|
1
|
Huo M, Li Y, Wu Y, Xie S, Chen D. Enzyme-free biosensor for ultrasensitive detection of mecA gene utilizing electrochemically controlled atom transfer radical polymerization triggered by copper nanoflowers enriched on DNA polymers. Talanta 2025; 284:127231. [PMID: 39577384 DOI: 10.1016/j.talanta.2024.127231] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/30/2024] [Revised: 11/01/2024] [Accepted: 11/15/2024] [Indexed: 11/24/2024]
Abstract
Herein, an ultrasensitive electrochemical biosensor is constructed to detect mecA gene by utilizing electrochemically controlled atom transfer radical polymerization (eATRP) triggered by copper nanoflowers enriched on DNA polymers. Firstly, specific capture and enrichment of mecA gene is achieved by using magnetic separation system, effectively weakening the interference of the complex matrix. Next, enzyme-free hybridization chain reaction is triggered in the presence of mecA gene to form long DNA polymers containing numerous active sites for subsequent binding to streptavidin-copper hybrid nanoflowers (SA@Cu HNFs). Then, numerous Cu(I) afforded by the reduction and dissolution of collected SA@Cu HNFs, as catalysts and signal transduction modulators, are applied to promote the click reaction between azide-modified DNA probes on the electrode surfaces and propargyl 2-bromoisobutyrate. Finally, plentiful electroactive polymers are continually grown in situ via eATRP, significantly boosting the signal output. Under optimal conditions, the biosensor can detect mecA gene as low as 0.06 fM, with a linear range from 0.1 fM to 10 pM. Moreover, the biosensor is high selective, and suitable for mecA gene detection in actual environment and food samples. Due to its ultra-sensitivity and cost-effectiveness, the developed strategy can achieve other genes detection by simply substituting the recognition element of target.
Collapse
Affiliation(s)
- Mengyue Huo
- State Key Laboratory of Agricultural Microbiology Core Facility, Huazhong Agricultural University, Wuhan, Hubei, 430070, China; National Reference Laboratory of Veterinary Drug Residues (HZAU), Wuhan, Hubei, 430070, China
| | - Yi Li
- State Key Laboratory of Agricultural Microbiology Core Facility, Huazhong Agricultural University, Wuhan, Hubei, 430070, China; National Reference Laboratory of Veterinary Drug Residues (HZAU), Wuhan, Hubei, 430070, China
| | - Yongshan Wu
- State Key Laboratory of Agricultural Microbiology Core Facility, Huazhong Agricultural University, Wuhan, Hubei, 430070, China
| | - Shuyu Xie
- State Key Laboratory of Agricultural Microbiology Core Facility, Huazhong Agricultural University, Wuhan, Hubei, 430070, China; National Reference Laboratory of Veterinary Drug Residues (HZAU), Wuhan, Hubei, 430070, China; Key Laboratory of Prevention & Control for African Swine Fever and Other Major Pig Diseases, Ministry of Agriculture and Rural Affairs, Wuhan, Hubei, 430070, China.
| | - Dongmei Chen
- National Reference Laboratory of Veterinary Drug Residues (HZAU), Wuhan, Hubei, 430070, China.
| |
Collapse
|
|
2
|
Konstantinovski M, van Geest C, Bruijning M, Kroon-de Keizer L, Wallinga J, van Burgel N, Veldkamp KE. Rate of nosocomial MRSA transmission evaluated via contact screening. Antimicrob Resist Infect Control 2024; 13:92. [PMID: 39192375 PMCID: PMC11348754 DOI: 10.1186/s13756-024-01448-8] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/27/2023] [Accepted: 08/05/2024] [Indexed: 08/29/2024] Open
Abstract
BACKGROUND The prevention of methicillin-resistant S. aureus (MRSA) transmission in the healthcare setting is a priority in Infection Control practices. A cornerstone of this policy is contact tracing of nosocomial contacts after an unexpected MRSA finding. The objective of this retrospective study was to quantify the rates of MRSA transmission in different clinical settings. METHODS This multi-centre study included MRSA contact screening results from two regional hospitals and one academic hospital. MRSA contact tracing investigations from 2000 until 2019 were reviewed and post-contact screening results were included of index patients with an MRSA-positive culture and their unprotected contacts. Available typing results were used to rule out incidental findings. RESULTS Of 27,377 contacts screened after MRSA exposure, 21,488 were Health Care Workers (HCW) and 4816 patients. Post-contact screening was initiated for a total of 774 index cases, the average number of screened contacts per index case was 35.7 (range 1 to 640). MRSA transmission was observed in 0.15% (41) of the contacts, 19 (0.09%) HCW and 22 (0.46%) patients. The number needed to screen to detect one MRSA transmission was 667. The highest risk of MRSA transmission occurred during patient-to-patient contacts, with transmission rates varying from 0.32 to 1.32% among the participating hospitals. No transmissions were detected in HCW (n=2834) in the outpatient setting, and the rate of transmissions among HCW contacts on the wards was 0.13% (19 of 15,874). Among 344 contacts of patients with contact precautions, no transmissions were detected. CONCLUSIONS Reconsidering current MRSA contact tracing practices may lead to a more targeted approach with a lower number needed to screen.
Collapse
Affiliation(s)
- Maria Konstantinovski
- Department of Medical Microbiology, Leiden University Medical Center, Leiden University, Leiden, The Netherlands.
- Department of Microbiology, Medical Laboratories, Reinier de Graaf Groep, Delft, The Netherlands.
| | - Crista van Geest
- Department of Microbiology, Haga Teaching Hospital, The Hague, The Netherlands
| | - Marguerite Bruijning
- Department of Medical Microbiology, Leiden University Medical Center, Leiden University, Leiden, The Netherlands
| | | | - Jacco Wallinga
- Department of Biomedical Data Sciences, Leiden University Medical Center, Leiden, The Netherlands
- Center for Infectious Disease Control, National Institute for Public Health and the Environment, Bilthoven, The Netherlands
| | - Nathalie van Burgel
- Department of Microbiology, Haga Teaching Hospital, The Hague, The Netherlands
| | - Karin-Ellen Veldkamp
- Department of Medical Microbiology, Leiden University Medical Center, Leiden University, Leiden, The Netherlands
| |
Collapse
|
|
3
|
Wang Q, Yang Q. Seizing the Hidden Assassin: Current Detection Strategies for Staphylococcus aureus and Methicillin-Resistant S. aureus. JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY 2024. [PMID: 39031091 DOI: 10.1021/acs.jafc.4c02421] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 07/22/2024]
Abstract
Staphylococcus aureus (S. aureus) is a kind of pathogenic bacteria which can lead to food poisoning, hospital, and community infections. S. aureus and methicillin-resistant S. aureus (MRSA) have become headaches for public health worldwide. Therefore, strengthening the detection of S. aureus and MRSA is a critical step to prevent and control its spread and infection. This review summarized multiple detection methods (electrochemical, optical, and other biosensors) for sensitive and efficient detection of nonresistant and resistant S. aureus. First, we have introduced the principle and methods of detection platform for S. aureus and MRSA. We also contrasted various detection strategies. Finally, the current situation and prospect of S. aureus and MRSA detection in the future are explored in depth, and its development direction of detection methods is also predicted. In this review, we found that although biosensors have shown tremendous brilliance in the field of monitoring, they are currently in the experimental stage. It can be certain that we are very close to entering the commercialization stage. The point-of care testing available to nonprofessionals will become a new direction. We firmly believe that the monitoring system will be more perfect and stable and public life will be healthier and safer.
Collapse
Affiliation(s)
- Qi Wang
- College of Food Science and Engineering, Qingdao Agricultural University, no. 700 Changcheng Road, Qingdao 266109, China
| | - Qingli Yang
- College of Food Science and Engineering, Qingdao Agricultural University, no. 700 Changcheng Road, Qingdao 266109, China
| |
Collapse
|
|
4
|
Omar RF, Boissinot M, Huletsky A, Bergeron MG. Tackling Infectious Diseases with Rapid Molecular Diagnosis and Innovative Prevention. Infect Dis Rep 2024; 16:216-227. [PMID: 38525764 PMCID: PMC10961803 DOI: 10.3390/idr16020017] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/23/2024] [Revised: 02/28/2024] [Accepted: 03/01/2024] [Indexed: 03/26/2024] Open
Abstract
Infectious diseases (IDs) are a leading cause of death. The diversity and adaptability of microbes represent a continuing risk to health. Combining vision with passion, our transdisciplinary medical research team has been focussing its work on the better management of infectious diseases for saving human lives over the past five decades through medical discoveries and innovations that helped change the practice of medicine. The team used a multiple-faceted and integrated approach to control infectious diseases through fundamental discoveries and by developing innovative prevention tools and rapid molecular diagnostic tests to fulfill the various unmet needs of patients and health professionals in the field of ID. In this article, as objectives, we put in context two main research areas of ID management: innovative infection prevention that is woman-controlled, and the rapid molecular diagnosis of infection and resistance. We also explain how our transdisciplinary approach encompassing specialists from diverse fields ranging from biology to engineering was instrumental in achieving success. Furthermore, we discuss our vision of the future for translational research to better tackle IDs.
Collapse
Affiliation(s)
- Rabeea F. Omar
- Centre de Recherche en Infectiologie de l’Université Laval, Axe Maladies Infectieuses et Immunitaires, Centre de Recherche du CHU de Québec-Université Laval, Québec City, QC G1V 4G2, Canada; (M.B.); (A.H.); (M.G.B.)
| | | | | | | |
Collapse
|
|
5
|
Duraiswamy S, Agarwalla S, Lok KS, Tse YY, Wu R, Wang Z. A multiplex Taqman PCR assay for MRSA detection from whole blood. PLoS One 2023; 18:e0294782. [PMID: 38011181 PMCID: PMC10681265 DOI: 10.1371/journal.pone.0294782] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/20/2023] [Accepted: 11/08/2023] [Indexed: 11/29/2023] Open
Abstract
Methicillin-resistant Staphylococcus aureus (MRSA) causes a wide range of hospital and community-acquired infections worldwide. MRSA is associated with worse clinical outcomes that can lead to multiple organ failure, septic shock, and death, making timely diagnosis of MRSA infections very crucial. In the present work, we develop a method that enables the positive enrichment of bacteria from spiked whole blood using protein coated magnetic beads, followed by their lysis, and detection by a real-time multiplex PCR directly. The assay targeted bacterial 16S rRNA, S. aureus (spa) and methicillin resistance (mecA). In addition, an internal control (lambda phage) was added to determine the assay's true negative. To validate this assay, staphylococcal and non-staphylococcal bacterial strains were used. The three-markers used in this study were detected as expected by monomicrobial and poly-microbial models of the S. aureus and coagulase-negative staphylococci (CoNS). The thermal cycling completed within 30 mins, delivering 100% specificity. The detection LoD of the pre-processing step was ∼ 1 CFU/mL from 2-5mL of whole blood and that of PCR was ∼ 1pg of NA. However, the combined protocol led to a lower detection limit of 100-1000 MRSA CFUs/mL. The main issue with the method developed is in the pre-processing of blood which will be the subject of our future study.
Collapse
Affiliation(s)
- Suhanya Duraiswamy
- Department of Chemical Engineering, Indian Institute of Technology Hyderabad, Telangana, India
| | - Sushama Agarwalla
- Department of Chemical Engineering, Indian Institute of Technology Hyderabad, Telangana, India
| | - Khoi Sheng Lok
- Singapore Institute of Manufacturing Technology (SIMTech), Agency for Science, Technology and Research (A*STAR), Singapore, Republic of Singapore
| | - Yee Yung Tse
- Singapore Institute of Manufacturing Technology (SIMTech), Agency for Science, Technology and Research (A*STAR), Singapore, Republic of Singapore
| | - Ruige Wu
- Singapore Institute of Manufacturing Technology (SIMTech), Agency for Science, Technology and Research (A*STAR), Singapore, Republic of Singapore
| | - Zhiping Wang
- Singapore Institute of Manufacturing Technology (SIMTech), Agency for Science, Technology and Research (A*STAR), Singapore, Republic of Singapore
| |
Collapse
|
|
6
|
Saeki M, Nirasawa S, Aung MS, Ono M, Urushibara N, Kobayashi N, Takahashi S. Detecting the performance of methicillin-resistant Staphylococcus aureus by a molecular diagnostic assay in positive blood culture: Influence of coexistence of mecA-positive bacteria and diversity in orfX-SCCmec junction region in methicillin-susceptible S. aureus. J Infect Chemother 2023:S1341-321X(23)00116-2. [PMID: 37178974 DOI: 10.1016/j.jiac.2023.05.006] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/01/2023] [Revised: 04/23/2023] [Accepted: 05/02/2023] [Indexed: 05/15/2023]
Abstract
BACKGROUND In blood cultures that test positive for staphylococcal bacteria, rapid identification of methicillin-resistant Staphylococcus aureus (MRSA) or methicillin-susceptible Staphylococcus aureus (MSSA) by molecular assay is useful for appropriate antimicrobial treatment of bloodstream infections. Although the Xpert MRSA/SA BC assay is widely available in clinical settings in Japan, its efficacy has not yet evaluated thoroughly. METHODS We retrospectively studied 100 blood culture cases positive for S. aureus at Sapporo Medical University Hospital between March 2019 to May 2022. Cycle threshold (CT) values for target genes from the Xpert MRSA/SA BC assay were compared to phenotypic results. Genotyping and genetic analysis of the orfX-SCCmec junction region was performed for selected isolates. RESULTS We analyzed 25 and 75 isolates assigned to MRSA and MSSA, respectively, using the Xpert MRSA/SA BC assay. Of these, 99 isolates from agar cultures showed compatible susceptibility to oxacillin. One genetically misidentified case of MRSA was found to be caused by the mixed growth of MSSA and methicillin-resistant S. hominis on agar culture. Of the 73 MSSA with pure growth on agar culture, 45 (61.6%) were found to be orfX-SCCmec-positive, spa-positive, and mecA-negative in this assay. These MSSA belong to diverse spa and coa types. CONCLUSION The Xpert MRSA/SA BC assay accurately identified MRSA and MSSA in positive blood cultures. However, over half of the MSSA isolates showed positive results for orfX-SCCmec, presumably due to genetic diversity in the orfX-associated region of MSSA. Therefore, the coexistence of MSSA and mecA-harboring coagulase-negative staphylococci may cause confusion about identification of MRSA.
Collapse
Affiliation(s)
- Masachika Saeki
- Division of Laboratory Medicine, Sapporo Medical University Hospital, Sapporo, Japan
| | - Shinya Nirasawa
- Division of Laboratory Medicine, Sapporo Medical University Hospital, Sapporo, Japan
| | - Meiji Soe Aung
- Department of Hygiene, Sapporo Medical University School of Medicine, Sapporo, Japan
| | - Mayumi Ono
- Division of Laboratory Medicine, Sapporo Medical University Hospital, Sapporo, Japan
| | - Noriko Urushibara
- Department of Hygiene, Sapporo Medical University School of Medicine, Sapporo, Japan
| | - Nobumichi Kobayashi
- Department of Hygiene, Sapporo Medical University School of Medicine, Sapporo, Japan
| | - Satoshi Takahashi
- Division of Laboratory Medicine, Sapporo Medical University Hospital, Sapporo, Japan; Department of Infection Control and Laboratory Medicine, Sapporo Medical University School of Medicine, Sapporo, Japan.
| |
Collapse
|
|
7
|
Development of a Real-Time Recombinase-Aided Amplification Method to Rapidly Detect Methicillin-Resistant Staphylococcus aureus. Microorganisms 2022; 10:microorganisms10122351. [PMID: 36557604 PMCID: PMC9784193 DOI: 10.3390/microorganisms10122351] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/04/2022] [Revised: 10/03/2022] [Accepted: 10/31/2022] [Indexed: 11/29/2022] Open
Abstract
Methicillin-resistant staphylococcus aureus (MRSA) is a major pathogen responsible for human hospital and community-onset diseases and severe invasive livestock infections. Rapid detection of MRSA is essential to control the spread of MRSA. Conventional identification methods and antibacterial susceptibility tests of MRSA are time-consuming. The commonly used qPCR assay also has the disadvantages of being complicated and expensive, restricting its application in resource-limited clinical laboratories. Here, a real-time fluorescent recombinase-assisted amplification (RAA) assay targeting the most conserved regions within the mecA gene of MRSA was developed and evaluated to detect MRSA. The detection limit of this assay was determined to be 10 copies/reaction of positive plasmids. The established RAA assay showed high specificity for MRSA detection without cross-reactivities with other clinically relevant bacteria. The diagnostic performance of real-time RAA was evaluated using 67 clinical S. aureus isolates from dairy farms, which were detected in parallel using the TaqMan probe qPCR assay. The results showed that 56 and 54 samples tested positive for MRSA by RAA and qPCR, respectively. The overall agreement between both assays was 97.01% (65/67), with a kappa value of 0.9517 (p < 0.001). Further linear regression analysis demonstrated that the detection results between the two assays were significantly correlated (R2 = 0.9012, p < 0.0001), indicating that this RAA assay possesses similar detection performance to the qPCR assay. In conclusion, our newly established RAA assay is a time-saving and convenient diagnostic tool suitable for MRSA detection and screening.
Collapse
|
|
8
|
Heng P, Liu J, Song Z, Wu C, Yu X, He Y. Rapid detection of Staphylococcus aureus using a novel multienzyme isothermal rapid amplification technique. Front Microbiol 2022; 13:1027785. [PMID: 36312945 PMCID: PMC9606696 DOI: 10.3389/fmicb.2022.1027785] [Citation(s) in RCA: 19] [Impact Index Per Article: 6.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/25/2022] [Accepted: 09/21/2022] [Indexed: 11/25/2022] Open
Abstract
Staphylococcus aureus is a common pathogen that causes various infections. Therefore, it is crucial to develop a fast and easy detection method for diagnosing and preventing S. aureus infections. In this study, MIRA assay was developed and validated (specificity; 100%) for the detection of S. aureus with nuc as the target gene. The reaction temperature and reaction time were then optimized, and the best reaction was at 40°C, 20 min. The assay could detect S. aureus in only 25 min. Additionally, the limit of detection of MIRA was 5 × 102 CFU/ml, 10-fold lower than that of the traditional PCR. Furthermore, this assay efficiently detected 219 S. aureus of 335 strains obtained from different bacterial samples (detection accuracy; 99.40%). In conclusion, this study provides a rapid and easy-to-operate method for the detection of S. aureus, and thus can be used for the timely diagnosis and prevention of S. aureus infection.
Collapse
Affiliation(s)
- Pengfei Heng
- State Key Laboratory of Southwestern Chinese Medicine Resources, College of Medical Technology, Chengdu University of Traditional Chinese Medicine, Chengdu, Sichuan, China
| | - Jiakai Liu
- Department of Ultrasound Medicine, The Second Affiliated Hospital of Chengdu Medical College, China National Nuclear Corporation 416 Hospital, Chengdu, Sichuan, China
| | - Zhen Song
- State Key Laboratory of Southwestern Chinese Medicine Resources, College of Medical Technology, Chengdu University of Traditional Chinese Medicine, Chengdu, Sichuan, China
| | - Chuan Wu
- State Key Laboratory of Southwestern Chinese Medicine Resources, College of Medical Technology, Chengdu University of Traditional Chinese Medicine, Chengdu, Sichuan, China
| | - Xiuzhong Yu
- Department of Laboratory Medicine, People’s Hospital of Xinjin District, Chengdu, Sichuan, China
| | - Yang He
- State Key Laboratory of Southwestern Chinese Medicine Resources, College of Medical Technology, Chengdu University of Traditional Chinese Medicine, Chengdu, Sichuan, China
- *Correspondence: Yang He,
| |
Collapse
|
|
9
|
Characterization of SCC mec Instability in Methicillin-Resistant Staphylococcus aureus Affecting Adjacent Chromosomal Regions, Including the Gene for Staphylococcal Protein A ( spa). Antimicrob Agents Chemother 2022; 66:e0237421. [PMID: 35254090 PMCID: PMC9017337 DOI: 10.1128/aac.02374-21] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022] Open
Abstract
Staphylococcal cassette chromosome mec (SCCmec) represents a sequence of clear clinical and diagnostic importance in staphylococci. At a minimum the chromosomal cassette contains the mecA gene encoding PBP2a but frequently also includes additional antibiotic resistance genes (e.g., ermA and aadC; macrolide and aminoglycoside resistance, respectively). Certain regions within SCCmec elements are hot spots for sequence instability due to cassette-specific recombinases and a variety of internal mobile elements. SCCmec changes may affect not only cassette stability but the integrity of adjacent chromosomal sequences (e.g., the staphylococcal protein A gene; spa). We investigated SCCmec stability in methicillin-resistant Staphylococcus aureus (MRSA) strains carrying one of four SCCmec types cultured in the absence of antimicrobial selection over a 3-month period. SCCmec rearrangements were first detected in cefoxitin-susceptible variants after 2 months of passage, and most commonly showed precise excision of the SCCmec element. Sequence analysis after 3 months revealed both precise SCCmec excision and a variety of SCCmec internal deletions, some including extensive adjacent chromosomal loss, including spa. No empty cassettes (i.e., loss of just mecA from SCCmec) were observed among the variants. SCCmec stability was influenced both by internal mobile elements (IS431) as well as the host cell environment. Genotypically similar clinical isolates with deletions in the spa gene were also included for purposes of comparison. The results indicate a role for host-cell influence and the IS431 element on SCCmec stability.
Collapse
|
|
10
|
Tenover FC, Tickler IA. Detection of Methicillin-Resistant Staphylococcus aureus Infections Using Molecular Methods. Antibiotics (Basel) 2022; 11:antibiotics11020239. [PMID: 35203841 PMCID: PMC8868555 DOI: 10.3390/antibiotics11020239] [Citation(s) in RCA: 7] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/13/2022] [Revised: 02/02/2022] [Accepted: 02/10/2022] [Indexed: 02/01/2023] Open
Abstract
The application of molecular detection methods for bacterial pathogens has dramatically improved the outcomes of septic patients, including those with methicillin-resistant Staphylococcus aureus (MRSA) infections. Molecular methods can be applied to a variety of clinical specimens including nasal swabs, growth in blood culture bottles, and wounds. While data show that the overall accuracy of molecular tests for MRSA is high, results can be confounded by the presence of multiple staphylococcal species in a specimen, insertions and deletions of DNA in and around the Staphylococcal Cassette Chromosome mec (SCCmec) element, and point mutations in mecA. Herein, we explore the complexities of molecular approaches to MRSA detection and the instances where phenotypic methods should be pursued to resolve discrepancies between genotypic and phenotypic results.
Collapse
|
|
11
|
Fritz B, Paschko E, Young W, Böhringer D, Wahl S, Ziemssen F, Egert M. Comprehensive Compositional Analysis of the Slit Lamp Bacteriota. Front Cell Infect Microbiol 2021; 11:745653. [PMID: 34869057 PMCID: PMC8635730 DOI: 10.3389/fcimb.2021.745653] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/29/2021] [Accepted: 11/01/2021] [Indexed: 11/17/2022] Open
Abstract
Slit lamps are routinely used to examine large numbers of patients every day due to high throughput. Previous, cultivation-based results suggested slit lamps to be contaminated with bacteria, mostly coagulase-negative staphylococci, followed by micrococci, bacilli, but also Staphylococcus aureus. Our study aimed at obtaining a much more comprehensive, cultivation-independent view of the slit lamp bacteriota and its hygienic relevance, as regularly touched surfaces usually represent fomites, particularly if used by different persons. We performed extensive 16S rRNA gene sequencing to analyse the bacteriota, of 46 slit lamps from two tertiary care centers at two sampling sites, respectively. 82 samples yielded enough sequences for downstream analyses and revealed contamination with bacteria of mostly human skin, mucosa and probably eye origin, predominantly cutibacteria, staphylococci and corynebacteria. The taxonomic assignment of 3369 ASVs (amplicon sequence variants) revealed 19 bacterial phyla and 468 genera across all samples. As antibiotic resistances are of major concern, we screened all samples for methicillin-resistant Staphylococcus aureus (MRSA) using qPCR, however, no signals above the detection limit were detected. Our study provides first comprehensive insight into the slit lamp microbiota. It underlines that slit lamps carry a highly diverse, skin-like bacterial microbiota and that thorough cleaning and disinfection after use is highly recommendable to prevent eye and skin infections.
Collapse
Affiliation(s)
- Birgit Fritz
- Faculty of Medical and Life Sciences, Institute of Precision Medicine, Microbiology and Hygiene Group, Furtwangen University, Villingen-Schwenningen, Germany
| | - Edita Paschko
- Faculty of Medical and Life Sciences, Institute of Precision Medicine, Microbiology and Hygiene Group, Furtwangen University, Villingen-Schwenningen, Germany
| | - Wayne Young
- Food Informatics Team, AgResearch Ltd., Palmerston North, New Zealand
| | - Daniel Böhringer
- Eye Center, Medical Center, Faculty of Medicine, University of Freiburg, Freiburg, Germany
| | - Siegfried Wahl
- Carl Zeiss Vision International GmbH, Aalen, Germany.,Institute for Ophthalmic Research, Eberhard-Karls University, Tuebingen, Germany
| | - Focke Ziemssen
- Center for Ophthalmology, Eberhard-Karls University, Tuebingen, Germany
| | - Markus Egert
- Faculty of Medical and Life Sciences, Institute of Precision Medicine, Microbiology and Hygiene Group, Furtwangen University, Villingen-Schwenningen, Germany
| |
Collapse
|
|
12
|
Ashokan A, Choo JM, Taylor SL, Lagana D, Shaw DR, Warner MS, Wesselingh SL, Rogers GB. Environmental dynamics of hospital microbiome upon transfer from a major hospital to a new facility. J Infect 2021; 83:637-643. [PMID: 34606783 DOI: 10.1016/j.jinf.2021.09.020] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/24/2021] [Revised: 07/23/2021] [Accepted: 09/07/2021] [Indexed: 01/04/2023]
Abstract
BACKGROUND Infection control is critical to safe hospital care. However, how bacteria within nosocomial environments relate to space utilisation and occupancy remains poorly understood. Our aim was to characterise the hospital microbiome in the context of the closure of a tertiary hospital and the opening of a new facility. METHODS Environmental swabs were collected from common and inpatient areas in the old and new hospitals during a 12-month transition period. Microbiota characteristics were determined by 16S rRNA gene sequencing and quantitative (q)PCR. Targeted assays were used to detect Methicillin-resistant Staphylococcus aureus (MRSA) and vanB-positive Vancomycin-Resistant Enterococci (VRE). RESULTS The transition to full occupancy in the new facility was associated with an increase in bacterial load (inpatient areas, 3 months p = 0.001; common areas, 6 months p = 0.039) and a change in microbiota composition (baseline-12 months, PERMANOVA p = 0.002). These changes were characterised by an increase in human microbiota-associated taxa, including Acinetobacter and Veillonella. Closure of the existing facility was associated with a decrease in bacterial load (p = 0.040). Detection of MRSA did not differ significantly between sites. CONCLUSIONS Occupancy is a major determinant of bacterial dispersion within hospital environments. Steady-state bacterial levels and microbiota composition provide a basis for assessment of infection control measures.
Collapse
Affiliation(s)
- Anushia Ashokan
- Microbiome and Host Health, South Australia Health and Medical Research Institute, Adelaide, SA, Australia; SAHMRI Microbiome Research Laboratory, Flinders University College of Medicine and Public Health, Adelaide, SA, Australia; Faculty of Health and Medical Sciences, University of Adelaide, North Terrace, Adelaide, SA, Australia; Department of Infectious Diseases, Royal Adelaide Hospital, Adelaide, SA, Australia.
| | - Jocelyn M Choo
- Microbiome and Host Health, South Australia Health and Medical Research Institute, Adelaide, SA, Australia; SAHMRI Microbiome Research Laboratory, Flinders University College of Medicine and Public Health, Adelaide, SA, Australia
| | - Steven L Taylor
- Microbiome and Host Health, South Australia Health and Medical Research Institute, Adelaide, SA, Australia; SAHMRI Microbiome Research Laboratory, Flinders University College of Medicine and Public Health, Adelaide, SA, Australia
| | - Diana Lagana
- Department of Infectious Diseases, Royal Adelaide Hospital, Adelaide, SA, Australia
| | - David R Shaw
- Department of Infectious Diseases, Royal Adelaide Hospital, Adelaide, SA, Australia
| | - Morgyn S Warner
- Department of Infectious Diseases, Royal Adelaide Hospital, Adelaide, SA, Australia; South Australia (SA) Pathology, North Terrace, Adelaide, SA, Australia
| | - Steve L Wesselingh
- South Australia Health and Medical Research Institute, Adelaide, SA, Australia
| | - Geraint B Rogers
- Microbiome and Host Health, South Australia Health and Medical Research Institute, Adelaide, SA, Australia; SAHMRI Microbiome Research Laboratory, Flinders University College of Medicine and Public Health, Adelaide, SA, Australia
| |
Collapse
|
|
13
|
Duquette-Lozeau K, Létourneau V, Lemieux J, Fournel S, Côté C, Godbout S, Duchaine C. Production of composted recycled manure solids from a Canadian dairy farm: Impact on microbial air quality in experimental conditions. JOURNAL OF THE AIR & WASTE MANAGEMENT ASSOCIATION (1995) 2021; 71:413-421. [PMID: 33030410 DOI: 10.1080/10962247.2020.1832620] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 04/28/2020] [Revised: 09/16/2020] [Accepted: 09/18/2020] [Indexed: 06/11/2023]
Abstract
Recycled manure solids (RMS) produced in dairy farms from fresh manure need to be sanitized before using them as bedding material. However, the impact on air quality of composting RMS remains unknown. Four RMS composting methods were tested during a 10-day aging of piles in experimental chambers: static windrow (SW), turned windrow (TW), SW following drum composting for 24 h (DC24) or SW following drum composting for 72 h (DC72). Air samples were collected using a SASS®3100 Dry Air Sampler on days 0 (pilling of the RMS), 5, and 10. Bacteria (16S rRNA genes), Penicillium/Aspergillus, A fumigatus, and 11 human pathogenic bacteria (e.g. Klebsiella pneumonia) were quantified by qPCR while endotoxins and dust particles were, respectively, measured by LAL assays and with a DustTrakTM DRX Aerosol Monitor. On day 0, RMS produced by SW and TW yielded the lowest concentrations of airborne bacteria, while DC24 resulted in the lowest levels of Penicillium/Aspergillus and dust particles. SW method led on day 5 to the lowest concentration of bacteria and Penicillium/Aspergillus, and DC24 and DC72 to the lowest concentration of airborne dust. On day 10, SW and TW piles were associated with the lowest levels of Penicillium/Aspergillus and dust particles. A significant difference was observed between concentration of airborne bacteria, Penicillium/Aspergillus and endotoxins before and during the turnover of TW piles. None of the studied human pathogens was detected in the air samples. Results of the present study suggest that SW and TW are the most promising methods for the production of composted RMS with respect to microbial air quality. However, the experimental chambers do not accurately represent commercial dairy barns and further research on these composting methods is necessary. Finally, the study highlights that bedding material and its management may be determinant factors for air quality in dairy barns.Implications: The research evaluated the impact on microbial air quality of composting recycled manure solids (RMS) produced from fresh cow manure. RMS need to be composted or sanitized before using them as bedding material for animals. The impact on animal health of RMS still needs to be confirmed, while the effect on air quality and the health of dairy farmers is unknown. In the present study, microbial air quality associated with four RMS composting methods was investigated. Data revealed that two methods resulted in lower aerosolization of dust particles, endotoxins, molds, and bacteria.
Collapse
Affiliation(s)
- Karine Duquette-Lozeau
- Département de biochimie, de microbiologie et de bio-informatique, Université Laval, Québec, Canada
| | - Valérie Létourneau
- Centre de recherche de l'Institut universitaire de cardiologie et de pneumologie de Québec, Université Laval, Québec, Canada
| | - Joanie Lemieux
- Département de biochimie, de microbiologie et de bio-informatique, Université Laval, Québec, Canada
| | - Sébastien Fournel
- Département des sols et de génie agroalimentaire, Université Laval, Québec, Canada
| | - Caroline Côté
- Research and Development Institute for the Agri-Environment (IRDA), Québec, Canada
| | - Stéphane Godbout
- Research and Development Institute for the Agri-Environment (IRDA), Québec, Canada
| | - Caroline Duchaine
- Département de biochimie, de microbiologie et de bio-informatique, Université Laval, Québec, Canada
- Centre de recherche de l'Institut universitaire de cardiologie et de pneumologie de Québec, Université Laval, Québec, Canada
| |
Collapse
|
|
14
|
Kasas S, Malovichko A, Villalba MI, Vela ME, Yantorno O, Willaert RG. Nanomotion Detection-Based Rapid Antibiotic Susceptibility Testing. Antibiotics (Basel) 2021; 10:287. [PMID: 33801939 PMCID: PMC7999052 DOI: 10.3390/antibiotics10030287] [Citation(s) in RCA: 21] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/16/2021] [Revised: 02/26/2021] [Accepted: 03/07/2021] [Indexed: 01/04/2023] Open
Abstract
Rapid antibiotic susceptibility testing (AST) could play a major role in fighting multidrug-resistant bacteria. Recently, it was discovered that all living organisms oscillate in the range of nanometers and that these oscillations, referred to as nanomotion, stop as soon the organism dies. This finding led to the development of rapid AST techniques based on the monitoring of these oscillations upon exposure to antibiotics. In this review, we explain the working principle of this novel technique, compare the method with current ASTs, explore its application and give some advice about its implementation. As an illustrative example, we present the application of the technique to the slowly growing and pathogenic Bordetella pertussis bacteria.
Collapse
Affiliation(s)
- Sandor Kasas
- Laboratory of Biological Electron Microscopy, Ecole Polytechnique Fédérale de Lausanne, 1015 Lausanne, Switzerland; (A.M.); (M.I.V.)
- Unité Facultaire d’Anatomie et de Morphologie (UFAM), CUMRL, University of Lausanne, 1005 Lausanne, Switzerland
- International Joint Research Group VUB-EPFL NanoBiotechnology and NanoMedicine (NANO), Vrije Universiteit Brussel, 1050 Brussels, Belgium;
| | - Anton Malovichko
- Laboratory of Biological Electron Microscopy, Ecole Polytechnique Fédérale de Lausanne, 1015 Lausanne, Switzerland; (A.M.); (M.I.V.)
- International Joint Research Group VUB-EPFL NanoBiotechnology and NanoMedicine (NANO), Vrije Universiteit Brussel, 1050 Brussels, Belgium;
| | - Maria Ines Villalba
- Laboratory of Biological Electron Microscopy, Ecole Polytechnique Fédérale de Lausanne, 1015 Lausanne, Switzerland; (A.M.); (M.I.V.)
- International Joint Research Group VUB-EPFL NanoBiotechnology and NanoMedicine (NANO), Vrije Universiteit Brussel, 1050 Brussels, Belgium;
| | - María Elena Vela
- Instituto de Investigaciones Fisicoquímicas Teóricas y Aplicadas (INIFTA), Facultad de Ciencias Exactas, Universidad Nacional de La Plata, and CONICET, Diagonal 113 y 64, 1900 La Plata, Argentina;
| | - Osvaldo Yantorno
- Centro de Investigación y Desarrollo en Fermentaciones Industriales (CINDEFI-CONICET-CCT La Plata), Facultad de Ciencias Exactas, Universidad Nacional de La Plata, 1900 La Plata, Argentina;
| | - Ronnie G. Willaert
- International Joint Research Group VUB-EPFL NanoBiotechnology and NanoMedicine (NANO), Vrije Universiteit Brussel, 1050 Brussels, Belgium;
- Research Group Structural Biology Brussels, Vrije Universiteit Brussel, 1050 Brussels, Belgium
| |
Collapse
|
|
15
|
Methicillin-Resistant Coagulase-Negative Staphylococci Carriage is a Protective Factor of Methicillin-Resistant Staphylococcus Aureus Nasal Colonization in HIV-Infected Patients: A Cross-Sectional Study. ACTA ACUST UNITED AC 2021; 2021:5717413. [PMID: 33505540 PMCID: PMC7815391 DOI: 10.1155/2021/5717413] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/05/2020] [Revised: 09/10/2020] [Accepted: 12/26/2020] [Indexed: 11/30/2022]
Abstract
Background Methicillin-resistant coagulase-negative Staphylococci (MRCoNS) is regarded as the repository of mecA gene for methicillin-resistant Staphylococcus aureus (MRSA) and may develop methicillin-susceptible Staphylococcus aureus (MSSA) to MRSA. Therefore, we aimed to explore whether MRCoNS carriage is a risk factor of MRSA colonization. Phenotypic characteristics were performed to further assess the associations between MRSA and MRCoNS. Methods This cross-sectional study was conducted in Guangzhou, China. Participants completed a questionnaire and provided a nasal swab for further analysis. The risk factors of MRSA colonization were analyzed using nonconditional logistic regression models. The phenotypic characteristics between MRSA and MRCoNS were compared by Chi-square test. Results Among the 1001 HIV-infected patients, a total of 119 (11.89%) participants were positive for MRSA, and 34.45% (41/119) of all MRSA carriers were positive for MRCoNS. We found MRCoNS carriage was a protective factor of MRSA colonization (adjusted odds ratio = 0.59, 95% confidence interval: 0.38–0.91). A significant difference in the proportions of antibiotic resistance between MRSA and MRCoNS isolates was found except for penicillin, clindamycin, tetracycline, and teicoplanin. The main STs and CC types of MRSA isolates in this population were ST188 (15.1%) and CC59 (17.6%), respectively. Conclusions HIV-infected patients remain a highly vulnerable population for MRSA colonization. Though who carried MRCoNS is less likely to have MRSA colonization, similarity of some antibiotic resistance between MRSA and MRCoNS was found in this study. Regular surveillance on the colonization and antibiotic patterns of MRSA and MRCoNS is still necessary.
Collapse
|
|
16
|
Rm R, Maroli N, J A, Ponmalai K, K K. Highly adaptable and sensitive FRET-based aptamer assay for the detection of Salmonella paratyphi A. SPECTROCHIMICA ACTA. PART A, MOLECULAR AND BIOMOLECULAR SPECTROSCOPY 2020; 243:118662. [PMID: 32810775 DOI: 10.1016/j.saa.2020.118662] [Citation(s) in RCA: 19] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 02/25/2019] [Revised: 05/01/2020] [Accepted: 06/26/2020] [Indexed: 06/11/2023]
Abstract
Here we demonstrate a facile and versatile fluorescence resonance energy transfer (FRET) based aptasensor for rapid detection of Salmonella paratyphi A. The assay shows a detection limit up to 10 cfu·mL-1 with no cross-reactivity with other bacterial species. Less than 8% of inter-assay coefficient variance and recovery rate between 85 and 102% attests the assay reliability. The advantages of FRET-based aptamer assay over the conventional immunoassay formats such as ELISA are the specificity, speed, reliability, and simplicity of the assay. The ssDNA aptamers specific towards pathogenic Salmonella paratyphi A were generated via whole-cell SELEX. The aptamer was conjugated onto quantum dot (QD) that served as the molecular beacon and graphene oxide (GO) was used as a fluorescence quencher. Thus the proposed method enables detection of target pathogen using FRET-based assay. Further interaction of aptamer with pathogen protein DNA gyrase was explored using classical molecular dynamics simulation.
Collapse
Affiliation(s)
- Renuka Rm
- Molecular Immunology Laboratory, DRDO-BU-CLS, Bharathiar University Campus, Coimbatore 641046, Tamil Nadu, India
| | - Nikhil Maroli
- Computational Biology Division, DRDO-BU CLS, Bharathiar University Campus, Coimbatore 641046, Tamil Nadu, India
| | - Achuth J
- Molecular Immunology Laboratory, DRDO-BU-CLS, Bharathiar University Campus, Coimbatore 641046, Tamil Nadu, India
| | - Kolandaivel Ponmalai
- Computational Biology Division, DRDO-BU CLS, Bharathiar University Campus, Coimbatore 641046, Tamil Nadu, India
| | - Kadirvelu K
- Molecular Immunology Laboratory, DRDO-BU-CLS, Bharathiar University Campus, Coimbatore 641046, Tamil Nadu, India.
| |
Collapse
|
|
17
|
Application of Antiviral Polyoxometalates to Living Environments—Antiviral Moist Hand Towels and Stationery Items. APPLIED SCIENCES-BASEL 2020. [DOI: 10.3390/app10228246] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/23/2022]
Abstract
Safe, secure, and environmentally friendly active substances should be developed. VB (virus block) refers to an antibacterial/antiviral mixture of two kinds of polyoxometalates (PMs), i.e., K11H[(VO)3(SbW9O33)2]·27H2O (VB2) and α-Na2[SbW9O33]9− (VB3), and polyhexamethylene biguanide (PHMB). VB was demonstrated to exert antiviral effects on cultured cells. The effects were maintained even in hygiene products or solids. The antiviral effects were analyzed by reverse transcription–polymerase chain reaction (RT–PCR), and the results were correlated with TCID50, potentially eliminating the need for handling infectious viruses. VB was demonstrated to be extremely effective (up to 99.99% inhibition) in cultured cells, with antibacterial/antiviral effects maintained in VB-containing hygiene products. VB was applied to solids, demonstrating their high applicability and versatility. VB withstands high temperatures regardless of materials because its effects are enhanced by more frequent contact with viruses and bacteria due to the increased surface area of the compound.
Collapse
|
|
18
|
Shanmugakani RK, Srinivasan B, Glesby MJ, Westblade LF, Cárdenas WB, Raj T, Erickson D, Mehta S. Current state of the art in rapid diagnostics for antimicrobial resistance. LAB ON A CHIP 2020; 20:2607-2625. [PMID: 32644060 PMCID: PMC7428068 DOI: 10.1039/d0lc00034e] [Citation(s) in RCA: 35] [Impact Index Per Article: 7.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/07/2023]
Abstract
Antimicrobial resistance (AMR) is a fundamental global concern analogous to climate change threatening both public health and global development progress. Infections caused by antimicrobial-resistant pathogens pose serious threats to healthcare and human capital. If the increasing rate of AMR is left uncontrolled, it is estimated that it will lead to 10 million deaths annually by 2050. This global epidemic of AMR necessitates radical interdisciplinary solutions to better detect antimicrobial susceptibility and manage infections. Rapid diagnostics that can identify antimicrobial-resistant pathogens to assist clinicians and health workers in initiating appropriate treatment are critical for antimicrobial stewardship. In this review, we summarize different technologies applied for the development of rapid diagnostics for AMR and antimicrobial susceptibility testing (AST). We briefly describe the single-cell technologies that were developed to hasten the AST of infectious pathogens. Then, the different types of genotypic and phenotypic techniques and the commercially available rapid diagnostics for AMR are discussed in detail. We conclude by addressing the potential of current rapid diagnostic systems being developed as point-of-care (POC) diagnostic tools and the challenges to adapt them at the POC level. Overall, this review provides an insight into the current status of rapid and POC diagnostic systems for AMR.
Collapse
Affiliation(s)
- Rathina Kumar Shanmugakani
- Institute for Nutritional Sciences, Global Health, and Technology, Cornell University, Ithaca, New York, USA
- Division of Nutritional Sciences, Cornell University, Ithaca, New York, USA
| | - Balaji Srinivasan
- Institute for Nutritional Sciences, Global Health, and Technology, Cornell University, Ithaca, New York, USA
- Division of Nutritional Sciences, Cornell University, Ithaca, New York, USA
| | - Marshall J. Glesby
- Division of Infectious Diseases, Department of Medicine, Weill Cornell Medicine, New York, New York, USA
| | - Lars F. Westblade
- Division of Infectious Diseases, Department of Medicine, Weill Cornell Medicine, New York, New York, USA
- Department of Pathology and Laboratory Medicine, Weill Cornell Medicine, New York, New York, USA
| | - Washington B. Cárdenas
- Laboratorio para Investigaciones Biomédicas, Escuela Superior Politécnica del Litoral, Guayaquil, Guayas, Ecuador
| | - Tony Raj
- St. John’s Research Institute, Bangalore, Karnataka, India
| | - David Erickson
- Institute for Nutritional Sciences, Global Health, and Technology, Cornell University, Ithaca, New York, USA
- Division of Nutritional Sciences, Cornell University, Ithaca, New York, USA
- Sibley School of Mechanical and Aerospace Engineering, Cornell University, Ithaca, New York, USA
| | - Saurabh Mehta
- Institute for Nutritional Sciences, Global Health, and Technology, Cornell University, Ithaca, New York, USA
- Division of Nutritional Sciences, Cornell University, Ithaca, New York, USA
| |
Collapse
|
|
19
|
Performance of the Hologic Panther Fusion® MRSA Assay for the nasal screening of methicillin-sensitive and methicillin-resistant Staphylococcus aureus carriage. Eur J Clin Microbiol Infect Dis 2020; 39:2169-2176. [PMID: 32643026 DOI: 10.1007/s10096-020-03968-8] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/12/2020] [Accepted: 06/25/2020] [Indexed: 01/07/2023]
Abstract
Staphylococcus aureus (SA) nasal carriage screening is usually based on either culture or molecular biology. The aim of the study was to evaluate the performance of the Panther Fusion® MRSA Assay (PF) that proposes a complete automation of the molecular screening for MSSA and MRSA carriage. Four hundred thirty-four nasal samples collected on ESwab™ were screened using PF. Results were compared with standard culture on BBL™ CHROMagar™ Staph aureus and chromID® MRSA agar. Discordant results were analyzed with additional techniques: Xpert SA Nasal Complete on GeneXpert (GX), culture on selective agar after 24 h in broth enrichment, and, if necessary, characterization of mec gene and SCCmec cassette using DNA microarray. The PF presented an overall agreement of 97.5% for SA detection and 97.9% for MRSA detection. Furthermore, 7.1% (31/434) of the samples were SA-negative in primary culture but SA-positive using PF and GX, confirming the greater sensitivity of molecular tests compared with culture. Of note, 4 out of 30 MRSA-positive samples were not detected due to an atypical SCCmec cassette, while 2 samples were falsely detected as MRSA due to co-colonization with a MSSA drop-out strain and a methicillin-resistant coagulase-negative staphylococcal strain. Considering all results, the PF instrument appears as a reliable and rapid (< 3 h) package for MSSA/MRSA nasal screening. This technology using random access capability and direct sampling of the primary container is innovative and corresponds therefore to a new step in complete molecular biology automation in bacteriology.
Collapse
|
|
20
|
McClure JA, Conly JM, Obasuyi O, Ward L, Ugarte-Torres A, Louie T, Zhang K. A Novel Assay for Detection of Methicillin-Resistant Staphylococcus aureus Directly From Clinical Samples. Front Microbiol 2020; 11:1295. [PMID: 32625187 PMCID: PMC7314949 DOI: 10.3389/fmicb.2020.01295] [Citation(s) in RCA: 14] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/25/2020] [Accepted: 05/20/2020] [Indexed: 12/16/2022] Open
Abstract
The timely detection of Methicillin-resistant Staphylococcus aureus (MRSA) is crucial for antimicrobial therapy and a key factor to limit the hospital spread of MRSA. Currently available commercial MRSA detection assays target the 3' end of the orfX gene and the right extremity of Staphylococcal Cassette Chromosome mec (SCCmec). These assays suffer from both false positive due to SCC-like elements that lack mecA and false negative results due to the inability to detect new or variant SCCmec cassettes with the existing primers. We developed a novel MRSA detection scheme, designed to circumvent issues present in the existing commercial assays. Our assay demonstrated specificity and accuracy, capable of detecting prototypic strains of SCCmec types I-XIII [C(t) values ranged 8.58-26.29]. Previous false positive isolates (N = 19) by Xpert MRSA nasal assay were accurately classified with our assay. Further validation with 218 randomly selected clinical isolates (73 MRSA, 75 MSSA, 43 MR-CoNS, and 27 MS-CoNS) confirmed its feasibility and practicality. Testing assay performance with 88 direct clinical swabs from 33 patients showed that the assay was 96.6% in agreement with clinical culture results. Our novel MRSA detection assay targets both the S. aureus specific sequence and the mecA/mecC genes simultaneously to overcome the false positive and false negative deficits of currently available commercial assays. The results validate our assay and confirmed its feasibility and practicality. The assay is not affected by SCCmec types and only needs modification if new mec homologs emerge and establishes a new platform for other emerging SCCmec types.
Collapse
Affiliation(s)
- Jo-Ann McClure
- Centre for Antimicrobial Resistance, Alberta Health Services/Alberta Precision Laboratories/University of Calgary, Calgary, AB, Canada
| | - John M Conly
- Centre for Antimicrobial Resistance, Alberta Health Services/Alberta Precision Laboratories/University of Calgary, Calgary, AB, Canada.,Department of Pathology and Laboratory Medicine, University of Calgary, Calgary, AB, Canada.,Department of Microbiology, Immunology and Infectious Diseases, University of Calgary, Calgary, AB, Canada.,Department of Medicine, University of Calgary, Calgary, AB, Canada.,The Calvin, Phoebe and Joan Snyder Institute for Chronic Diseases, University of Calgary, Calgary, AB, Canada
| | - Osahon Obasuyi
- Department of Pathology and Laboratory Medicine, University of Calgary, Calgary, AB, Canada
| | - Linda Ward
- Alberta Health Services, Calgary, AB, Canada
| | - Alejandra Ugarte-Torres
- Department of Medicine, University of Calgary, Calgary, AB, Canada.,Alberta Health Services, Calgary, AB, Canada
| | - Thomas Louie
- Department of Medicine, University of Calgary, Calgary, AB, Canada.,Alberta Health Services, Calgary, AB, Canada
| | - Kunyan Zhang
- Centre for Antimicrobial Resistance, Alberta Health Services/Alberta Precision Laboratories/University of Calgary, Calgary, AB, Canada.,Department of Pathology and Laboratory Medicine, University of Calgary, Calgary, AB, Canada.,Department of Microbiology, Immunology and Infectious Diseases, University of Calgary, Calgary, AB, Canada.,Department of Medicine, University of Calgary, Calgary, AB, Canada.,The Calvin, Phoebe and Joan Snyder Institute for Chronic Diseases, University of Calgary, Calgary, AB, Canada
| |
Collapse
|
|
21
|
Wang Y, Zhao X, Zhang M, Sun X, Bai J, Peng Y, Li S, Han D, Ren S, Wang J, Han T, Gao Y, Ning B, Gao Z. A fluorescent amplification strategy for high-sensitive detection of 17 β-estradiol based on EXPAR and HCR. Anal Chim Acta 2020; 1116:1-8. [PMID: 32389184 DOI: 10.1016/j.aca.2020.04.010] [Citation(s) in RCA: 16] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/29/2020] [Revised: 04/01/2020] [Accepted: 04/02/2020] [Indexed: 01/14/2023]
Abstract
Environmental endocrine disruptors in the environment and food, especially 17 β-estradiol (E2), are important factors affecting the growth and development of organisms. In this research, we constructed a fluorescence strategy for two-step amplification that combined two currently popular methods, exponential amplification reaction (EXPAR) and hybridization chain reaction (HCR). E2 competed with the complementary DNA (cDNA) to bind the aptamer modified on the magnetic beads. The free complementary strand in the supernatant was used as a trigger sequence to activate EXPAR, producing a large amount of short single-stranded DNA (ssDNA). The amplified ssDNA can trigger the second HCR amplification, producing many long double-stranded DNA (dsDNA) analogues. According to the principle of fluorescence resonance energy transfer, the carboxyfluorescein (FAM) signals in H1 and H2 hairpins were quenched by black hole quencher (BHQ-1). After the addition of E2 and initiation of amplification, the initially quenched fluorescent signal would be restored. This strategy with a detection limit of 0.37 pg mL-1 (S/N = 3) showed a good linear relationship in the range of 0.4-800 pg mL-1. In addition, the recovery rates of the method for milk and water samples were 98.55%-116.95% and 92.32%-107.00%, respectively. This is the first report of the combined detection of EXPAR and HCR, providing a reference for rapid and highly sensitive detection using multiple isothermal amplification methods.
Collapse
Affiliation(s)
- Yu Wang
- Tianjin Key Laboratory of Risk Assessment and Control Technology for Environment and Food Safety, Institute of Environmental and Operational Medicine, Academy of Military Medical Science, Academy of Military Science, Tianjin, 300050, PR China
| | - Xudong Zhao
- Tianjin Key Laboratory of Risk Assessment and Control Technology for Environment and Food Safety, Institute of Environmental and Operational Medicine, Academy of Military Medical Science, Academy of Military Science, Tianjin, 300050, PR China
| | - Man Zhang
- Tianjin Key Laboratory of Risk Assessment and Control Technology for Environment and Food Safety, Institute of Environmental and Operational Medicine, Academy of Military Medical Science, Academy of Military Science, Tianjin, 300050, PR China; School of Medical Instrument and Food Engineering, University of Shanghai for Science and Technology. Shanghai, 200093, PR China
| | - Xuan Sun
- Tianjin Key Laboratory of Risk Assessment and Control Technology for Environment and Food Safety, Institute of Environmental and Operational Medicine, Academy of Military Medical Science, Academy of Military Science, Tianjin, 300050, PR China; College of Life Science and Technology, Huazhong Agricultural University, Wuhan, 430070, PR China
| | - Jialei Bai
- Tianjin Key Laboratory of Risk Assessment and Control Technology for Environment and Food Safety, Institute of Environmental and Operational Medicine, Academy of Military Medical Science, Academy of Military Science, Tianjin, 300050, PR China
| | - Yuan Peng
- Tianjin Key Laboratory of Risk Assessment and Control Technology for Environment and Food Safety, Institute of Environmental and Operational Medicine, Academy of Military Medical Science, Academy of Military Science, Tianjin, 300050, PR China
| | - Shuang Li
- Tianjin Key Laboratory of Risk Assessment and Control Technology for Environment and Food Safety, Institute of Environmental and Operational Medicine, Academy of Military Medical Science, Academy of Military Science, Tianjin, 300050, PR China
| | - Dianpeng Han
- Tianjin Key Laboratory of Risk Assessment and Control Technology for Environment and Food Safety, Institute of Environmental and Operational Medicine, Academy of Military Medical Science, Academy of Military Science, Tianjin, 300050, PR China
| | - Shuyue Ren
- Tianjin Key Laboratory of Risk Assessment and Control Technology for Environment and Food Safety, Institute of Environmental and Operational Medicine, Academy of Military Medical Science, Academy of Military Science, Tianjin, 300050, PR China
| | - Jiang Wang
- Tianjin Key Laboratory of Risk Assessment and Control Technology for Environment and Food Safety, Institute of Environmental and Operational Medicine, Academy of Military Medical Science, Academy of Military Science, Tianjin, 300050, PR China
| | - Tie Han
- Tianjin Key Laboratory of Risk Assessment and Control Technology for Environment and Food Safety, Institute of Environmental and Operational Medicine, Academy of Military Medical Science, Academy of Military Science, Tianjin, 300050, PR China
| | - Yifei Gao
- School of Chemistry, University of New South Wales, Sydney, Australia
| | - Baoan Ning
- Tianjin Key Laboratory of Risk Assessment and Control Technology for Environment and Food Safety, Institute of Environmental and Operational Medicine, Academy of Military Medical Science, Academy of Military Science, Tianjin, 300050, PR China
| | - Zhixian Gao
- Tianjin Key Laboratory of Risk Assessment and Control Technology for Environment and Food Safety, Institute of Environmental and Operational Medicine, Academy of Military Medical Science, Academy of Military Science, Tianjin, 300050, PR China.
| |
Collapse
|
|
22
|
Complete Genome Sequence of a Staphylococcus aureus Isolate from a Nasopharyngeal Swab from a Mine Worker in South Africa. Microbiol Resour Announc 2019; 8:8/50/e01075-19. [PMID: 31831607 PMCID: PMC6908792 DOI: 10.1128/mra.01075-19] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
Here, we present the complete genome sequence of Staphylococcus aureus NP66, isolated from a South African mine worker.
Collapse
|
|
23
|
Athamanolap P, Hsieh K, O'Keefe CM, Zhang Y, Yang S, Wang TH. Nanoarray Digital Polymerase Chain Reaction with High-Resolution Melt for Enabling Broad Bacteria Identification and Pheno-Molecular Antimicrobial Susceptibility Test. Anal Chem 2019; 91:12784-12792. [PMID: 31525952 DOI: 10.1021/acs.analchem.9b02344] [Citation(s) in RCA: 64] [Impact Index Per Article: 10.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/19/2022]
Abstract
Toward combating infectious diseases caused by pathogenic bacteria, there remains an unmet need for diagnostic tools that can broadly identify the causative bacteria and determine their antimicrobial susceptibilities from complex and even polymicrobial samples in a timely manner. To address this need, a microfluidic and machine-learning-based platform that performs broad bacteria identification (ID) and rapid yet reliable antimicrobial susceptibility testing (AST) is developed. Specifically, this platform builds on "pheno-molecular AST", a strategy that transforms nucleic acid amplification tests (NAATs) into phenotypic AST through quantitative detection of bacterial genomic replication, and utilizes digital polymerase chain reaction (PCR) and digital high-resolution melt (HRM) to quantify and identify bacterial DNA molecules. Bacterial species are identified using integrated experiment-machine learning algorithm via HRM profiles. Digital DNA quantification allows for rapid growth measurement that reflects susceptibility profiles of each bacterial species within only 30 min of antibiotic exposure. As a demonstration, multiple bacterial species and their susceptibility profiles in a spiked-in polymicrobial urine specimen were correctly identified with a total turnaround time of ∼4 h. With further development and clinical validation, this platform holds the potential for improving clinical diagnostics and enabling targeted antibiotic treatments.
Collapse
Affiliation(s)
- Pornpat Athamanolap
- Department of Biomedical Engineering , Johns Hopkins School of Medicine , Baltimore , Maryland 21205 , United States
| | | | - Christine M O'Keefe
- Department of Biomedical Engineering , Johns Hopkins School of Medicine , Baltimore , Maryland 21205 , United States
| | - Ye Zhang
- Department of Biomedical Engineering , Johns Hopkins School of Medicine , Baltimore , Maryland 21205 , United States
| | - Samuel Yang
- Department of Emergency Medicine , Stanford University , Stanford , California 94304 , United States
| | - Tza-Huei Wang
- Department of Biomedical Engineering , Johns Hopkins School of Medicine , Baltimore , Maryland 21205 , United States.,The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins , Baltimore , Maryland 21287 , United States
| |
Collapse
|
|
24
|
Wang M, Yang H, Wu Y, Fu Z. Fluorescent analysis of Staphylococcus aureus by using daptomycin and immunoglobulin G for dual sites affinity. SPECTROCHIMICA ACTA. PART A, MOLECULAR AND BIOMOLECULAR SPECTROSCOPY 2019; 215:340-344. [PMID: 30852281 DOI: 10.1016/j.saa.2019.02.088] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 11/23/2018] [Revised: 02/13/2019] [Accepted: 02/18/2019] [Indexed: 06/09/2023]
Abstract
A dual sites affinity protocol was developed for fluorescent analysis of Staphylococcus aureus (S. aureus) by employing daptomycin and immunoglobulin G (IgG) as the recognition elements. Pig IgG immobilized on microplate was employed as the first recognition element to capture S. aureus owing to the fact that the Fc segment of mammal IgG can selectively bind with protein A on the surface of the target bacteria. Meanwhile, fluorescein isothiocyanate-conjugated daptomycin was employed as the second recognition element as well as the signal tracer for the target bacteria utilizing the binding capability of daptomycin to Gram-positive bacteria. S. aureus can be analyzed within a concentration range of 5.0 × 103-5.0 × 108 CFU mL-1 with a detection limit of 3.6 × 103 CFU mL-1. The analytical process can be accomplished within 1.5 h by using a pre-coated microplate. The dual sites affinity protocol can exclude the interference led by Gram-negative bacteria and other common Gram-positive bacteria. We have successfully applied it to analyze S. aureus in spiked lake water and physiological saline injection samples, and the recovery values ranged from 88.0% to 120.0%. The results demonstrate its application potential for environmental sanitation and drug safety control.
Collapse
Affiliation(s)
- Mengyao Wang
- Key Laboratory of Luminescence and Real-Time Analytical Chemistry (Ministry of Education), College of Pharmaceutical Sciences, Southwest University, Chongqing 400716, China
| | - Honglin Yang
- Key Laboratory of Luminescence and Real-Time Analytical Chemistry (Ministry of Education), College of Pharmaceutical Sciences, Southwest University, Chongqing 400716, China
| | - Yue Wu
- Key Laboratory of Luminescence and Real-Time Analytical Chemistry (Ministry of Education), College of Pharmaceutical Sciences, Southwest University, Chongqing 400716, China
| | - Zhifeng Fu
- Key Laboratory of Luminescence and Real-Time Analytical Chemistry (Ministry of Education), College of Pharmaceutical Sciences, Southwest University, Chongqing 400716, China.
| |
Collapse
|
|
25
|
Wang H, Hecht S, Kline D, Leber AL. Staphylococcus aureus and methicillin resistance detection directly from pediatric samples using PCR assays with differential cycle threshold values for corroboration of methicillin resistance. J Microbiol Methods 2019; 159:167-173. [PMID: 30826439 DOI: 10.1016/j.mimet.2019.01.009] [Citation(s) in RCA: 12] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/15/2018] [Revised: 01/15/2019] [Accepted: 01/20/2019] [Indexed: 01/13/2023]
Abstract
Staphylococcus aureus is a major human pathogen, causing a variety of nosocomial and community-acquired infections. While S. aureus usually grows well, there are situations where it cannot be isolated in culture, such as patients who have received prior antimicrobial therapy. There are commercially available tests for molecular identification of S. aureus and methicillin resistance; however, they often have limited utility due to restrictive specimen requirements, lack of data in pediatric populations and issues with specificity for methicillin resistance detections. Our objective was to evaluate the performance of laboratory-developed PCR assays that detect S. aureus and methicillin resistance directly from various specimen types. We developed two real-time PCR assays: 1) a singleplex assay targeting the nucA gene and 2) a multiplex PCR assay (mecA/SCC-orf PCRs) that detects the mecA gene and the conjunction region where SCCmec elements insert into the genome. A total of 538 pediatric specimens, including specimens from the lower respiratory tract (n = 149), abscess/wounds (n = 245), tissue and body fluids (n = 144), were tested and the results compared with culture and susceptibility testing. The nucA PCR is sensitive and specific for detection of S. aureus when compared with culture with an overall agreement of 93.1% and sensitivity and specificity of 93.5% and 93.0%, respectively. Among those culture-confirmed and nucA PCR positive specimens (n = 145), concordance between mecA/SCC-orf PCRs, using cycle threshold values for corroboration, and conventional methods was 98.6% and the sensitivity and specificity were 97.3% and 100%, respectively. The assays' performance suggests they are rapid, reliable tools to detect and differentiate between methicillin susceptible and methicillin resistant S. aureus in our pediatric patient population providing diagnostic impact when used in conjunction with culture.
Collapse
Affiliation(s)
- Huanyu Wang
- Department of Pathology and Laboratory Medicine, Nationwide Children's Hospital and The Ohio State University, Columbus, OH, United States of America
| | - Shaina Hecht
- Department of Pediatrics, Division of Pediatric Infectious Diseases, Nationwide Children's Hospital and The Ohio State University, Columbus, OH, United States of America
| | - David Kline
- Department of Biomedical Informatics, The Ohio State University, Columbus, OH, United States of America
| | - Amy L Leber
- Department of Pathology and Laboratory Medicine, Nationwide Children's Hospital and The Ohio State University, Columbus, OH, United States of America.
| |
Collapse
|
|
26
|
Nemr CR, Smith SJ, Liu W, Mepham AH, Mohamadi RM, Labib M, Kelley SO. Nanoparticle-Mediated Capture and Electrochemical Detection of Methicillin-Resistant Staphylococcus aureus. Anal Chem 2019; 91:2847-2853. [PMID: 30676721 DOI: 10.1021/acs.analchem.8b04792] [Citation(s) in RCA: 57] [Impact Index Per Article: 9.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/14/2023]
Abstract
The spread of antibiotic-resistant bacteria poses a global threat to public health. Conventional bacterial detection and identification methods often require pre-enrichment and/or sample preprocessing and purification steps that can prolong diagnosis by days. Methicillin-resistant Staphylococcus aureus (MRSA) is one of the most widespread antibiotic-resistant bacteria and is the leading cause of hospital-acquired infections. Here, we have developed a method to specifically capture and detect MRSA directly from patient nasal swabs with no prior culture and minimal processing steps using a microfluidic device and antibody-functionalized magnetic nanoparticles. Bacteria are captured based on antibody recognition of a membrane-bound protein marker that confers β-lactam antibiotic resistance. MRSA identification is then achieved by the use of a strain-specific antibody functionalized with alkaline phosphatase for electrochemical detection. This approach ensures that only those bacteria of the target strain and resistance profile are measured. The method has a limit of detection of 845 CFU/mL and excellent discrimination against high concentrations of common nontarget nasal flora with a turnaround time of under 4.5 h. This detection method was successfully validated using clinical nasal swab specimens ( n = 30) and has the potential to be tailored to various bacterial targets.
Collapse
|
|
27
|
Zhang Y, Shi S, Xing J, Tan W, Zhang C, Zhang L, Yuan H, Zhang M, Qiao J. A novel colorimetric sensing platform for the detection ofS. aureuswith high sensitivity and specificity. RSC Adv 2019; 9:33589-33595. [PMID: 35528901 PMCID: PMC9073649 DOI: 10.1039/c9ra05304b] [Citation(s) in RCA: 16] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/11/2019] [Accepted: 10/11/2019] [Indexed: 12/19/2022] Open
Abstract
In this study, a novel colorimetric sensing platform was developed for the detection of S. aureus using dog immunoglobulin G (IgG) as the capture antibody and chicken anti-protein A immunoglobulin Y labeled with horseradish peroxidase (HRP-IgY) as the detection antibody. Dog IgG labeled with magnetic beads was used to capture S. aureus through the interaction between the Fc region of dog IgG and Staphylococcal protein A (SPA). HRP-IgY was introduced to recognize the residual SPA on the surface of S. aureus and to create a sandwich format, after which a soluble 3,3′,5,5′-tetramethylbenzidine (TMB) substrate was added. A stop solution was utilized to cease the enzymatic chromogenic reaction, and then optical density was read at 450 nm. Under optimal conditions, the proposed method displayed a low detection limit of 1.0 × 103 CFU mL−1 and a wide linear range of 3.1 × 103 to 2.0 × 105 CFU mL−1. This detection method exhibited high specificity against other foodborne bacteria. The recovery rates ranged from 95.2% to 129.2%. To our knowledge, this is the first report to employ dog IgG and chicken IgY as an antibody pair to detect S. aureus. This technique exhibits high application potential for S. aureus monitoring in various kinds of samples. Utilization of dog IgG and chicken anti-protein A IgY as an antibody pair for sensitive and selective detection of S. aureus.![]()
Collapse
Affiliation(s)
- Yun Zhang
- Henan Key Laboratory of Immunology and Targeted Therapy
- Henan Collaborative Innovation Center of Molecular Diagnosis and Laboratory Medicine
- School of Laboratory Medicine
- Xinxiang Medical University
- Xinxiang 453003
| | - Shuyou Shi
- Henan Key Laboratory of Immunology and Targeted Therapy
- Henan Collaborative Innovation Center of Molecular Diagnosis and Laboratory Medicine
- School of Laboratory Medicine
- Xinxiang Medical University
- Xinxiang 453003
| | - Jiajia Xing
- School of International Education
- Xinxiang Medical University
- Xinxiang 453003
- PR China
| | - Wenqing Tan
- Henan Key Laboratory of Immunology and Targeted Therapy
- Henan Collaborative Innovation Center of Molecular Diagnosis and Laboratory Medicine
- School of Laboratory Medicine
- Xinxiang Medical University
- Xinxiang 453003
| | - Chenguang Zhang
- Henan Key Laboratory of Immunology and Targeted Therapy
- Henan Collaborative Innovation Center of Molecular Diagnosis and Laboratory Medicine
- School of Laboratory Medicine
- Xinxiang Medical University
- Xinxiang 453003
| | - Lin Zhang
- School of Innovation and Entrepreneurship
- Xinxiang Medical University
- Xinxiang 453003
- PR China
| | - Huan Yuan
- Henan Key Laboratory of Immunology and Targeted Therapy
- Henan Collaborative Innovation Center of Molecular Diagnosis and Laboratory Medicine
- School of Laboratory Medicine
- Xinxiang Medical University
- Xinxiang 453003
| | - Miaomiao Zhang
- Henan Key Laboratory of Immunology and Targeted Therapy
- Henan Collaborative Innovation Center of Molecular Diagnosis and Laboratory Medicine
- School of Laboratory Medicine
- Xinxiang Medical University
- Xinxiang 453003
| | - Jinjuan Qiao
- Department of Medical Laboratory
- Weifang Medical University
- Weifang 261053
- PR China
| |
Collapse
|
|
28
|
Using multiplex PCR as a diagnostic tool to detect methicillin resistant Staphylococcus aureus. JOURNAL OF SURGERY AND MEDICINE 2018. [DOI: 10.28982/josam.415215] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/07/2023] Open
|
|
29
|
Huang TH, Tzeng YL, Dickson RM. FAST: Rapid determinations of antibiotic susceptibility phenotypes using label-free cytometry. Cytometry A 2018; 93:639-648. [PMID: 29733508 DOI: 10.1002/cyto.a.23370] [Citation(s) in RCA: 20] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/28/2017] [Revised: 01/25/2018] [Accepted: 03/15/2018] [Indexed: 11/08/2022]
Abstract
Sepsis, a life-threatening immune response to blood infections (bacteremia), has a ∼30% mortality rate and is the 10th leading cause of US hospital deaths. The typical bacterial loads in adult septic patients are ≤100 bacterial cells (colony forming units, CFU) per ml blood, while pediatric patients exhibit only ∼1000 CFU/ml. Due to the low numbers, bacteria must be propagated through ∼24-hours blood cultures to generate sufficient CFUs for diagnosis and further analyses. Herein, we demonstrate that, unlike other rapid post-blood culture antibiotic susceptibility tests (ASTs), our phenotypic approach can drastically accelerate ASTs for the most common sepsis-causing gram-negative pathogens by circumventing long blood culture-based amplification. For all blood isolates of multi-drug resistant pathogens investigated (Escherichia coli, Klebsiella pneumoniae, and Acinetobacter nosocomialis), effective antibiotic(s) were readily identified within the equivalent of 8 hours from initial blood draw using <0.5 mL of adult blood per antibiotic. These methods should drastically improve patient outcomes by significantly reducing time to actionable treatment information and reduce the incidence of antibiotic resistance. © 2018 International Society for Advancement of Cytometry.
Collapse
Affiliation(s)
- Tzu-Hsueh Huang
- School of Chemistry & Biochemistry and Petit Institute of Bioengineering and Bioscience, Georgia Institute of Technology, Atlanta, Georgia, 30332-0400
| | - Yih-Ling Tzeng
- Division of Infectious Diseases, Emory University School of Medicine, Atlanta, Georgia, 30322
| | - Robert M Dickson
- School of Chemistry & Biochemistry and Petit Institute of Bioengineering and Bioscience, Georgia Institute of Technology, Atlanta, Georgia, 30332-0400
| |
Collapse
|
|
30
|
El-Bouri K, Johnston S, Rees E, Thomas I, Bome-Mannathoko N, Jones C, Reid M, Ben-Ismaeil B, Davies AP, Harris LG, Mack D. Comparison of bacterial identification by MALDI-TOF mass spectrometry and conventional diagnostic microbiology methods: agreement, speed and cost implications. Br J Biomed Sci 2018. [DOI: 10.1080/09674845.2012.12002436] [Citation(s) in RCA: 21] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/17/2022]
Affiliation(s)
- K. El-Bouri
- Public Health Wales Microbiology Laboratory ABM Swansea, Singleton Hospital, Abertawe-Bro Morgannwg University Health Board, Swansea, United Kingdom
| | - S. Johnston
- Public Health Wales Microbiology Laboratory ABM Swansea, Singleton Hospital, Abertawe-Bro Morgannwg University Health Board, Swansea, United Kingdom
| | - E. Rees
- Public Health Wales Microbiology Laboratory ABM Swansea, Singleton Hospital, Abertawe-Bro Morgannwg University Health Board, Swansea, United Kingdom
| | - I. Thomas
- Public Health Wales Microbiology Laboratory ABM Swansea, Singleton Hospital, Abertawe-Bro Morgannwg University Health Board, Swansea, United Kingdom
| | - N. Bome-Mannathoko
- Medical Microbiology and Infectious Diseases, Institute of Life Science, School of Medicine, Swansea University, Swansea, United Kingdom
| | - C. Jones
- Medical Microbiology and Infectious Diseases, Institute of Life Science, School of Medicine, Swansea University, Swansea, United Kingdom
| | - M. Reid
- Public Health Wales Microbiology Laboratory ABM Swansea, Singleton Hospital, Abertawe-Bro Morgannwg University Health Board, Swansea, United Kingdom
- Medical Microbiology and Infectious Diseases, Institute of Life Science, School of Medicine, Swansea University, Swansea, United Kingdom
| | - B. Ben-Ismaeil
- Public Health Wales Microbiology Laboratory ABM Swansea, Singleton Hospital, Abertawe-Bro Morgannwg University Health Board, Swansea, United Kingdom
| | - A. P. Davies
- Public Health Wales Microbiology Laboratory ABM Swansea, Singleton Hospital, Abertawe-Bro Morgannwg University Health Board, Swansea, United Kingdom
- Medical Microbiology and Infectious Diseases, Institute of Life Science, School of Medicine, Swansea University, Swansea, United Kingdom
| | - L. G. Harris
- Medical Microbiology and Infectious Diseases, Institute of Life Science, School of Medicine, Swansea University, Swansea, United Kingdom
| | - D. Mack
- Public Health Wales Microbiology Laboratory ABM Swansea, Singleton Hospital, Abertawe-Bro Morgannwg University Health Board, Swansea, United Kingdom
- Medical Microbiology and Infectious Diseases, Institute of Life Science, School of Medicine, Swansea University, Swansea, United Kingdom
| |
Collapse
|
|
31
|
Schumacher A, Vranken T, Malhotra A, Arts JJC, Habibovic P. In vitro antimicrobial susceptibility testing methods: agar dilution to 3D tissue-engineered models. Eur J Clin Microbiol Infect Dis 2018; 37:187-208. [PMID: 28871407 PMCID: PMC5780537 DOI: 10.1007/s10096-017-3089-2] [Citation(s) in RCA: 42] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/15/2017] [Accepted: 07/20/2017] [Indexed: 12/22/2022]
Abstract
In the field of orthopaedic surgery, bacterial invasion of implants and the resulting periprosthetic infections are a common and unresolved problem. Antimicrobial susceptibility testing methods help to define the optimal treatment and identify antimicrobial resistance. This review discusses proven gold-standard techniques and recently developed models for antimicrobial susceptibility testing, while also providing a future outlook. Conventional, gold-standard methods, such as broth microdilution, are still widely applied in clinical settings. Although recently developed methods based on microfluidics and microdroplets have shown advantages over conventional methods in terms of testing speed, safety and the potential to provide a deeper insight into resistance mechanisms, extensive validation is required to translate this research to clinical practice. Recent optical and mechanical methods are complex and expensive and, therefore, not immediately clinically applicable. Novel osteoblast infection and tissue models best resemble infections in vivo. However, the integration of biomaterials into these models remains challenging and they require a long tissue culture, making their rapid clinical implementation unlikely. A method applicable for both clinical and research environments is difficult to realise. With a continuous increase in antimicrobial resistance, there is an urgent need for methods that analyse recurrent infections to identify the optimal treatment approaches. Graphical abstract Timeline of published and partly applied antimicrobial susceptibility testing methods, listed according to their underlying mechanism, complexity and application in research or clinics.
Collapse
Affiliation(s)
- A Schumacher
- Department of Instructive Biomaterials Engineering, MERLN Institute for Technology-Inspired Regenerative Medicine, Maastricht University, Universiteitssingel 40, Room C3.577, 6229 ER, Maastricht, Netherlands.
- Science and Technology Faculty, University of Twente, Drienerlolaan 5, 7522 NB, Enschede, The Netherlands.
| | - T Vranken
- Department of Orthopaedic Surgery, CAPHRI Care and Public Health Research Institute, Maastricht University Medical Centre, Maastricht, The Netherlands
| | - A Malhotra
- Department of Instructive Biomaterials Engineering, MERLN Institute for Technology-Inspired Regenerative Medicine, Maastricht University, Universiteitssingel 40, Room C3.577, 6229 ER, Maastricht, Netherlands
| | - J J C Arts
- Department of Orthopaedic Surgery, CAPHRI Care and Public Health Research Institute, Maastricht University Medical Centre, Maastricht, The Netherlands
- Orthopaedic Biomechanics Group, Department of Biomedical Engineering, Eindhoven University of Technology (TU/e), Eindhoven, The Netherlands
| | - P Habibovic
- Department of Instructive Biomaterials Engineering, MERLN Institute for Technology-Inspired Regenerative Medicine, Maastricht University, Universiteitssingel 40, Room C3.577, 6229 ER, Maastricht, Netherlands
| |
Collapse
|
|
32
|
Nolte FS. Molecular Microbiology. PRINCIPLES AND APPLICATIONS OF MOLECULAR DIAGNOSTICS 2018. [PMCID: PMC7150357 DOI: 10.1016/b978-0-12-816061-9.00005-9] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Subscribe] [Scholar Register] [Indexed: 11/18/2022]
Abstract
Background Nucleic acid (NA) amplification techniques are now commonly used to diagnose and manage patients with infectious diseases. The growth in the number of Food and Drug Administration–approved test kits and analyte-specific reagents has facilitated the use of this technology in clinical laboratories. Technological advances in NA amplification techniques, automation, NA sequencing, and multiplex analysis have reinvigorated the field and created new opportunities for growth. Simple, sample-in, answer-out molecular test systems are now widely available that can be deployed in a variety of laboratory and clinical settings. Molecular microbiology remains the leading area in molecular pathology in terms of both the numbers of tests performed and clinical relevance. NA-based tests have reduced the dependency of the clinical microbiology laboratory on more traditional antigen detection and culture methods and created new opportunities for the laboratory to impact patient care. Content This chapter reviews NA testing as it applies to specific pathogens or infectious disease syndromes, with a focus on those diseases for which NA testing is now considered the standard of care and highlights the unique challenges and opportunities that these tests present for clinical laboratories.
Collapse
|
|
33
|
Accurate Detection of Methicillin-Resistant Staphylococcus aureus in Mixtures by Use of Single-Bacterium Duplex Droplet Digital PCR. J Clin Microbiol 2017; 55:2946-2955. [PMID: 28724560 DOI: 10.1128/jcm.00716-17] [Citation(s) in RCA: 35] [Impact Index Per Article: 4.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/02/2017] [Accepted: 07/14/2017] [Indexed: 12/16/2022] Open
Abstract
Accurate and rapid identification of methicillin-resistant Staphylococcus aureus (MRSA) is needed to screen MRSA carriers and improve treatment. The current widely used duplex PCR methods are not able to differentiate MRSA from coexisting methicillin-susceptible S. aureus (MSSA) or other methicillin-resistant staphylococci. In this study, we aimed to develop a direct method for accurate and rapid detection of MRSA in clinical samples from open environments, such as nasal swabs. The new molecular assay is based on detecting the cooccurrence of nuc and mecA markers in a single bacterial cell by utilizing droplet digital PCR (ddPCR) with the chimeric lysin ClyH for cell lysis. The method consists of (i) dispersion of an intact single bacterium into nanoliter droplets, (ii) temperature-controlled release of genomic DNA (gDNA) by ClyH at 37°C, and (iii) amplification and detection of the markers (nuc and mecA) using standard TaqMan chemistries with ddPCR. Results were analyzed based on MRSA index ratios used for indicating the presence of the duplex-positive markers in droplets. The method was able to achieve an absolute limit of detection (LOD) of 2,900 CFU/ml for MRSA in nasal swabs spiked with excess amounts of Escherichia coli, MSSA, and other mecA-positive bacteria within 4 h. Initial testing of 104 nasal swabs showed that the method had 100% agreement with the standard culture method, while the normal duplex qPCR method had only about 87.5% agreement. The single-bacterium duplex ddPCR assay is rapid and powerful for more accurate detection of MRSA directly from clinical specimens.
Collapse
|
|
34
|
Guardabassi L, Damborg P, Stamm I, Kopp PA, Broens EM, Toutain PL. Diagnostic microbiology in veterinary dermatology: present and future. Vet Dermatol 2017; 28:146-e30. [PMID: 28133869 DOI: 10.1111/vde.12414] [Citation(s) in RCA: 22] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 11/13/2016] [Indexed: 12/31/2022]
Abstract
BACKGROUND The microbiology laboratory can be perceived as a service provider rather than an integral part of the healthcare team. OBJECTIVES The aim of this review is to discuss the current challenges of providing a state-of-the-art diagnostic veterinary microbiology service including the identification (ID) and antimicrobial susceptibility testing (AST) of key pathogens in veterinary dermatology. METHODS The Study Group for Veterinary Microbiology (ESGVM) of the European Society of Clinical Microbiology and Infectious Diseases (ESCMID) identified scientific, technological, educational and regulatory issues impacting the predictive value of AST and the quality of the service offered by microbiology laboratories. RESULTS The advent of mass spectrometry has significantly reduced the time required for ID of key pathogens such as Staphylococcus pseudintermedius. However, the turnaround time for validated AST methods has remained unchanged for many years. Beyond scientific and technological constraints, AST methods are not harmonized and clinical breakpoints for some antimicrobial drugs are either missing or inadequate. Small laboratories, including in-clinic laboratories, are usually not adequately equipped to run up-to-date clinical microbiologic diagnostic tests. CONCLUSIONS AND CLINICAL IMPORTANCE ESGVM recommends the use of laboratories employing mass spectrometry for ID and broth micro-dilution for AST, and offering assistance by expert microbiologists on pre- and post-analytical issues. Setting general standards for veterinary clinical microbiology, promoting antimicrobial stewardship, and the development of new, validated and rapid diagnostic methods, especially for AST, are among the missions of ESGVM.
Collapse
Affiliation(s)
- Luca Guardabassi
- Department of Biomedical Sciences, Ross University School of Veterinary Medicine, PO Box 334, Basseterre, St Kitts and Nevis, West Indies.,Department of Veterinary Disease Biology, Faculty of Health and Medical Sciences, University of Copenhagen, Stigbøjlen 4, 1870, Frederiksberg, Denmark
| | - Peter Damborg
- Department of Veterinary Disease Biology, Faculty of Health and Medical Sciences, University of Copenhagen, Stigbøjlen 4, 1870, Frederiksberg, Denmark
| | - Ivonne Stamm
- IDEXX Vet·Med·Labor, Moerikestrasse 28/3, D-71636, Ludwigsburg, Germany
| | - Peter A Kopp
- IDEXX Vet·Med·Labor, Moerikestrasse 28/3, D-71636, Ludwigsburg, Germany
| | - Els M Broens
- Department of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, Utrecht University, Yalelaan 1, 3584 CL, Utrecht, The Netherlands
| | - Pierre-Louis Toutain
- UMR 1331 Toxalim INRA/INP, Ecole Nationale Vétérinaire de Toulouse, 23 Chemin des Capelles, BP 87614, 31076, Toulouse Cedex 3, France
| | | |
Collapse
|
|
35
|
Herma M, Petersdorf S, Henrich B. Methicillin-resistant Staphylococcus aureus Screening PCR adapted to locally emerging variants-Evaluation of novel SCCmec primers. Int J Med Microbiol 2017; 307:209-215. [PMID: 28495466 DOI: 10.1016/j.ijmm.2017.05.001] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/02/2016] [Revised: 04/13/2017] [Accepted: 05/02/2017] [Indexed: 12/01/2022] Open
Abstract
Infections with multi-resistant bacteria, such as Methicillin-resistant Staphylococcus aureus (MRSA), represent a world-wide health-care problem. The original MRSA Screening TaqMan PCR was based on the detection of the SCCmec-orfX-junction as described by the group of Huletsky in 2004. In the recent years, this assay increasingly failed to detect new MRSA variants in swab specimens. In this work, we analyzed the usefulness of 17 additional SCCmec primers to increase PCR sensitivity by testing 290 collected samples with negative PCR results and positive MRSA culture in a retrospective analysis, and 380 samples of the daily routine diagnostics. Sequencing of the PCR products revealed that locally new MRSA variants became detectable by nine of these forward primers. Four primers were solely responsible for the detection of 85.4% (117/123) of the PCR products: F13 (n=76), F11 (n=6), F14 (n=15) and F25 (n=8). These four primers were integrated in the Screening PCR and the novel primer collection was validated by testing 71 MRSA isolates, which covered SCCmec types I to VI, 50 MSSA isolates and 100 swab specimens. The sensitivity of MRSA Screening PCR increased from 93% to 98.6% without affecting the detection of the common MRSA strains. Phylogenetic analysis of the PCR products suggests that the adapted MRSA Screening PCR is able to detect SCCmec types I-X, including CA- and LA-MRSA variants by the SCCmec primers F11 and F25.
Collapse
Affiliation(s)
- Miriam Herma
- Institute of Medical Microbiology and Hospital Hygiene, University Hospital, Heinrich-Heine-University, Düsseldorf, Germany
| | - Sabine Petersdorf
- Institute of Medical Microbiology and Hospital Hygiene, University Hospital, Heinrich-Heine-University, Düsseldorf, Germany
| | - Birgit Henrich
- Institute of Medical Microbiology and Hospital Hygiene, University Hospital, Heinrich-Heine-University, Düsseldorf, Germany.
| |
Collapse
|
|
36
|
Koivusalo M, Vermeiren C, Yuen J, Reeve C, Gadbois S, Katz K. Canine scent detection as a tool to distinguish meticillin-resistant Staphylococcus aureus. J Hosp Infect 2017; 96:93-95. [DOI: 10.1016/j.jhin.2017.03.005] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/16/2016] [Accepted: 03/02/2017] [Indexed: 10/20/2022]
|
|
37
|
Bissonnette L, Bergeron MG. Portable devices and mobile instruments for infectious diseases point-of-care testing. Expert Rev Mol Diagn 2017; 17:471-494. [PMID: 28343420 DOI: 10.1080/14737159.2017.1310619] [Citation(s) in RCA: 17] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/19/2022]
Abstract
INTRODUCTION Rapidity, simplicity, and portability are highly desirable characteristics of tests and devices designed for performing diagnostics at the point of care (POC), either near patients managed in healthcare facilities or to offer bioanalytical alternatives in external settings. By reducing the turnaround time of the diagnostic cycle, POC diagnostics can reduce the dissemination, morbidity, and mortality of infectious diseases and provide tools to control the global threat of antimicrobial resistance. Areas covered: A literature search of PubMed and Google Scholar, and extensive mining of specialized publications, Internet resources, and manufacturers' websites have been used to organize and write this overview of the challenges and requirements associated with the development of portable sample-to-answer diagnostics, and showcase relevant examples of handheld devices, portable instruments, and less mobile systems which may or could be operated at POC. Expert commentary: Rapid (<1 h) diagnostics can contribute to control infectious diseases and antimicrobial resistant pathogens. Portable devices or instruments enabling sample-to-answer bioanalysis can provide rapid, robust, and reproducible testing at the POC or close from it. Beyond testing, to realize some promises of personalized/precision medicine, it will be critical to connect instruments to healthcare data management systems, to efficiently link decentralized testing results to the electronic medical record of patients.
Collapse
Affiliation(s)
- Luc Bissonnette
- a Centre de recherche en infectiologie de l'Université Laval, Axe maladies infectieuses et immunitaires, Centre de recherche du CHU de Québec-Université Laval , Québec City , Québec , Canada
| | - Michel G Bergeron
- a Centre de recherche en infectiologie de l'Université Laval, Axe maladies infectieuses et immunitaires, Centre de recherche du CHU de Québec-Université Laval , Québec City , Québec , Canada.,b Département de microbiologie-infectiologie et d'immunologie , Faculté de médecine, Université Laval , Québec City , Québec , Canada
| |
Collapse
|
|
38
|
Seidel C, Peters S, Eschbach E, Feßler AT, Oberheitmann B, Schwarz S. Development of a nucleic acid lateral flow immunoassay (NALFIA) for reliable, simple and rapid detection of the methicillin resistance genes mecA and mecC. Vet Microbiol 2017; 200:101-106. [DOI: 10.1016/j.vetmic.2016.08.009] [Citation(s) in RCA: 19] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/23/2016] [Revised: 07/22/2016] [Accepted: 08/13/2016] [Indexed: 11/29/2022]
|
|
39
|
Nielsen XC, Madsen TV, Engberg J. Evaluation of Xpert MRSA Gen 3 and BD MAX MRSA XT for meticillin-resistant Staphylococcus
aureus screening in a routine diagnostic setting in a low-prevalence area. J Med Microbiol 2017; 66:90-95. [DOI: 10.1099/jmm.0.000411] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/18/2022] Open
Affiliation(s)
- Xiaohui Chen Nielsen
- Department of Clinical Microbiology, Slagelse Hospital, Ingemannsvej 46, DK-4200 Slagelse, Denmark
| | - Tina Vasehus Madsen
- Department of Clinical Microbiology, Slagelse Hospital, Ingemannsvej 46, DK-4200 Slagelse, Denmark
| | - Jørgen Engberg
- Department of Clinical Microbiology, Slagelse Hospital, Ingemannsvej 46, DK-4200 Slagelse, Denmark
| |
Collapse
|
|
40
|
Bakthavatchalam YD, Nabarro LEB, Veeraraghavan B. Evolving Rapid Methicillin-resistant Staphylococcus aureus Detection: Cover All the Bases. J Glob Infect Dis 2017; 9:18-22. [PMID: 28250621 PMCID: PMC5330039 DOI: 10.4103/0974-777x.199997] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/04/2022] Open
Abstract
The dissemination of methicillin-resistant (MR) Staphylococcus aureus (SA) in community and health-care settings is of great concern and associated with high mortality and morbidity. Rapid detection of MRSA with short turnaround time can minimize the time to initiate appropriate therapy and further promote infection control. Early detection of MRSA directly from clinical samples is complicated by the frequent association of MRSA with methicillin-susceptible SA (MSSA) and coagulase-negative Staphylococcus (CoNS) species. Infection associated with true MRSA or MSSA is differentiated from CoNS, requires target specific primers for the presence of SA and mec A or nuc or fem A gene for confirmation of MR. Recently, livestock-associated MRSA carrying mec C variant complicates the epidemiology of MRSA further. Several commercial rapid molecular kits are available with a different combination of these targets for the detection of MRSA or MSSA. The claimed sensitivity and specificity of the currently available commercial kits is varying, because of the different target combination used for detection of SA and MR.
Collapse
Affiliation(s)
| | - Laura E B Nabarro
- Department of Clinical Microbiology, Christian Medical College, Vellore, Tamil Nadu, India; Department of Infectious Disease, Public Health England, London, UK
| | - Balaji Veeraraghavan
- Department of Clinical Microbiology, Christian Medical College, Vellore, Tamil Nadu, India
| |
Collapse
|
|
41
|
Dubourg G, Fournier PE. Advances in Diagnostic Microbiology. Infect Dis (Lond) 2017. [DOI: 10.1016/b978-0-7020-6285-8.00161-1] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 11/26/2022] Open
|
|
42
|
Hell M, Bauer JW, Laimer M. [Molecular diagnosis of methicillin-resistent Staphylococcus aureus: Methods and efficacy]. Hautarzt 2016; 67:6-15. [PMID: 26016829 DOI: 10.1007/s00105-015-3643-8] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/21/2022]
Abstract
Methicillin-resistant Staphylococcus aureus (MRSA) isolates are a serious public health problem whose ever-increasing rate is commensurate with the pressure it is exerting on the healthcare system. At present, more than 20% of clinical S. aureus isolates in German hospitals are methicillin-resistant, in Austria less than 10%. Strategies from low-prevalence countries show that this development is not necessarily inevitable. In the Scandinavian countries and the Netherlands, thanks to a rigorous prevention programme, MRSA prevalence has been kept at an acceptably low level (< 1-3%). Central to these search-and-destroy control strategies is an admission screening using several MRSA swabs taken from mucocutaneous colonisation sites of high-risk patients (MRSA surveillance). It has also been reported that the speed with which MRSA carriage is detected has an important role, as it is a key component of any effective strategy to prevent the pathogen from spreading. Since MRSA culturing involves a 2-3 day delay before the final results are available, rapid detection techniques (commonly referred to as MRSA rapid tests) using polymerase chain reaction (PCR) methods and, most recently, rapid culturing methods have been developed. The implementation of rapid tests reduces the time of detection of MRSA carriers from 48-72 to 2-5 h. Clinical evaluation data have shown that MRSA can thus be detected with very high sensitivity. Specificity, however, is sometimes impaired due to false-positive PCR signals occurring in mixed flora specimens. In order to rule out false-positive PCR results, a culture screen must always be carried out simultaneously. The data provide preliminary evidence that a PCR assay can reduce nosocomial MRSA transmission in high-risk patients or high-risk areas, whereas an approach that screens all patients admitted to the hospital is probably not effective. Information concerning the cost effectiveness of rapid MRSA tests is still sparse and thus the issue remains debated.
Collapse
Affiliation(s)
- M Hell
- Zentrum für Krankenhaushygiene und Infektionskontrolle, Paracelsus Medizinische Privatuniversität Salzburg, Müllner Hauptstrasse 48, 5020, Salzburg, Österreich.
| | - J W Bauer
- Universitätsklinik für Dermatologie, Paracelsus Medizinische, Privatuniversität Salzburg, Salzburg, Österreich
| | - M Laimer
- Universitätsklinik für Dermatologie, Paracelsus Medizinische, Privatuniversität Salzburg, Salzburg, Österreich
| |
Collapse
|
|
43
|
Han HW, Chang HC, Chang TC. Identification of Staphylococcus spp. and detection of mecA by an oligonucleotide array. Diagn Microbiol Infect Dis 2016; 86:23-9. [PMID: 27342780 DOI: 10.1016/j.diagmicrobio.2016.06.003] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/17/2016] [Revised: 05/31/2016] [Accepted: 06/01/2016] [Indexed: 01/15/2023]
Abstract
Phenotypic identification of coagulase-negative staphylococci (CoNS) is difficult and many staphylococcal species carry mecA. This study developed an array that was able to detect mecA and identify 30 staphylococcal species by targeting the internal transcribed spacer regions. A total of 129 target reference strains (30 species) and 434 clinical isolates of staphylococci were analyzed. Gene sequencing of 16S rRNA, gap or tuf genes was the reference method for species identification. All reference strains (100%) were correctly identified, while the identification rates of clinical isolates of S. aureus and CoNS were 98.9% and 98%, respectively. The sensitivity and specificity for mecA detection were 99% and 100%, respectively, in S. aureus isolates, and both values were 100% in isolates of CoNS. The assay takes 6 h from a purified culture isolate, and so far it has not been performed directly on patient samples.
Collapse
Affiliation(s)
- Huan Wen Han
- Institute of Biomedical Engineering, College of Engineering, National Cheng Kung University, Tainan, Taiwan
| | - Hsien Chang Chang
- Institute of Biomedical Engineering, College of Engineering, National Cheng Kung University, Tainan, Taiwan; Medical Device Innovation Center, National Cheng Kung University, Tainan, Taiwan.
| | - Tsung Chain Chang
- Department of Medical Laboratory Science and Biotechnology, College of Medicine, National Cheng Kung University, Tainan, Taiwan.
| |
Collapse
|
|
44
|
Vos MC, Verbrugh HA. MRSA: We Can Overcome, But Who Will Lead the Battle? Infect Control Hosp Epidemiol 2016; 26:117-20. [PMID: 16955947 DOI: 10.1086/503507] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/03/2022]
|
|
45
|
Comparative evaluation of two fully-automated real-time PCR methods for MRSA admission screening in a tertiary-care hospital. Eur J Clin Microbiol Infect Dis 2016; 35:1475-8. [PMID: 27259711 DOI: 10.1007/s10096-016-2687-8] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/19/2016] [Accepted: 05/18/2016] [Indexed: 10/21/2022]
Abstract
We evaluated two fully-automated real-time PCR systems, the novel QIAGEN artus MRSA/SA QS-RGQ and the widely used BD MAX MRSA assay, for their diagnostic performance in MRSA admission screening in a tertiary-care university hospital. Two hundred sixteen clinical swabs were analyzed for MRSA DNA using the BD MAX MRSA assay. In parallel, the same specimens were tested with the QIAGEN artus MRSA/SA QS-RGQ. Automated steps included lysis of bacteria, DNA extraction, real-time PCR and interpretation of results. MRSA culture was additionally performed as a reference method for MRSA detection. Sensitivity values were similar for both assays (80 %), while the QIAGEN artus MRSA/SA QS-RGQ reached a slightly higher specificity (95.8 % versus 90.0 %). Positive (PPVs) and negative predictive values (NPVs) were 17.4 % and 99.4 % for the BD MAX MRSA assay and 33.3 % and 99.5 % for the QIAGEN artus MRSA/SA QS-RGQ, respectively. Total turn-around time (TAT) for 24 samples was 3.5 hours for both assays. In conclusion, both assays represent reliable diagnostic tools due to their high negative predictive values, especially for the rapid identification of MRSA negative patients in a low prevalence MRSA area.
Collapse
|
|
46
|
Liu Y, Zhang J, Ji Y. PCR-based Approaches for the Detection of Clinical Methicillin-resistant Staphylococcus aureus. Open Microbiol J 2016; 10:45-56. [PMID: 27335617 PMCID: PMC4899539 DOI: 10.2174/1874285801610010045] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/13/2015] [Revised: 10/20/2015] [Accepted: 10/22/2015] [Indexed: 11/22/2022] Open
Abstract
Staphylococcus aureus is an important pathogen that can cause a variety of infections, including superficial and systematic infections, in humans and animals. The persistent emergence of multidrug resistant S. aureus, particularly methicillin-resistant S. aureus, has caused dramatically economic burden and concerns in the public health due to limited options of treatment of MRSA infections. In order to make a correct choice of treatment for physicians and understand the prevalence of MRSA, it is extremely critical to precisely and timely diagnose the pathogen that induces a specific infection of patients and to reveal the antibiotic resistant profile of the pathogen. In this review, we outlined different PCR-based approaches that have been successfully utilized for the rapid detection of S. aureus, including MRSA and MSSA, directly from various clinical specimens. The sensitivity and specificity of detections were pointed out. Both advantages and disadvantages of listed approaches were discussed. Importantly, an alternative approach is necessary to further confirm the detection results from the molecular diagnostic assays.
Collapse
Affiliation(s)
- Ying Liu
- Shanghai Vocational College of Agriculture and Forestry, Shanghai, China; Department of Veterinary Biomedical Science, College of Veterinary Medicine, University of Minnesota, Saint Paul, United States
| | - Jiang Zhang
- Shanghai Vocational College of Agriculture and Forestry, Shanghai, China
| | - Yinduo Ji
- Department of Veterinary Biomedical Science, College of Veterinary Medicine, University of Minnesota, Saint Paul, United States
| |
Collapse
|
|
47
|
Dinarelli S, Girasole M, Kasas S, Longo G. Nanotools and molecular techniques to rapidly identify and fight bacterial infections. J Microbiol Methods 2016; 138:72-81. [PMID: 26806415 DOI: 10.1016/j.mimet.2016.01.005] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/21/2015] [Revised: 01/13/2016] [Accepted: 01/13/2016] [Indexed: 12/22/2022]
Abstract
Reducing the emergence and spread of antibiotic-resistant bacteria is one of the major healthcare issues of our century. In addition to the increased mortality, infections caused by multi-resistant bacteria drastically enhance the healthcare costs, mainly because of the longer duration of illness and treatment. While in the last 20years, bacterial identification has been revolutionized by the introduction of new molecular techniques, the current phenotypic techniques to determine the susceptibilities of common Gram-positive and Gram-negative bacteria require at least two days from collection of clinical samples. Therefore, there is an urgent need for the development of new technologies to determine rapidly drug susceptibility in bacteria and to achieve faster diagnoses. These techniques would also lead to a better understanding of the mechanisms that lead to the insurgence of the resistance, greatly helping the quest for new antibacterial systems and drugs. In this review, we describe some of the tools most currently used in clinical and microbiological research to study bacteria and to address the challenge of infections. We discuss the most interesting advancements in the molecular susceptibility testing systems, with a particular focus on the many applications of the MALDI-TOF MS system. In the field of the phenotypic characterization protocols, we detail some of the most promising semi-automated commercial systems and we focus on some emerging developments in the field of nanomechanical sensors, which constitute a step towards the development of rapid and affordable point-of-care testing devices and techniques. While there is still no innovative technique that is capable of completely substituting for the conventional protocols and clinical practices, many exciting new experimental setups and tools could constitute the basis of the standard testing package of future microbiological tests.
Collapse
Affiliation(s)
- S Dinarelli
- Istituto di Struttura della Materia, Consiglio Nazionale delle Ricerche, Rome, Italy
| | - M Girasole
- Istituto di Struttura della Materia, Consiglio Nazionale delle Ricerche, Rome, Italy
| | - S Kasas
- Ecole Polytechnique Fédérale de Lausanne, Laboratoire de Physique de la Matière Vivante, Lausanne, Switzerland; Département des Neurosciences Fondamentales, Université de Lausanne, Lausanne, Switzerland
| | - G Longo
- Istituto di Struttura della Materia, Consiglio Nazionale delle Ricerche, Rome, Italy.
| |
Collapse
|
|
48
|
Overview of Molecular Diagnostics in Multiple-Drug-Resistant Organism Prevention: Focus on Multiple-Drug-Resistant Gram-Negative Bacterial Organisms. Mol Microbiol 2016. [DOI: 10.1128/9781555819071.ch17] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022]
|
|
49
|
|
|
50
|
Østergaard C, Hansen SG, Møller JK. Rapid first-line discrimination of methicillin resistant Staphylococcus aureus strains using MALDI-TOF MS. Int J Med Microbiol 2015; 305:838-47. [DOI: 10.1016/j.ijmm.2015.08.002] [Citation(s) in RCA: 31] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/15/2015] [Revised: 08/06/2015] [Accepted: 08/06/2015] [Indexed: 01/10/2023] Open
|