigated the impact and cost-effectiveness of a multiplex PCR (mPCR) respiratory panel for pediatric respiratory infections in a Japanese community hospital. METHODS: We replaced RATs with an mPCR respiratory panel (FilmArray®) for admitted pediatric respiratory infections on March 26, 2018. We compared the days of antimicrobial therapy (DOT) and length of stay (LOS) during the mPCR period (March 2018 to April 2019) with those of the RAT period (March 2012 to March 2018). RESULTS: During the RAT and mPCR periods, 1132 and 149 patients were analyzed. The DOT/case was 12.82 vs 8.56 (p < 0.001), and the LOS was 8.18 vs 6.83 days (p = 0.032) in the RAT and mPCR groups, respectively. The total costs during admissions were ∖258,824 ($2331.7) and ∖243,841 ($2196.8)/case, respectively. Pathogen detection rates were 30.2% vs 87.2% (p < 0.001). CONCLUSION: Compared to conventional RATs, the mPCR test contributed to a reduction in the DOT and LOS in a Japanese community hospital for admission-requiring pediatric respiratory infections. However, a proper stewardship program is essential to further reduce the unnecessary usage of antimicrobials.

Escherichia coli H-Genotyping PCR: a Complete and Practical Platform for Molecular H Typing.

In Escherichia coli, more than 180 O groups and 53 H types have been recognized. The O:H serotyping of E. coli strains is an effective method for identifying strains with pathogenic potential and classifying them into clonal groups. In particular, the serotyping of Shiga toxin-producing E. coli (STEC) strains provides valuable information to evaluate the routes, sources, and prevalence of agents in outbreak investigations and surveillance. Here, we present a complete and practical PCR-based H-typing system, E. coli H-genotyping PCR, consisting of 10 multiplex PCR kits with 51 single PCR primer pairs. Primers were designed based on a detailed comparative analysis of sequences from all H-antigen (flagellin)-encoding genes, fliC and its homologs. The specificity of this system was confirmed by using all H type reference strains. Additionally, 362 serotyped wild strains were also used to evaluate its practicality. All 277 H-type-identified isolates gave PCR products that corresponded to the results of serological H typing. Moreover, 76 nonmotile and nine untypeable strains could be successfully subtyped into any H type by the PCR system. The E. coli H-genotyping PCR developed here allows broader, rapid, and low-cost subtyping of H types and will assist epidemiological studies as well as surveillance of pathogenic E. coli.

Nuclear and Wolbachia-based multimarker approach for the rapid and accurate identification of tsetse species.

BACKGROUND: Tsetse flies (Diptera: Glossinidae) are solely responsible for the transmission of African trypanosomes, causative agents of sleeping sickness in humans and nagana in livestock. Due to the lack of efficient vaccines and the emergence of drug resistance, vector control approaches such as the sterile insect technique (SIT), remain the most effective way to control disease. SIT is a species-specific approach and therefore requires accurate identification of natural pest populations at the species level. However, the presence of morphologically similar species (species complexes and sub-species) in tsetse flies challenges the successful implementation of SIT-based population control. RESULTS: In this study, we evaluate different molecular tools that can be applied for the delimitation of different Glossina species using tsetse samples derived from laboratory colonies, natural populations and museum specimens. The use of mitochondrial markers, nuclear markers (including internal transcribed spacer 1 (ITS1) and different microsatellites), and bacterial symbiotic markers (Wolbachia infection status) in combination with relatively inexpensive techniques such as PCR, agarose gel electrophoresis, and to some extent sequencing provided a rapid, cost effective, and accurate identification of several tsetse species. CONCLUSIONS: The effectiveness of SIT benefits from the fine resolution of species limits in nature. The present study supports the quick identification of large samples using simple and cost effective universalized protocols, which can be easily applied by countries/laboratories with limited resources and expertise.

Should We Be Testing for Baseline Integrase Resistance in Patients Newly Diagnosed With Human Immunodeficiency Virus?

Background: Current guidelines recommend genotype resistance testing at diagnosis to guide initial selection of antiretroviral therapy (ART). Many standard resistance genotypes exclude testing for resistance to integrase inhibitors ("IR testing"), although this class of drugs is a component of most recommended first-line regimens. Methods: We compared the 96-week clinical outcomes and cost-effectiveness of 2 strategies: no IR testing vs IR testing performed at human immunodeficiency virus (HIV) diagnosis. The base case prevalence of transmitted integrase strand transfer inhibitor (INSTI)-resistant (INSTI-R) virus is estimated at 0.1%. With no IR testing, all patients start dolutegravir (DTG)-based ART after genotype; 12-week suppression rates are 90% (INSTI-susceptible [INSTI-S] virus) and 35% (INSTI-R virus). Those not suppressed at 12 weeks undergo IR testing; if diagnosed with INSTI-R virus, they change to ritonavir-boosted darunavir (DRV/r)-based ART. With IR testing, all patients are diagnosed with INSTI-S/INSTI-R virus prior to ART initiation and start DTG- or DRV/r-based regimens, respectively. Costs include IR tests (175 US dollars [USD]) and ART (41100-44900 USD/year). We examined the impact of key parameters in sensitivity analyses. Results: IR testing resulted in worse clinical outcomes compared to no IR testing and increased costs by 200 USD/person/year. Prevalence of transmitted INSTI-R virus did not affect the favored strategy. No IR testing remained clinically preferred unless DTG suppression of INSTI-R virus was <20% or 96-week DRV/r suppression was >92%. If quality of life was worse with DRV/r- than DTG-based ART, no IR testing was clinically preferred over an even broader range of parameters. Conclusions: In patients with newly diagnosed HIV, IR testing is projected to result in worse outcomes and is not cost-effective. Pretreatment assessment for INSTI resistance should not be recommended in treatment guidelines.

A novel and rapid diagnostic method for discriminating between feces of sika deer and Japanese serow by loop-mediated isothermal amplification.

Severe damages to natural vegetation, agriculture, and forestry caused by overpopulation of sika deer (Cervus nippon) have markedly increased in Japan in recent years. To devise a population management plan of sika deer, information on the distribution and population size of the animal in each region is indispensable. An easy and effective method to obtain this information is to count the fecal pellets in the field. However, the habitat of sika deer in Japan overlaps that of Japanese serow (Capricornis crispus). Additionally, it is difficult to discriminate between the feces of both animals. Here, we present a rapid and precise diagnostic method for discriminating between the feces of sika deer and Japanese serow using loop-mediated isothermal amplification (LAMP) targeting cytochrome b gene in the mitochondrial DNA. Our results showed that the LAMP can discriminate between the feces of sika deer and Japanese serow, and the method is simpler and more sensitive than the conventional molecular diagnostic method. Since LAMP method does not require special skills for molecular biology techniques, even the field researchers who have never done a molecular experiment can easily carry out the protocol. In addition, the entire protocol, from DNA extraction from fecal pellet to identification of species, takes only about 75 min and does not require expensive equipment. Hence, this diagnostic method is simple, fast, and accessible to anyone. As such, the method can be a useful tool to estimate distribution and population size of sika deer.

New clustered regularly interspaced short palindromic repeat locus spacer pair typing method based on the newly incorporated spacer for Salmonella enterica.

A clustered regularly interspaced short palindromic repeat (CRISPR) typing method has recently been developed and used for typing and subtyping of Salmonella spp., but it is complicated and labor intensive because it has to analyze all spacers in two CRISPR loci. Here, we developed a more convenient and efficient method, namely, CRISPR locus spacer pair typing (CLSPT), which only needs to analyze the two newly incorporated spacers adjoining the leader array in the two CRISPR loci. We analyzed a CRISPR array of 82 strains belonging to 21 Salmonella serovars isolated from humans in different areas of China by using this new method. We also retrieved the newly incorporated spacers in each CRISPR locus of 537 Salmonella isolates which have definite serotypes in the Pasteur Institute's CRISPR Database to evaluate this method. Our findings showed that this new CLSPT method presents a high level of consistency (kappa = 0.9872, Matthew's correlation coefficient = 0.9712) with the results of traditional serotyping, and thus, it can also be used to predict serotypes of Salmonella spp. Moreover, this new method has a considerable discriminatory power (discriminatory index [DI] = 0.8145), comparable to those of multilocus sequence typing (DI = 0.8088) and conventional CRISPR typing (DI = 0.8684). Because CLSPT only costs about $5 to $10 per isolate, it is a much cheaper and more attractive method for subtyping of Salmonella isolates. In conclusion, this new method will provide considerable advantages over other molecular subtyping methods, and it may become a valuable epidemiologic tool for the surveillance of Salmonella infections.

High-resolution melt PCR analysis for genotyping of Ureaplasma parvum isolates directly from clinical samples.

Ureaplasma sp. infection in neonates and adults underlies a variety of disease pathologies. Of the two human Ureaplasma spp., Ureaplasma parvum is clinically the most common. We have developed a high-resolution melt (HRM) PCR assay for the differentiation of the four serovars of U. parvum in a single step. Currently U. parvum strains are separated into four serovars by sequencing the promoter and coding region of the multiple-banded antigen (MBA) gene. We designed primers to conserved sequences within this region for PCR amplification and HRM analysis to generate reproducible and distinct melt profiles that distinguish clonal representatives of serovars 1, 3, 6, and 14. Furthermore, our HRM PCR assay could classify DNA extracted from 74 known (MBA-sequenced) test strains with 100% accuracy. Importantly, HRM PCR was also able to identify U. parvum serovars directly from 16 clinical swabs. HRM PCR performed with DNA consisting of mixtures of combined known serovars yielded profiles that were easily distinguished from those for single-serovar controls. These profiles mirrored clinical samples that contained mixed serovars. Unfortunately, melt curve analysis software is not yet robust enough to identify the composition of mixed serovar samples, only that more than one serovar is present. HRM PCR provides a single-step, rapid, cost-effective means to differentiate the four serovars of U. parvum that did not amplify any of the known 10 serovars of Ureaplasma urealyticum tested in parallel. Choice of reaction reagents was found to be crucial to allow sufficient sensitivity to differentiate U. parvum serovars directly from clinical swabs rather than requiring cell enrichment using microbial culture techniques.

Qualitative Analysis To Ascertain Genotypic Identity of or Differences between Mycobacterium tuberculosis Isolates in Laboratories with Limited Resources.

Mycobacterium tuberculosis is currently genotyped using mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) typing, although the high cost of this technique restricts its implementation in resource-limited settings. We designed a MIRU-VNTR format, MLP3 (MIRU-VNTR length polymorphism triplex), that is based on the qualitative comparison of 5 nonfluorescent 3-band fingerprints in conventional electrophoresis and minimizes costs and technical demands. MLP3 successfully resolved cross-contamination alerts, discriminated reinfections from reactivations, clarified suspected microepidemics, and tracked transmission events of high epidemiological interest.

Real-time PCR genotyping of Neisseria gonorrhoeae isolates using 14 informative single nucleotide polymorphisms on gonococcal housekeeping genes.

OBJECTIVES: Neisseria gonorrhoeae multilocus sequence typing (MLST) is a key tool used to investigate the macroepidemiology of gonococci exhibiting antimicrobial resistance (AMR). However, the utility of MLST is undermined by the high workload and cost associated with DNA sequencing of seven housekeeping genes. In this study, we investigated single nucleotide polymorphism (SNP)-based profiling as a means of circumventing these problems. METHODS: A total of 14 SNPs were selected following in silico analysis of available N. gonorrhoeae MLST sequence data. Real-time PCR methods were developed for characterization of each SNP and applied to 86 N. gonorrhoeae isolates exhibiting a range of ceftriaxone MICs. Twenty-one isolates had previously been characterized by MLST. The ability of the real-time PCR methods to generate SNP profiles and of the 14 SNP profiles to predict MLST types were assessed. RESULTS: In silico analysis of the 217 different MLST types available on the Neisseria web site showed 181 different 14 SNP profiles (Simpson's index of diversity = 0.998). When the real-time PCR methods were applied to the isolates, 29 different 14 SNP profiles were obtained for 83 isolates. Predicted MLST types were consistent with those for the 21 isolates previously characterized by MLST. For 46 isolates with raised ceftriaxone MICs (≥ 0.03 mg/L), there were 14 different 14 SNP profiles observed, with two profiles accounting for more than half of these isolates. CONCLUSIONS: The 14 SNP real-time PCR profiling approach is a simple and cost-effective alternative to N. gonorrhoeae MLST and could be used to complement current typing schemes in N. gonorrhoeae AMR investigations.

Analysis of strain relatedness using high resolution melting in a case of recurrent candiduria.

BACKGROUND: Several genotyping protocols have been described to study Candida albicans strains with different sensitivity values. In this study we have analyzed the genetic relatedness and the antifungal susceptibility of several Candida albicans strains isolated from a patient who from suffered recurrent candiduria for a period of five years. Strains were genotyped using Microsatellite Length Polymorphism (MLP) with three microsatellite markers (HIS 3, EF 3 and CDC 3), and a new method based on high resolution melting (HRM) was developed to analyze the microsatellite region. This method was compared with the conventional technique that uses capillary electrophoresis. RESULTS: MICs of the isolates showed the existence of fluconazole susceptible and resistant strains. An inter-colony test using single concentration (8 and 16 mg/l) of fluconazole revealed the coexistence of both fluconazole susceptible and resistant strains. Both genotyping analysis methods showed that all the patient's isolates had a clonal origin. HRM analysis method developed was able to accurately establish strain relatedness and presented a discriminatory power of 0.77. CONCLUSIONS: Although HRM analysis method presented a lower discriminatory power compared to methods based on capillary electrophoresis, it provided a more cost-effective and suitable alternative for genotyping C. albicans in a clinical laboratory.

Evaluation of MALDI-TOF mass spectrometry for the identification of medically-important yeasts in the clinical laboratories of Dijon and Lille hospitals.

Conventional identification (CI) of yeasts is based on morphological, biochemical and/or immunological methods. Matrix-assisted laser desorption/ionization - time of flight (MALDI-TOF or MT-MS) mass spectrometry has been proposed as a new method for the identification of microorganisms. This prospective study compared the performance of MT-MS and CI for the identification of yeasts isolated from clinical samples. Sequencing of the internal transcribed spacer (ITS) regions of ribosomal DNA was used as the reference method in the analysis of a total of 1207 yeast isolates. Concordance between MT-MS and CI was observed for 1105 isolates (91.5%), while 74 isolates (6.1%) were misidentified. Molecular identification revealed that 73 of these 74 isolates were identified correctly by MT-MS and CI correctly identified the last one. Concordance between the two techniques was excellent for the medically-important species (98-100%), including the identification of closely-related species (Candida albicans/C. dubliniensis; C. inconspicua/C. norvegensis; C. parapsilosis/C. metapsilosis/C. orthopsilosis). Only 2.3% of isolates belonging to C. famata, C. lambica and C. magnoliae or to Geotrichum spp. and Trichosporon spp. were not identified by MT-MS. This investigation highlights the potential of MT-MS-based yeast identification as a reliable, time and cost-efficient alternative to CI.

A PCR-based intergenic spacer region-capillary gel electrophoresis typing method for identification and subtyping of Nocardia species.

While 16S rRNA sequence-based identification of Nocardia species has become the gold standard, it is not without its limitations. We evaluated a novel approach encompassing the amplification of the Nocardia 16S-23S rRNA intergenic spacer (IGS) region followed by fragment analysis by capillary gel electrophoresis (CGE) of the amplified product for species identification of Nocardia. One hundred forty-five Nocardia isolates (19 species) and four non-Nocardia aerobic actinomycetes were studied. Reproducibility testing was performed in a subset (21%) of isolates. Ninety-five different electropherograms were identified, with heterogeneity within species being a general observation. Among common Nocardia species (e.g., Nocardia cyriacigeorgica, N. nova, N. farcinica), 2 or 3 dominant electropherogram subgroups were typical. While only a minority (8/19; 42%) of the different Nocardia species contained isolates displaying unique fragment sizes that were predictive of a particular species, virtually all isolates (142/145; 98%) could be assigned to the correct species using IGS-CGE typing based on the number and size of amplified fragments. The median number of fragments for each isolate was 2 (range, 1 to 5) with only a minority (17%) having a single fragment detected. The majority (93%) of amplified fragments were between 408 and 461 bp. The technique was also non-operator dependent, highly reproducible, and quicker and less expensive than 16S sequencing. In summary, PCR-based IGS-CGE typing is relatively simple, accurate, reproducible, and cost-effective and offers a potential alternative to 16S rRNA sequencing for identifying and subtyping Nocardia isolates.

Microfluidic-chip-based multiple-locus variable-number tandem-repeat fingerprinting with new primer sets for methicillin-resistant Staphylococcus aureus.

The detection of outbreaks of methicillin-resistant Staphylococcus aureus (MRSA) infections and a rapid and accurate identification of sources and routes of transmission should be conducted in hospital settings as early and swiftly as possible. In this study, we investigated the application potential of a new approach based on multiple-locus variable-number tandem-repeat fingerprinting (MLVF) and microfluidics technology for a rapid discrimination of MRSA lineages in outbreak settings. A total of 206 nonrepetitive MRSA isolates recovered from infected patients at the University Medical Center Groningen between 2000 and 2010 were tested. The results obtained by MLVF using microcapillary electrophoresis with newly designed primers were compared to those obtained by spa typing and multiple-locus variable-number tandem-repeat analysis (MLVA). The discriminatory power was 0.980 (107 patterns), 0.969 (85 allelic profiles), and 0.959 (66 types) for MLVF, MLVA, and spa typing, respectively. All methods tested showed a good concordance of results calculated by the adjusted Rand's coefficient method. Comparisons of data obtained by the three approaches allowed us to propose an 88% cutoff value for the similarity between any two MLVF patterns, which can be used in S. aureus epidemiological studies, including analyses of outbreaks and strain transmission events. Of the three tested methods, MLVF is the cheapest, fastest, and easiest to perform. MLVF applied to microfluidic polymer chips is a rapid, cheap, reproducible, and highly discriminating tool to determine the clonality of MRSA isolates and to trace the spread of MRSA strains over periods of many years. Although spa typing should be used due to its portability of data, MLVF has a high added value because it is more discriminatory.

Characterization of genetic diversity of Bacillus anthracis in France by using high-resolution melting assays and multilocus variable-number tandem-repeat analysis.

Using high-resolution melting (HRM) analysis, we developed a cost-effective method to genotype a set of 13 phylogenetically informative single-nucleotide polymorphisms (SNPs) within the genome of Bacillus anthracis. SNP discrimination assays were performed in monoplex or duplex and applied to 100 B. anthracis isolates collected in France from 1953 to 2009 and a few reference strains. HRM provided a reliable and cheap alternative to subtype B. anthracis into one of the 12 major sublineages or subgroups. All strains could be correctly positioned on the canonical SNP (canSNP) phylogenetic tree, except the divergent Pasteur vaccine strain ATCC 4229. We detected the cooccurrence of three canSNP subgroups in France. The dominant B.Br.CNEVA sublineage was found to be prevalent in the Alps, the Pyrenees, the Auvergne region, and the Saône-et-Loire department. Strains affiliated with the A.Br.008/009 subgroup were observed throughout most of the country. The minor A.Br.001/002 subgroup was restricted to northeastern France. Multiple-locus variable-number tandem-repeat analysis using 24 markers further resolved French strains into 60 unique profiles and identified some regional patterns. Diversity found within the A.Br.008/009 and B.Br.CNEVA subgroups suggests that these represent old, ecologically established clades in France. Phylogenetic relationships with strains from other parts of the world are discussed.

A multi locus variable number of tandem repeat analysis (MLVA) scheme for Streptococcus agalactiae genotyping.

BACKGROUND: Multilocus sequence typing (MLST) is currently the reference method for genotyping Streptococcus agalactiae strains, the leading cause of infectious disease in newborns and a major cause of disease in immunocompromised children and adults. We describe here a genotyping method based on multiple locus variable number of tandem repeat (VNTR) analysis (MLVA) applied to a population of S. agalactiae strains of various origins characterized by MLST and serotyping. RESULTS: We studied a collection of 186 strains isolated from humans and cattle and three reference strains (A909, NEM316 and 2603 V/R). Among 34 VNTRs, 6 polymorphic VNTRs loci were selected for use in genotyping of the bacterial population. The MLVA profile consists of a series of allele numbers, corresponding to the number of repeats at each VNTR locus. 98 MLVA genotypes were obtained compared to 51 sequences types generated by MLST. The MLVA scheme generated clusters which corresponded well to the main clonal complexes obtained by MLST. However it provided a higher discriminatory power. The diversity index obtained with MLVA was 0.960 compared to 0.881 with MLST for this population of strains. CONCLUSIONS: The MLVA scheme proposed here is a rapid, cheap and easy genotyping method generating results suitable for exchange and comparison between different laboratories and for the epidemiologic surveillance of S. agalactiae and analyses of outbreaks.

Adapting spa typing for national laboratory-based surveillance of methicillin-resistant Staphylococcus aureus.

Laboratory-based surveillance of methicillin-resistant Staphylococcus aureus (MRSA) monitors the baseline occurrence of different genotypes and identifies strains and transmission chains responsible for outbreaks. The consequences of substituting pulsed-field gel electrophoresis (PFGE) with spa typing as a first-line typing method were analyzed by typing 589 strains isolated between 1997 and 2006, with a focus on both short- and long-term correspondence between the PFGE and spa typing results. The study, covering these ten years, included all Finnish MRSA blood isolates and representatives of the two most prevalent MRSA strains (PFGE types FIN-4 and FIN-16) in Finland. In addition, all sporadic isolates from 2006 were included. spa typing was more expensive but approximately four times faster to perform than PFGE. Nearly 90% of FIN-4 and FIN-16 isolates showed consistent spa types, t172 and t067, respectively. spa typing predicted the PFGE result of the blood isolates by a Wallace coefficient of 0.9009, recognized internationally successful strains (t041, t067) to be common also in Finland, and identified a separate cluster of isolates, also related in time and place among the FIN-4 strains. Additional typing by another method was needed to provide adequate discrimination or to characterize isolates with a newly recognized spa type in Finland.

Real-time PCR followed by high-resolution melting analysis - a new robust approach to evaluate SCCmec typing of methicillin-resistant Staphylococcus aureus.

AIM: We designed a novel approach based on real-time PCR followed by high-resolution melting (HRM) analysis to determine the Staphylococcal cassette chromosome mec (SCCmec) types of methicillin-resistant Staphylococcus aureus  strains, which we compared against the results of conventional multiplex PCR SCCmec typing. METHODS: Multiplex PCR (for ccr and mec gene complexes) was carried out as conventional method. The HRM analysis was then designed using standard strains of each SCCmec type. RESULTS: The M-PCR results included types III (33.33%), IV (43.33%) and V (23.33%). HRM analysis was able to distinguish all five types, which were used to set up the protocol with a sensitivity and specificity of 100% compared with the conventional method. CONCLUSION: This novel method for SCCmec typing has high specificity and sensitivity and can be conducted in a shorter period of time at lower costs.

Technical and economic efficiency of methods for extracting genomic DNA from Meloidogyne javanica.

Plant parasitic nematodes reduce the production of agricultural crops. Species diagnosis is essential to predict losses, determine economic damage levels and develop integrated pest management programs. DNA extraction techniques need to be improved for precise and rapid molecular diagnosis of nematodes. The objective of the present study was to evaluate the efficiency of DNA extraction and amplification by PCR, cost and execution time by Chelex, Worm Lysis Buffer Method (WLB), Holterman Lysis Buffer Method (HLB) and FastDNA methods for nematodes of the Meloidogyne genus. The qualitative and quantitative efficiency of DNA extraction varied between methods. The band size of the amplified PCR product with WLB, Chelex and HLB methods was 590 bp. Extraction with the FastDNA is not recommended for DNA extraction from nematodes because it results in a low DNA concentration without bands in PCR amplification, besides presenting high cost. The efficiency of the WLB method to extracting DNA from Meloidogyne javanica was greater, ensuring a higher concentration and purity of the extracted material and guaranteeing lower costs and greater ease of PCR amplification.

Simple and economical method for identification and speciation of Staphylococcus epidermidis and other coagulase negative Staphylococci and its validation by molecular methods.

Coagulase-negative staphylococci (CoNS) have been increasingly recognized as a clinically important group of species that can cause several opportunistic nosocomial infections. There are at least 47 known species of Staphylococci and to differentiate all these species >40 biochemical tests need to be performed. The present study was able to refine the CoNS identification process by using only five tests to identify S. epidermidis from the rest and used six other tests to identify eleven other clinically significant CoNS species. A total of 242 CoNS isolates were collected from tertiary care hospitals and included in the study. The five-biochemical test scheme devised based on mathematical probability derived from a computer algorithm included fermentation of mannitol, maltose, mannose, trehalose and novobiocin susceptibility to differentiate S. epidermidis from other CoNS species. The remaining CoNS isolates other than S. epidermidis were further characterized with the help of six additional tests, which identified another eleven species. Species-specific PCR and 16SrDNA sequencing were used to confirm and validate the identification scheme. Species-specific PCR and 16SrDNA sequencing showed 100% agreement with non-divergent phenotypic test results, indicating that the five selected assays are highly specific for identifying S. epidermidis. In conclusion, this study used only 11 tests to identify most of the clinically significant CoNS that can reduce cost and time. This scheme is easy to perform in any laboratory with basic resources, the results of this study were validated using more accurate molecular methods such as PCR and 16S rDNA typing to confirm the utility of the proposed scheme.

Evaluation of the FilmArray Blood Culture Identification Panel on Detection of Pathogenic Microorganisms in Positive Blood Cultures: the First Clinical Report in Japan.

FilmArray (FA) is a multiplex PCR-based desktop microbial detection system. The blood culture identification (BCID) panel is an adaptable panel for FA, which diagnoses sepsis and/or systemic infections by detecting 14 bacterial species, 4 bacterial genera, 1 bacterial family, 5 yeast species, and 3 antimicrobial resistance genes (mecA, Klebsiella pneumoniae carbapenemase [KPC], and vanA/B) in positive blood cultures within 1 h. We retrospectively evaluated the FA-BCID panel using 54 positive blood cultures, in which 57 bacterial and 3 yeast strains were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The FA-BCID panel revealed 59 microorganisms in 53 samples; this performance was similar to that of MALDI-TOF MS analysis; however, 1 bacterium in 1 sample was not detected. In addition, mecA genes were detected in 12 Staphylococcus species, which all manifested methicillin resistance in susceptibility testing, whereas genes KPC and vanA/B were not detected, in agreement with the results of antimicrobial susceptibility testing. Although more information on antimicrobial resistance, including activity of IMP-metallo-ß-lactamases, is required in Japan, the FA-BCID panel can detect pathogenic microorganisms in positive blood cultures rapidly, and this method could be beneficial for proper treatment of sepsis and/or systemic infections, especially in small hospitals.