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IFCC Curriculum

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The IFCC eAcademy is an open educational resource containing distance learning material created and/or reviewed by IFCC experts for the continuous professional development of members of IFCC member organisations. This new educational tool is being developed by the C-DL and C-Iel. Suggestions for topics, content or contributions may be made using the ‘Contact Us’ form on the IFCC website.

The IFCC Curriculum was developed by the C-DL to be a guide for IFCC member societies in their development of syllabuses for postgraduate trainees in laboratory medicine. It is also intended to provide a resource for trainees in planning their private study in preparation for academic and professional qualifications.  Phase one of the curriculum addresses concepts related to Laboratory Organisation and Management and Blood Sciences (Clinical Chemistry and Immunology). Future additions to the curriculum will expand the scope to other disciplines within laboratory medicine.

It aims to give some learning objectives in all fields of lab medicine (analytical and clinical).

Click here to download the IFCC Curriculum.

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IFCC’s Forthcoming Congresses – February Issue

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Calendar of IFCC Congresses/Conferences and Regional Federation’s Congresses

Apr 18 – 21, 2018 15th Arab Conference of Clinical Biology and Laboratory Medicine   Ramallah, PS
Jul 02 – 04, 2018 1st  IFCC, EFLM, AFCB Conference “Laboratory Medicine: Meeting the needs of Mediterranean Nations”  Rome, IT
Apr 10 – 13, 2019 COLABLIOCLI Regional Congress 2019 Panama, PA
May 19 – 23, 2019 XXIII IFCC-EFLM EUROMEDLAB – BARCELONA 2019 Barcelona, ES
Nov 17 – 20, 2019 APFCB Regional Congress 2019 Jaipur, IN
May 24 – 28, 2020 XXIV IFCC WORLDLAB – SEOUL 2020 Seoul, KR

 
May 16 – 20, 2021 XXIV IFCC-EFLM EUROMEDLAB – MUNICH 2021 Munich, DE
 May 21 – 25, 2023 XXV IFCC-EFLM WORLDLAB-EUROMEDLAB  ROME 2023

Calendar of events with IFCC auspices

Feb 28 – 02, 2018 3rd Turkish in vitro Diagnostic (IVD) Symposium Izmir, TR
Mar 05 – 09, 2018 3rd Winter School of Cell Analysis in Immunology Saint Etienne, FR
Mar 15 – 17, 2018 College of Chemical Pathologists of Sri Lanka Annual Academic Sessions 2018 Colombo, LK
Mar 16 – 17, 2018 23rd Annual Conference and Continuing Professional Development (ACCPD) Bahir Dar, ET
Apr 18 – 21, 2018 The 10th International Palestinan Conference of Laboratory Medicine and the 15th Arab Conference of Clinical Biology Ramallah, PS
May 09 – 12, 2018 9th Congress of the Croatian Society of Medical Biochemistry & Laboratory Medicine Zagreb, HR
May 09 – 12, 2018 2nd Congress of Romanian Association of Laboratory Medicine Bucharest, RO
May 23 – 25, 2018 21st Serbian Congress of Medical Biochemistry and Laboratory Medicine with international participations Belgrade, SRB
May 23 – 25, 2018 14th EFLM Symposium for Balkan Region Belgrade, SRB
May 24 – 25, 2018 XVI Meeting of the SEQCML Scientific Committee Madrid, ES
Jun 03 – 06, 2018 CSCC 2018 Annual Conference Ottawa, CA
Jun 06 – 08, 2018 Focus 2018 – Annual Meeting of ACB Manchester, UK
Jun 12 – 15, 2018 XXXVI Nordic Congress of Clinical Chemistry Helsinki, FI
Jun 18 – 19, 2018 2nd EFLM Strategic Conference “The end of Laboratory Medicine as we know it? Handling disruption of Laboratory Medicine in digital health” Mannheim, DE
Jun 21 – 22, 2018 7th International Symposium on Critical Care Testing and Blood Gases Antibes, FR
Jun 30 – July 03, 2018 International Society for Enzymology Conference Naxos, GR
Jul 13 – 14, 2018 Turning Science Into Caring (TSIC) Shanghai, CN
Sep 26 – 29, 2018 15th Annual Meeting of the German Society for Clinical Chemistry and Laboratory Medicine – The foundation for diagnosis and therapy Mannheim, DE
Sep 30 – Oct 03, 2018 Santorini Conference “Systems medicine and personalised health & therapy” – “The odyssey from hope to practice”. Santorini, GR
Oct 03 – 05, 2018 26th BCLF Meeting and 6th National Congress of MSMBLM Skopje, MK
Oct 30, 2018 International Conference on Laboratory Medicine “LABORATORY MEDICINE: 25 YEARS ON” Padova, IT

When Rapid Blood Culture Identification Results Don’t Correlate: Clinical Correlation Needed

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Because the PCR only provides the organism identification (sometimes only to the family-level, i.e.; Enterobacteriaceae), laboratories must continue to culture the positive blood for definitive identification and/or antimicrobial susceptibility results. So what do you do when the results don’t correlate?

The Issue

From time to time, the PCR result is not going to correlate with the direct Gram stain or with the culture results. Although this is an issue one would fully anticipate, what do you do when this happens? Do you take some sort of action to arbitrate? Do you report the results as is?

First of all, the PCR assays do not detect all organisms. They only detect the most common bloodstream pathogens. Therefore, one should fully expect to observe cases in which the Gram stain would be positive, but the PCR results would be negative (scenario 1).  This is not a surprise.

Additionally, one should also assume that the PCR will occasionally detect organisms that were present at the lower limit of detection of the Gram stain. An example of this would be that the Gram stain is positive for one morphology (i.e.; Gram-positive cocci), but the PCR is positive for two organisms (i.e.; Staphylococcus and a Proteus species). Most of these cases tend to correlate with culture. In other words, although the second organism was not originally observed in the Gram stain, it was detected via PCR and then it also subsequently grew in culture (scenario 2).

Another type of discordant result laboratories sometimes experience is when the organism detected via PCR does not grow in culture for whatever reason. Similar to scenario 2 stated above, except that the culture is also negative for the second organism (scenario 3). Perhaps the patient was treated with antibiotics and the organism is no longer viable for culture? Perhaps a sampling or processing error was to blame?

The Solution

Depending on the scenario and how much work you want to do, you can either repeat testing or try an alternative method. Take scenario 2 for example. If the PCR detects two organisms and the Gram stain is only positive for one, then review of the original Gram stain is warranted. It is possible that the Gram-negative was somehow missed. Our eyes tend to go to the darker, more obvious structures. Perhaps the Gram-negative organism was faintly stained and it was overlooked? It is also possible that the Gram-positive is present in much lower numbers and only Gram-negative organism was originally observed. If the Gram stain result remains the same after review (only one organism observed), then there is nothing much left to do except to wait for the culture. That being said, an alternative method, such as acridine orange can be utilized in this type of scenario (two different cell morphologies). Acridine orange is a fluorescent stain that improves organism detection, as it is more sensitive than the Gram stain (1, 2).

If only the Proteus is growing (and the Staphylococcus isn’t from scenario 2) and we normally subculture positive blood to blood, chocolate, and MacConkey agars, then perhaps including an additional media that inhibits Gram-negative growth would be beneficial.

Scenario 3 can be a little more difficult to solve because you can’t make a non-viable organism grow. It just is what it is. [Spoiler alert: in next month’s blog I plan to write about when you should change your thinking from true-positive to false-positive.]

Regardless of why the result is discrepant, our laboratory appends a comment to the discordant result which says, “Clinical correlation needed.” This lets the clinician know that the results are abnormal and that they must use other relevant information to make a definitive diagnosis. In addition to the comment, we also make sure the discrepancy is notified to laboratory technical leadership (i.e.; Doctoral Director, Technical Lead/Specialist). This allows us to keep track of discrepancies as they may become important to know about in the future (see next month’s blog).

The Conclusion

In terms of organism detection, nucleic assays (i.e., NAATs) can provide superior sensitivity over antigen and culture-based methods of organism detection (i.e., sensitivity = PCR > culture > Gram). From the laboratory perspective, other potential benefits of utilizing nucleic acid detection methodologies include decreased TAT, simplified workflows, and reduced hands-on time. In terms of patient care, many have noted improved outcomes due to increased sensitivity and decreased time to result.

Although advances in technology can significantly improve analytical performance, they can also add complexity to the post-analytical process. Making sense of the results can sometimes lead to confusion. It is important to know the product’s limitations and what your risk(s) is. This should already be known and included in your Individualized Quality Control Plan (IQCP). Lastly, guiding the clinician to proper result interpretation is also important to maintain valuable patient care.

References

  1. Mirrett, S., Lauer, B.A., Miller, G.A., Reller, L.B. 1981. Comparison of Acridine Orange, Methylene Blue, and Gram Stains for Blood Cultures. J. Clin. Microbiol. 15(4): 562-566.
  2. Lauer, B.A., Reller, L.B., and Mirrett, S. 1981. Comparison of Acridine Orange and Gram Stains for Detection of Microorganisms in Cerebrospinal Fluid and Other Clinical Specimens. J. Clin. Microbiol. 14(2): 201-205.

 

Author: Raquel Martinez, PhD, D(ABMM), was named an ASCP 40 Under Forty TOP FIVE honoree for 2017. She is one of two System Directors of Clinical and Molecular Microbiology at Geisinger Health System in Danville, Pennsylvania. Her research interests focus on infectious disease diagnostics, specifically rapid molecular technologies for the detection of bloodstream and respiratory virus infections, and antimicrobial resistance, with the overall goal to improve patient outcomes.

Source: LaBlogatory

MALDI TOF MS Quality Control in Clinical Microbiology

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Since a large number of clinical microbiology laboratories have adopted MALDI-TOFMS as a primary method, labs absolutely need to implement sufficient quality control (QC) practices to en-sure they report accurate identifications.

Until recently, few guidelines for using MALDI-TOF MS in clinical microbiology existed. However, in April 2017, the Clinical and Laboratory Standards Institute (CLSI) published a document, Methods for the Identification of Cultured Microorganisms Using Matrix-Assisted Laser Desorption/Ionization Time-of-Flight (M58-Ed1), which lays out comprehensive recommendations for MALDI-TOF MS in clinical microbiology laboratories. This CLSI document, as well as recommendations from instrument manufacturers and regulatory agencies, all agree on the need for robust internal and external QC designed to account for the unique performance and limitations of MALDI-TOF MS.

Internal QC

Laboratories must perform internal QC before using MALDI-TOF MS to identify microorganisms. Internal QC consists of an automatic instrument calibration using a manufacturer-specified calibration standard. Depending on the system, calibrators include a manufactured extract of Escherichia coli (E. coli) or a specific E. coli calibration strain. Laboratories should ensure that they follow manufacturers’ specifications for preparing, using, and storing calibrators.

During calibration, the calibrator generates and automatically analyzes a mass spectrum to check the spectrum baseline and ensure the expected calibration peaks are present. Laboratories use these parameters to confirm their instrument settings are appropriate and their instruments will automatically adjust if necessary. The calibrator spectrum is also run against the reference database to ensure the correct identification is given with a level of confidence that meets the manufacturer’s specifications. Laboratories must perform calibration before every run.

In addition to ensuring a successful calibration, the College of American Pathologists (CAP) Microbiology Checklist requires that labs run a calibrator control each day of patient testing, when a new target is used, or more often if the manufacturer recommends it. The CAP checklist also requires that labs maintain a written procedure for operating and calibrating the instrument as well as all calibration records.

It is important for labs to not only document calibration results but also promptly investigate calibration failures. Spectral acquisition cannot occur until calibration is successful. Calibration failures of-ten result from user error, typically due to improper application of the calibrator. Labs can assess potential user error by reapplying and reanalyzing their calibrator. Calibration failure also occurs when the calibrator has been prepared improperly or when problems crop up with the matrix, reagents, target, or instrument.

External QC

Laboratories should perform external QC using appropriate positive and negative controls. While most manufacturers do not, CAP requires that positive controls (either an appropriate control microorganism or calibrator) be tested each day of patient testing.

For positive controls, labs should test well-characterized strains using the same methodology they use for patient isolates. For example, yeast typically require extraction prior to analysis, so labs should process yeast QC organisms using the same extraction methodology. Most laboratories should, at a minimum, test a bacterial QC organism on each day of testing. If laboratories are using MALDI-TOF MS to identify yeast, mycobacteria, Nocardia, or molds, appropriate QC organisms for each organism type should be run each day they test for these microorganisms. Labs must obtain correct, high-confidence identifications for all QC organisms. If a lab fails to identify a QC organism, it must investigate and suspend patient testing until the problem is resolved. If a Food and Drug Administration (FDA)-cleared platform is used, manufacturers may recommend specific American Type Culture Collection strains for use as positive controls.

CAP also requires that labs use manufacturer-recommended control microorganisms for FDA-approved platforms. While there are no specific QC organism recommendations for laboratories operating research-use-only platforms, laboratory directors should ensure appropriate control organisms are tested each day. Results of QC testing should be documented and periodically reviewed to assess not only instrument performance but also testing consistency among users.

Labs should also include a negative control with each run. Typically the negative control consists of reagents spotted directly on the target plate or slide. Matrix should be applied to a random blank spot on each target plate or slide to ensure there is no reagent contamination and, for systems that use a reusable target plate, to ensure that the target plate has been adequately cleaned between runs.

CAP requires labs that operate platforms with reusable targets test a blank negative control to ensure adequate cleaning of the target. If an extraction is performed, the reagents used for the extraction can be spotted and overlaid with matrix to ensure no false positive results are produced due to reagent contamination. Because of the implications of reporting organism identifications directly from blood cultures, labs should test lysis buffers and other reagents used for sample preparation to ensure they are free of contamination. Currently, there are no regulatory requirements for testing reagents on a routine basis.

Ensuring Spectral Quality

Once controls are satisfactory, testing of patient samples can begin. To ensure they produce high quality spectra, labs must follow recommendations for optimal culture conditions and sample preparation, as well as manufacturers’ recommendations for approved media types. If necessary, labs should validate additional media types. They should also use fresh isolates whenever possible. Spectral quality depends on placing an optimal quantity of microorganism on the target plate, and special spotting techniques and extractions might be necessary to identify certain microorganisms. Labs should consider analyzing all isolates in duplicate and have procedures in place to help resolve discordant results between spots.

In addition, since MALDI-TOF MS cannot identify all organisms in polymicrobial cultures, labs should ensure cultures are pure. Ensuring purity is particularly important when microorganisms are identified directly from liquid cultures: Labs should report results as preliminary until purity can be confirmed. A robust training and competency assessment program is also essential to ensure testing staff are competent in performing identifications of commonly encountered microorganisms and in using and maintaining the instrument.

Reporting Identifications

Another important QC consideration involves interpreting and reporting of MALDI-TOF MS identifications. Spectral databases differ in composition depending on the manufacturer and whether they are FDA cleared. Users also can develop custom databases. While manufacturers validate identifications from their FDA cleared platforms and these identifications have been cleared for reporting, laboratories still must determine how to report them. Laboratories face choices such as reporting the identification to the genus, species, or complex level.

Reporting also may differ based on specimen source. For example, reporting species-level identifications for coagulase-negative staphylococci from certain sites may lead to clinicians attributing a higher degree of significance to a culture result.

A major challenge for laboratories operating research-use-only platforms is how to report unfamiliar or uncommon identifications. If these laboratories do not independently validate them, such identifications should always be confirmed with supplemental testing. However, validating targets for rarely encountered organisms is especially difficult due to problems obtaining an adequate number of isolates for validation studies.

In addition, labs should familiarize themselves with the identification limitations of their MALDI-TOF MS platform by reviewing technical bulletins, comments provided by the software, and from periodic review of the scientific literature. Unless they extensively validate lower confidence thresholds, labs should adhere to the manufacturer-recommended thresholds for genus and species-level identifications.

In light of the numerous considerations for interpreting and reporting of MALDI-TOF MS identifications, labs should develop reporting guidelines for their bench technologists to ensure accuracy and consistency in reporting. Labs also should participate in external proficiency testing programs to en-sure they are proficient in correctly generating and reporting identifications.

MALDI-TOF Limitations

While MALDI-TOF MS is a robust system for microorganism identification, it is not infallible. For ex-ample, MALDI-TOF MS cannot discriminate reliably between closely related microorganisms, and incorrect identifications can occur from user error (such as spotting the organism on the wrong spot on the target plate), analysis of mixed cultures, and other reasons.

Due to these limitations, labs should consider MALDI-TOF MS results as one component of the overall testing system for identifying microorganisms. Results should always be reviewed by a trained microbiologist and correlated with other characteristics, including growth requirements, colo-ny morphology, and Gram-stain. In addition, labs should ensure they maintain their instruments and update their database in keeping with manufacturers’ recommendations and retain all maintenance records in the laboratory.

As more laboratories abandon traditional methods in favor of MALDI-TOF MS, those that make the switch absolutely need to follow best practices for quality control by adhering to recommendations given by instrument manufacturers, regulatory agencies, and other guidelines.

Author: Lori Bourassa, PhD, MPH, D(ABMM), is an assistant professor and assistant director of the clinical microbiology division at the University of Washington Medical Center in Seattle. 

Source: aacc.org

[podcast] Pradeep Dabla: IFCC-TFYS and activities- let’s join hands together

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Dr. Pradeep Dabla, chair of the IFCC TFYS, presents the Complimentary Educational Webinar: Integration and Impact of High-Sensitive Troponin Testing in Improving Patient Care.

The IFCC Task Force Young Scientists would like to invite you to our fourth educational webinar for scientists and laboratorians, brought to you by the IFCC Task Force Young Scientists. This educational program focuses on highly sensitive Troponin assays that have been recently introduced in Europe. By attending this session, attendees will gain a better understanding of these new Troponin assays, how the test is being integrated, and the impact on clinical practice and the laboratory.

Thursday, February 15th, 2018 – 9:00 a.m. EST

 

For more information login to and join us for webinar: ifcc.org/task-force-young-scientists

Faecal Haemoglobin: Newer Approaches to Screening and Diagnosis of Colorectal

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Presenter: Callum G. Fraser (UK)

Moderator: Sally C Benton (UK)

Colorectal cancer (CRC) is still a very important health problem world-wide. It is the third most common cancer and the fourth leading cause of cancer-related death. Fortunately, many screening programmes have now been set up to detect neoplasia in those who do not have any symptoms of bowel disease. These programmes lead to early detection of CRC and its precursors, which significantly improves outcomes. There are a number of strategies available for CRC screening, but faecal immunochemical test for haemoglobin (FIT) are now considered as the best currently available non-invasive investigation and are being widely established. FIT are available in two formats, qualitative FIT and quantitative FIT: the former are often used in opportunistic screening, whereas the latter are widely used in population-based programmatic screening. Quantitative FIT have many advantages, a major benefit being that the faecal haemoglobin concentration (f-Hb) can be estimated. This Webinar will explore the diagnostic accuracy of FIT, and factors affecting f-Hb, including age, sex and socioeconomic status. The difficulties in selecting the f-Hb cut-off to be used for CRC screening will be addressed in detail.

However, most CRC are not detected through screening programmes, but when a person with symptoms seeks medical advice. The symptoms of serious bowel disease are very common presentations in primary care, but most with these do not have serious disease. Generally, people with symptoms are referred to secondary care for colonoscopy, which is inadequate in many countries. A particular problem is that the number of patients with symptoms being referred from primary care is increasing rapidly, in large part due to local, regional and national campaigns raising awareness of the need to get symptoms investigated. This Webinar will discuss the significant evidence now available that demonstrates that triage using FIT at a low cut-off around 10 μg Hb/g faeces has the potential to correctly rule-out CRC and avoid colonoscopy in many symptomatic patients. Importantly, secondary care referral following a positive FIT allows the identification of other significant bowel pathology in patients who are found not to have CRC, mainly inflammatory bowel disease. This very recent use of FIT is an excellent example of translation of research into clinical practice: the first study investigating the use of quantitative FIT using numerical data on f-Hb was published on-line in 2012 and, only five years later, a national guideline has recommended its use in routine clinical practice.

IFCC’s Forthcoming Congresses – January Issue

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Calendar of IFCC Congresses/Conferences and Regional Federation’s Congresses

 Jul 02 – 04, 2018   1st  IFCC, EFLM, AFCB Conference “Laboratory Medicine: Meeting the needs of Mediterranean Nations”  Rome, IT
May 19 – 23, 2019 XXIII IFCC-EFLM EUROMEDLAB – BARCELONA 2019 Barcelona, ES
May 24 – 28, 2020   XXIV IFCC WORLDLAB – SEOUL 2020 Seoul, KR

 
 May 16 – 20, 2021    XXIV IFCC-EFLM EUROMEDLAB – MUNICH 2021 Munich, DE
 May 21 – 25, 2023    XXV IFCC-EFLM WORLDLAB-EUROMEDLAB  ROME 2023 Rome, IT

Calendar of events with IFCC auspices

Feb 08 – 09, 2018 International Congress on Quality in Laboratory Medicine Helsinki, FI
Feb 28 -Mar 02, 2018 3rd Turkish in vitro Diagnostic (IVD) Symposium Izmir, TR
Mar 05 – 09, 2018 3rd Winter School of Cell Analysis in Immunology Saint-Etienne, FR
Mar 15 – 17, 2018 College of Chemical Pathologists of Sri Lanka Annual Academic Sessions 2018 Colombo, LK
Apr 18 – 21, 2018 The 10th International Palestinan Conference of Laboratory Medicine and the 15th Arab Conference of Clinical Biology Ramallah, PS
May 09 – 12, 2018 2nd Congress of Romanian Association of Laboratory Medicine Bucarest, RO
May 23 – 25, 2018 14th EFLM Symposium for Balkan Region Belgrade, SRB
May 23 – 25, 2018 21st Serbian Congress of Medical Biochemistry and Laboratory Medicine with international participations Belgrade, SRB
Jun 06 – 08, 2018 Focus 2018 – Annual Meeting of ACB Manchester, UK
Jun 12 – 15, 2018 XXXVI Nordic Congress of Clinical Chemistry Helsinki, FI
Jun 18 – 19, 2018 2nd EFLM Strategic Conference “The end of Laboratory Medicine as we know it? Handling disruption of Laboratory Medicine in digital health” Mannheim, DE
Jun 21 – 22, 2018 7th International Symposium on Critical Care Testing and Blood Gases Antibes, FR
Jun 30 – Jul 03, 2018 International Society for Enzymology Conference Naxos, GR
Jul 13 – 14, 2018 Turning Science Into Caring (TSIC) Shanghai, CN
Sep 26 – 29, 2018 15th Annual Meeting of the German Society for Clinical Chemistry and Laboratory Medicine – The foundation for diagnosis and therapy Mannheim, DE
Sep 30 – Oct 03 , 2018 Santorini Conference “Systems medicine and personalised health & therapy” – “The odyssey from hope to practice”. Thira Santoirini, GR
Oct 03 – 05, 2018 26th BCLF Meeting and 6th National Congress of MSMBLM Skopje, MK

Complete Genes May Pass from Food to Human Blood

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Our bloodstream is considered to be an environment well separated from the outside world and the digestive tract. According to the standard paradigm large macromolecules consumed with food cannot pass directly to the circulatory system. During digestion proteins and DNA are thought to be degraded into small constituents, amino acids and nucleic acids, respectively, and then absorbed by a complex active process and distributed to various parts of the body through the circulation system. Here, based on the analysis of over 1000 human samples from four independent studies, we report evidence that meal-derived DNA fragments which are large enough to carry complete genes can avoid degradation and through an unknown mechanism enter the human circulation system. In one of the blood samples the relative concentration of plant DNA is higher than the human DNA. The plant DNA concentration shows a surprisingly precise log-normal distribution in the plasma samples while non-plasma (cord blood) control sample was found to be free of plant DNA.

Authors: Sándor Spisák1,2*, Norbert Solymosi3,4, Péter Ittzés3, András Bodor3, Dániel Kondor3, Gábor Vattay3, Barbara K. Barták5, Ferenc Sipos5, Orsolya Galamb5, Zsolt Tulassay1,5, Zoltán Szállási2, Simon Rasmussen6, Thomas Sicheritz-Ponten6, Søren Brunak6, Béla Molnár1,5, István Csabai3,7

  1. Molecular Medicine Research Group, Hungarian Academy of Sciences, Budapest, Hungary,
  2. Children’s Hospital, Harvard Medical School, Boston, Massachusetts, United
    States of America,
  3. Department of Physics of Complex Systems, Eo¨tvo¨s University, Budapest, Hungary,
  4. Department of Animal Hygiene, Herd Health and Veterinary Ethology, Szent Istva´n University, Budapest, Hungary,
  5. 2nd Department of Internal Medicine, Semmelweis University, Budapest, Hungary,
  6. Center for Biological Sequence Analysis, Technical University of Denmark, Lyngby, Denmark,
  7. Department of Physics and Astronomy, The Johns Hopkins University, Baltimore, Maryland, United States of America

Citation: Complete Genes May Pass from Food to Human Blood. PLoS ONE 8(7): e69805. https://doi.org/10.1371/journal.pone.0069805

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Frequency and Mechanisms of Spontaneous Fosfomycin Nonsusceptibility Observed upon Disk Diffusion Testing of Escherichia coli

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Fosfomycin maintains activity against most Escherichia coli clinical isolates, but the growth of E. coli colonies within the zone of inhibition around the fosfomycin disk is occasionally observed upon susceptibility testing. We aimed to estimate the frequency of such nonsusceptible inner colony mutants and identify the underlying resistance mechanisms. Disk diffusion testing of fosfomycin was performed on 649 multidrug-resistant E. coli clinical isolates collected between 2011 and 2015. For those producing inner colonies inside the susceptible range, the parental strains and their representative inner colony mutants were subjected to MIC testing, whole-genome sequencing, reverse transcription-quantitative PCR (qRT-PCR), and carbohydrate utilization studies. Of the 649 E. coli clinical isolates, 5 (0.8%) consistently produced nonsusceptible inner colonies. Whole-genome sequencing revealed the deletion of uhpT encoding hexose-6-phosphate antiporter in 4 of the E. coli inner colony mutants, while the remaining mutant contained a nonsense mutation in uhpA.

The expression of uhpT was absent in the mutant strains with uhpT deletion and was not inducible in the strain with the uhpA mutation, unlike in its parental strain. All 5 inner colony mutants had reduced growth on minimal medium supplemented with glucose-6-phosphate. In conclusion, fosfomycin-nonsusceptible inner colony mutants can occur due to the loss of function or induction of UhpT but are rare among multidrugresistant E. coli clinical strains. Considering that these mutants carry high biological costs, we suggest that fosfomycin susceptibility of strains that generate inner colony mutants can be interpreted on the basis of the zone of inhibition without accounting for the inner colonies.

Authors: Aaron E. Lucas,a Ryota Ito,a,b Mustapha M. Mustapha,a Christi L. McElheny,a Roberta T. Mettus,a Sarah L. Bowler,a Serena F. Kantz,a Marissa P. Pacey,a A. William Pasculle,c Vaughn S. Cooper,d,e Yohei Doia,b,e

  1. Division of Infectious Diseases, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA
  2. Department of Microbiology, Fujita Health University, Toyoake, Aichi, Japan
  3. Clinical Microbiology Laboratory, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania, USA
  4. Department of Molecular Microbiology and Genetics, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA
  5. Center for Innovative Antimicrobial Therapy, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA

Download, and learn the genetic basis for this decision in the report here: J.-Clin.-Microbiol.-2018-Lucas

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