gms | German Medical Science

GMS Zeitschrift zur Förderung der Qualitätssicherung in medizinischen Laboratorien

Gesellschaft zur Förderung der Qualitätssicherung in medizinischen Laboratorien e. V. (INSTAND e. V.)

ISSN 1869-4241

Bacterial and fungal genome detection PCR/NAT: discussion of the November 2015 distribution for external quality assessment of nucleic acid-based protocols in diagnostic medical microbiology by INSTAND e.V.

Report

  • corresponding author Udo Reischl - Institute for Clinical Microbiology and Hygiene, University Hospital Regensburg, Germany
  • Wulf Schneider - Institute for Clinical Microbiology and Hygiene, University Hospital Regensburg, Germany
  • Martin Ehrenschwender - Institute for Clinical Microbiology and Hygiene, University Hospital Regensburg, Germany
  • Andreas Hiergeist - Institute for Clinical Microbiology and Hygiene, University Hospital Regensburg, Germany
  • Matthias Maaß - Labor Dr. Heidrich und Kollegen MVZ GmbH, Hamburg, Germany
  • Eberhard Straube - Institute of Medical Microbiology, University Hospital of the Friedrich Schiller University of Jena, Germany
  • Dimitrios Frangoulidis - Bundeswehr Institute of Microbiology, Munich, Germany
  • Gregor Grass - Bundeswehr Institute of Microbiology, Munich, Germany
  • Heiner von Buttlar - Bundeswehr Institute of Microbiology, Munich, Germany
  • Wolf Splettstößer - Bundeswehr Institute of Microbiology, Munich, Germany
  • Volker Fingerle - Bavarian State Office for Health and Food Safety, Oberschleissheim, Germany
  • Andreas Sing - Bavarian State Office for Health and Food Safety, Oberschleissheim, Germany
  • Enno Jacobs - Institute for Medical Microbiology and Hygiene, Technical University of Dresden, Germany
  • Ingrid Reiter-Owona - Institute for Medical Microbiology, Immunology and Parasitology (IMMIP), University of Bonn, Germany
  • Martin Kaase - National Reference Laboratory for multidrug-resistant gram-negative bacteria, Department for Medical Microbiology, Ruhr-University Bochum, Germany

GMS Z Forder Qualitatssich Med Lab 2015;6:Doc05

doi: 10.3205/lab000020, urn:nbn:de:0183-lab0000201

This is the English version of the article.
The German version can be found at: http://www.egms.de/de/journals/lab/2015-6/lab000020.shtml

Published: December 22, 2015

© 2015 Reischl et al.
This is an Open Access article distributed under the terms of the Creative Commons Attribution 4.0 License. See license information at http://creativecommons.org/licenses/by/4.0/.


Abstract

This contribution provides a report on the recent proficiency testing scheme “Bacterial and Fungal Genome Detection (PCR/NAT)”. It summarizes some benchmarks and the overall assessment of results reported by all of the participating laboratories.

This highly requested scheme for external quality assessment (EQAS) of molecular diagnostic methods in the field of medical microbiology was launched in 2002 by the German Society of Hygiene and Microbiology (DGHM) and is now organized by INSTAND e.V., Düsseldorf, Germany. This segment of the INSTAND e.V. proficiency testing program is open for diagnostic laboratories worldwide. The concept of this EQAS scheme, which is in accordance to the ”Directive of the German Medical Association for quality assurance of medical laboratory examinations“ (RiLiBÄK), part B3, is based on two validation rounds per year (spring and autumn) and a permanently expanding coverage of relevant bacterial or fungal pathogens.

Briefly, next to “simply negative” samples the corresponding sets of quality control (QC) specimens may contain some strong-positive samples, samples spiked with clinical variants or species closely related to the target organisms. Further information as well as the statistically documented and discussed results of the past rounds of this proficiency testing scheme “Bacterial and Fungal Genome Detection (PCR/NAT)” can be found at the homepage of INSTAND e.V. (http://www.instandev.de). Although the preferred language of these documents is German, we are aiming to provide at least a brief discussion of the results and some key issues in English and keep the tables in a bilingual version.


Brief discussion of the current results

For the growing number of international participants we provide a brief discussion of the current results in an English version.


Examination results November 2015

RV 530: Neisseria gonorrhoeae & Chlamydia trachomatis (GO & CT)

Despite the relatively low amounts of C. trachomatis and N. gonorrhoeae target organisms in the current set of QC samples, the availability of well-established commercial or in-house NAT-assays has led to a high portion of correct results.

The current set of QC samples contained two samples with different amounts of C. trachomatis (~1x104 IFU/mL in sample # 1525301 and ~1x105 IFU/mL in sample # 1525304) and two samples with different amounts of N. gonorrhoeae target organisms (~105 CFU/mL in sample # 1525302 and ~104 CFU/mL in sample # 1525301).

Despite relatively low amounts of C. trachomatis target cells in the positive samples # 1525301 and # 1525304, only 1 false-negative result was observed among the Chlamydia trachomatis-specific results, reported by the 202 participants. Among the N. gonorrhoeae-specific results, false-negative results were reported by only 2 of the 202 participants for samples # 1525301 and # 1525302, which contained N. gonorrhoeae target organisms in an amount of 1x105 CFU/mL and 1x104 CFU/mL respectively. Also 7 false-positive results for the two GO-negative samples were reported by participants.

Since the amount of target organisms in CT-positive samples # 1525301, # 1525304, and NG-positive samples # 1525301 and # 1525302 could not be considered as “extremely low”, false negative results should encourage the participants to review and optimize their CT- and GO-specific NAT-based assays. Inhibition controls were included by all of the 202 participants and no inhibitory events were reported. Overall, a very good diagnostic performance and no noticeable issues regarding sensitivity and specificity were observed for the C. trachomatis- and N. gonorrhoeae-specific NAT assays used by the 202 participants

Tables 4 to 7 (Attachment 1 [Attach. 1], p. 2-3) were include this time to enable a detailed evaluation of the C. trachomatis- and GO-specific NAT components of combined GO/CT test systems. In Tables 4 and 5 (Attachment 1 [Attach. 1], p. 2) only the C. trachomatis (CT)-specific results and in the Tables 6 and 7 (Attachment 1 [Attach. 1], p. 3) only the Neisseria gonorrhoeae (GO)-specific results are presented and evaluated statistically.

RV 531: Chlamydia trachomatis

The current set of QC samples contained two positive samples: # 1525312 with ~1x104 IFU/mL of C. trachomatis target organisms and sample # 1525314 with ~1x105 IFU/mL of C. trachomatis target organisms. Samples # 1525311 and # 1525313 contained no target organisms but only human cells and E. coli cells.

As depicted in Tab. 2 (Attachment 1 [Attach. 1], p. 4), the reported results were generally correct for the two positive samples.

For the C. trachomatis-negative samples # 1525311 and # 1525313 containing only non-infectious human cells and E. coli, 4 false-positive results and 2 false-negative results were observed among the 99 participants.

Assuming a sequential processing of the 4 individual samples of the current set, a contamination event of the “negative” sample 1 by target organism or PCR products of the positive samples “2” or “4” is unlikely in the current sample constellation. So false positive results should encourage the affected participants to review and optimize their DNA extraction procedure and their CT-specific NAT-based test system.

This striking match of the current results with observations and accuracy rates in the last years can be considered as an evidence for a high reliability and consistency of the applied assays and overall sample processing.

Run controls were performed by all of the 99 participants and inhibition events were not observed this time. In this context, it should be noted, that we have not added putative inhibitory substances into the samples of the current distribution.

Overall, a very good diagnostic performance and no noticeable issues regarding sensitivity and specificity were observed for the C. trachomatis-specific NAT assays used by the 99 participants.

RV 532: Bordetella pertussis

The current set of QC samples contained one sample with a relatively high amount of Bordetella pertussis (# 1525322; 1x104 CFU/mL), and three samples negative for the respective target organism: one negative sample containing Bordetella parapertussis (# 1525324; 1x105 CFU/mL), one negative sample containing Bordetella bronchiseptica (# 1525321 with ~1x105 CFU/mL), as well as one sample containing only human cells and Escherichia coli (# 1525323).

The availability of well-established commercial or in-house NAT-assays has led to a high portion of correct results. Nearly all of the 146 participants reported correct positive results for the sample # 1525322 (B. pertussis, 1x104 CFU/mL). Two of the participating laboratories observed false-negative results for B. pertussis DNA in sample # 1525322. The amount of 104 CFU/mL of B. pertussis target organisms is significantly above the previously observed lower limit of detection for the corresponding PCR/NAT assays or test systems. False-negative or questionable results should therefore lead to re-evaluations of the assay sensitivity.

Samples # 1525321 and # 1525324 which contained ~1x105 CFU/mL of Bordetella bronchiseptica and Bordetella parapertussis, respectively, were correctly tested negative by 142 of the 146 participants but 6 of the participating laboratories observed false-positive results for B. pertussis DNA. Sample # 1525323 contained only E. coli. All but two participants correctly reported this sample as negative for Bordetella pertussis. The false-positivity issue is probably due to contamination events in the course of sample preparation or low analytical specifity of the used PCR/NAT test systems. For sample # 1525311, one result was classified as “questionable” by one participant. For questionable results, certificates are only issued when correct results are reported by the participant for the remaining 3 samples of RV 532.

For the detection of B. pertussis, most participants used self-developed (in-house) test systems with inhibition and/or positive controls. Therefore, 57 participating laboratories used IS481 insertion sequence, 8 the pertussis toxin coding gene and 2 ribosomal genes. Run controls were performed by 144 of 146 participants and no inhibition events were observed with the samples of the current distribution.

RV 533: Helicobacter pylori

The current set of QC samples contained three samples with a Clarithromycin-resistant Helicobacter pylori isolated from a patient in the course of an antibiotic therapy failure study in a kind of dilution series. Sample # 1525333 contained approximately 1x106 CFU/mL, sample # 1525334 approximately 1x105 CFU/mL and sample # 1525331 approximately 1x104 CFU/mL of the respective target organisms.

The availability of well evaluated NAT-based assays and the relatively high amount of target organisms in the three Helicobacter pylori-positive samples (#1525331: ~1x104 CFU/mL, #1525333: ~1x106 CFU/mL, and # 1525334: ~1x105 CFU/mL) led to positive predictive values of 100%. Also for the Helicobacter pylori-negative sample #1525332 correctly negative PCR/NAT-results were reported by all the participating laboratories.

As noted in the description of RV 533, clarithromycin resistance testing in the examined H. pylori isolates could be performed by participants on a voluntary basis. This molecular resistance testing is usually based on amplification and sequencing of characteristic regions within the H. pylori 23 S rDNA or the use of hybridization probes based qPCR assays. Results for clarithromycin resistance were reported by 34 of the 37 participants. With one exception, the results were correct.

RV 534: EHEC/STEC

As discussed previously, the challenge in NAT-based detection of EHEC/STEC is not the detection of small amounts of target organisms, but the sophisticated analysis and typing of different Shiga toxin genes and other putative pathogenic factors (such as the eae gene encoding intimin or the hlyA gene encoding enterohemolysin).

The current set of QC samples contained two samples positive for EHEC: # 1525343 (E. coli, 5x104 CFU/mL, clinical isolate, stx2c-positive, eae-positive, hlyA-positive and O157-positive) and # 1525342 (Escherichia coli, 1x105 CFU/mL, clinical isolate,stx2d-positive). The other two EHEC-negative samples contained an ETEC strain (sample # 1525341; 5x105 CFU/mL) and an eae- and hlyA-negative E. coli K12 strain (# 1525344).

All but 3 participants correctly reported negative results for sample # 1525344, containing only E. coli K12. The other “EHEC-negative” sample (#1525341), containing a significant amount of an LT- and ST-positive ETEC isolate was also reported PCR-negative by all but two participants. For the EHEC/STEC positive samples # 1525342 and 1525343, the availability of well-established NAT-based assays and strategies for molecular differentiation resulted in consistently high accuracy rates. Sample # 1525342 was correctly reported positive by 118 of the 124 participants and 117 of the 124 participants detected the target organisms in the EHEC/STEC-positive sample # 1525343 correctly.

As in most of the participating laboratories, a NAT-based detection of shiga toxin coding genes is used primarily as a culture confirmation test most future positive samples will contain relatively high amounts of target organisms. The focus will remain more on the analytical specificity of the used test systems and less on the lower detection limit obtained. Partial or complete shiga toxin subtyping, eae-, and hlyA-detection techniques were performed by 104 of the 124 participating laboratories. With one exception, the reported results were correct. None of the participants observed significant inhibition of the NAT-reaction.

RV 535: Borrelia burgdorferi

Due to numerous requests, here a short note for our participants outside Europe: as this proficiency testing panel is designed for a specific and sensitive detection of B. burgdorferi sensu lato DNA, the positive samples do not necessarily contain suspensions of “prototype” isolates of B. burgdorferi sensu stricto and in many of the bi-annual rounds of our external quality assessment scheme (EQAS) also other B. burgdorferi genotypes or genospecies will be present in individual samples.

The current set of QC samples contained three samples with B. burgdorferi sensu stricto organisms in our proprietary matrix: sample # 1525353 (1x104 genome equivalents/mL) and samples # 1525351 and # 1525354 (1x103 genome equivalents/mL). Sample # 1525352 contained about 1x105 genome equivalents/mL of Borrelia garinii OspA type 8 organisms.

With the exception of 15 and 17 false-negative results for samples # 1525351 and # 1525354 (containing a relatively low amount of 1x103 organisms/mL of Borrelia target organisms), one false-negative result for sample # 1525352 (1x105 organisms/mL) and 7 false-negative results for sample # 1525353 (1x104 organisms/mL) all participants reported correct results for the four positive samples. Regarding the low amount for samples # 1525351 and # 1525354 and the use of identical strains, the sample # 1525354 was set as an educational sample and not included in the evaluation. The false-negative results should prompt re-evaluation of the assay sensitivity.

Approximately half of the participating laboratories used self-developed (in-house) tests with inhibition and/or positive controls. None of the participants noted significant inhibition of the NAT-reaction. There were also no significant differences in test performance between commercially available kits and in-house assays for the diagnostic detection of Borrelia burgdorferi by PCR/NAT techniques.

RV 536: Legionella pneumophila

Due to numerous requests: this test is designed exclusively for the testing of NAT-based methods and protocols for direct detection of low amounts of Legionella pneumophila from appropriate clinical specimen (such as respiratory specimens for example). Individual samples may contain relatively small amounts of the corresponding target organism. For this reason, participation is promising only for diagnostic laboratories, which have established a highly sensitive and specific PCR-/NAT-based method for the detection of L. pneumophila DNA or who want to evaluate their method with the help of an external quality control. The current set of QC samples contained only one positive sample with Legionella pneumophila serogroup 2 (# 1525362; ~1x105 CFU/mL) next to two samples containing Legionella micdadei (# 1525363; ~1x106 CFU/mL and # 1525364; ~1x104 CFU/mL). Sample # 1525361 contained no target organisms but only human cells and E. coli cells.

The L. pneumophila-positive (~1x105 CFU/mL) sample # 1525362 was correctly tested positive by all of the 104 participating laboratories. Both of the L. micdadei-positive samples (# 1525363 with ~1x106 CFU/mL and # 1525364 with ~1x104 CFU/mL) were correctly tested negative by 94 and 98 participating laboratories, respectively. Sample # 1525361, which contained only E. coli, was classified as “questionable” by one participant and the remaining 103 participants reported correct negative PCR/NAT results for L. pneumophila DNA. All but one participant included inhibition controls in their test systems. Only one inhibition of the PCR-/NAT-reactions was reported for sample 1525363.

RV 537: Salmonella enterica

The current set of QC samples contained two samples with Salmonella enterica serovar Enteritidis (sample # 1525371 with 5x105 CFU/mL, and sample # 1525373 with 5x104 CFU/mL). Sample # 1525372 contained Salmonella enterica serovar Grumpensis (with 5x104 CFU/mL) and sample # 1525374 contained no target organisms but only human cells and E. coli cells.

All participants reported correct results for samples #1525371 and #1525374. Sample #1525372, containing ~5x104 CFU/mL Salmonella enterica serovar Grumpensis, as well as sample #1525373, containing ~5x104 CFU/mL Salmonella enterica serovar Enteritidis was correctly identified as “positive” by 19 of the 20 participants. Reporting a false-negative result for this sample should prompt a thorough re-evaluation of the performance of the test system. Inhibitory events in the PCR-/NAT-reaction were not detected by any of the participants.

RV 538: Listeria spp.

The current set of QC samples contained a sample without the corresponding target organisms (# 1525381; only E. coli cells), and three samples positive for L. monocytogenes (# 1525382, # 1525383 and # 1525384). The Listeria monocytogenes-containing samples (# 1525382 with 1x105 CFU/mL of L. monocytogenes and # 1525383 with 5x105 CFU/mL of L. monocytogenes) were correctly reported positive by all participants. In addition, the “negative” E. coli containing sample # 1525381 was also identified as negative by all laboratories. Most of the participants used very sensitive Listeria monocytogenes-specific assays, which is reflected by the high number of correctly positive results for sample # 1525384, containing only 1x103 CFU/mL of L. monocytogenes.

It should be noted that participants using L. monocytogenes-specific PCR/NAT-assays may indicate the corresponding results by the accessory code number 71. In this case, (false) negative results for non-Listeria monocytogenes species do not negatively affect issuing the corresponding QC certificates. In sum, the current results indicate a remarkably high analytical sensitivity of the current L. monocytogenes-specific PCR assays.

RV 539: MRSA

The concept of this proficiency testing series is designed to determine the analytical sensitivity and specificity of NAT-based assays for the direct detection of MRSA DNA in typical clinical sample material. With the development and composition of the corresponding sample materials we want to mimic the situation of processing clinical samples like wound or nasal swabs, so the lyophilized samples usually contain low amounts of target organisms in a background of human cells and other components. It is therefore important to note that NAT assays designed mainly for MRSA culture confirmation purposes may fail due to the low number of MRSA organisms in individual samples of the QC set.

Sample # 1525391 of the current set contained a relatively high number of a mecC-positive and methicillin-resistant S. aureus isolate (MRSA, PVL-negative, spa:t10009; ~1x105 CFU/mL) and sample # 1525393 contained a typical MRSA isolate (MRSA, PVL-negative; ~1x105 CFU/mL). Sample # 1525392 of the current set contained relatively high amounts of a mecA dropout MSSA isolate and only E. coli and human cells were present in sample # 1525394 of the current distribution.

The MRSA-negative sample # 1525394 was correctly reported negative by 307 of the 314 participants. Only one participant reported a false-positive result, presumably due to intra-laboratory contamination events from the highly positive sample # 1525393 during the sample preparation, amplification or detection. The MRSA-positive sample # 1525393 was correctly reported positive by 310 of the 314 participants. Three false-negative results were reported for MRSA sample # 1525393 and one participant classified his/her result as “questionable”.

Affected participants are encouraged to analyse and optimize their NAT-based assays, because the amount of MRSA target organisms in sample # 1525393 (1x105 CFU/mL) was not abnormally low.

The apparently “bad” performances for the MRSA-positive sample # 1525391 and for the MRSA-negative sample # 1525392 are qickly explained on closer inspection. Sample # 1525391 contained a mecC-positive S. aureus isolate which was correctly detected and classified as MRSA only by 115 of the 314 participants.

Sample # 1525392 contained one of the yet still relatively rare S. aureus strains, that belongs to the group of so-called mecA dropout MSSA isolates: Oxacillin sensitive S. aureus strains which contain the MRSA-typical SCCmec cassette, but significant parts or the entire mecA gene are deleted on genomic level. Consequently only 167 of the 314 participants reported correct negative MRSA results for this tricky sample. Compared to the some previous rounds of PCR/NAT external quality assessment, a much better diagnostic performance was observed for these variant S. aureus genetic constellations. The mecC-positive S. aureus isolate, sent out formerly in the May 2014 distribution, was detected by 19% of the participants at that time whereas 36% of participants reported correctly MRSA positive results in the current distribution. The mecA dropout variant, sent out formerly in the November 2012 distribution, was detected by 30% of the participants at that time whereas 53% of participants reported correctly MRSA negative results in the current distribution.

This situation nicely reflects the various (and obviously successful) efforts of diagnostic companies and in-house assay development teams to continuously improve and adopt their protocols to the current challenges of direct PCR/NAT testing for MRSA.

Also, an optional molecular detection of putative pathogenicity factor PVL (Panton-Valentine Leukocidin) or its coding gene lukF/S-PV was inquired. Corresponding results were reported by 85 of the 314 participating laboratories and within the current distribution, the results for the molecular PVL testing were correct in all but two cases. Additional information can be found at Linde and Lehn [2] or Witte et al. [3]. A well evaluated protocol for the detection of PVL-positive PVL isolate can be found at Reischl et al. [4].

In addition, commercial real-time PCR assays reliably targeting PVL-genes in MRSA and MSSA isolates are available in the meantime (for example: r-biopharm and TIB Molbiol).

RV 540: Chlamydia pneumoniae

The concept of this proficiency testing series is designed to determine the analytical sensitivity and specificity of NAT-based assays for the direct detection of C. pneumoniae in typical (clinical) sample material. With the development and composition of the corresponding sample materials we intended to mimic the situation of processing typical clinical samples like BAL or other respiratory specimens. So the lyophilized samples usually contain low amounts of target organisms in a natural background of human cells and other components. As a consequence, diagnostic assays designed for C. pneumoniae antigen detection in clinical specimens or other serological assays will fail due to the low number of C. pneumoniae infected cells in individual samples of the QC set.

The current set of QC samples contained two samples positive for C. pneumoniae. Sample # 1525403 was spiked with ~1x106 IFU/mL of C. pneumoniae, whereas sample # 1525401 contained an approximately hundred fold lower amount of C. pneumoniae (~1x104 IFU/mL). Sample # 1525402 contained significant numbers of Mycoplasma pneumoniae (~1x105 genome copies/mL). Only E. coli and human cells were present in sample # 1525404.

As depicted in Tab. 2 (Attachment 1 [Attach. 1], p. 13), with one exception all participants reported correct results for the positive samples # 1525401 and # 1525403. For the “negative” sample 1525402, containing Mycoplasma pneumoniae, all laboratories reports correct negative results. However, for the second “negative” sample # 1525404 (containing E. coli), we received 3 false-positive and 1 questionable report. Most probably, this is due to (cross-)contamination during sample processing and extraction, as cross-reactivity of NAT/PCR-based assays specific for Chlamydia pneumoniae with Escherichia coli is unlikely. Certainly, a false-positive result should prompt investigations and improvement of the diagnostic workflow.

RV 541: Mycoplasma pneumoniae

General note to our participants: the concept of this proficiency testing series is designed to determine the analytical sensitivity and specificity of NAT-based assays for the direct detection of M. pneumoniae in typical sample material. With the development and composition of the corresponding sample materials we aim to mimic the situation of processing typical clinical specimens like BAL or other respiratory materials. Therefore, the lyophilized samples may contain low amounts of target organisms in a natural background of human cells and other components typically present in patient specimens. As a consequence, diagnostic assays designed for M. pneumoniae antigen detection in clinical specimens or other serological assays will fail due to the low number of M. pneumoniae infected cells in individual samples of the RV 541 distributions.

The current set of QC samples contained only one positive sample. A relatively high amount of M. pneumoniae (~1x106 genome copies/mL) was present in sample # 1525411.

Samples # 1525414 and # 1525413 were designed to monitor assay specificity: they contained a considerable amount of M. genitalium (~106 genome copies/mL) and M. salivarium (~105 genome copies/mL) respectively, as a related species to the target organism. The set was completed by sample # 1525412, which contained only human cells and a considerable amount of E. coli organisms.

With the exception of one laboratory, all 133 participants correctly reported sample # 1525411 as positive. The Mycoplasma salivarium (# 1525413) and Mycoplasma genitalium (# 1525414) containing samples were correctly reported “negative” by 129 and 125 of the 133 participants, respectively. The false-positive results in these cases may indicate lacking species-specificity of the test systems and trigger further investigations. Additionally, “the negative” sample # 1525412, containing E. coli, was wrongly reported positive by 3 of the 133 participants. As a (cross-) contamination is the most probable explanation for a false-positive result in this sample, the diagnostic workflow should be re-assessed.

RV 542: Coxiella burnetii & Bacillus anthracis

General note to our participants: the concept of this proficiency testing series is designed to determine the analytical sensitivity and specificity of NAT-based assays for the direct detection of C. burnetii DNA and/or Bacillus anthracis DNA in typical sample material. With the development and composition of the corresponding sample materials we aim to mimic the situation of processing typical clinical samples. So the lyophilized samples may contain low amounts of target organisms in a natural background of human cells and other components typically present in patient specimens.

The current set of QC samples contained two samples with different amounts of Coxiella burnetii organisms (~5x103 genome copies/mL in sample # 1525421 and ~5x104 genome copies/mL in sample # 1525422), one sample with ~1x105 genome copies/mL of Bacillus anthracis (sample # 1525422) and one sample with ~1x106 genome copies/mL of a Bacillus anthracis Pasteur Strain (sample # 1525424). Sample # 1525423 contained only human cells and a considerable amount of E. coli organisms.

For convenient data presentation and analysis, we decided to depict the PCR/NAT-results for each target organisms within this combined EQAS scheme in two separate tables: please see Tables 2 and 3 (Attachment 1 , p. 15) for the Coxiella burnetii-specific results and Tables 4 and 5 (Attachment 1 , p. 16) for the Bacillus anthracis-specific results.

Coxiella burnetii: The relatively high amount (5x104 genome copies/mL) of C. burnetii organisms in sample # 1525422 was correctly reported by all participants, as well as the ten-fold lower concentration of the pathogen in sample #1525421. The two “negative” samples (#1525423 contained only E. coli and #1525424 contained only B. anthracis) were correctly reported negative by all participants. Overall, there were no noticeable problems with the current set of QC samples and a good correlation with the expected results was observed.

Bacillus anthracis: The results for this newly introduced EQAS scheme are easily discussed. All participants correctly reported negative results for the samples # 1525421 and # 1525423 which did not contain the target organism. The “positive” sample # 1525422 containing ~1x105 genome copies/mL was correctly reported by 18 of the 19 participants. Additionally, we received one questionable result, which should definitely prompt investigations regarding sample workup and performance of the test system used by the reporting laboratory, as the amount of the target organism in the sample was pretty high.

With the exception of one participant, the second positive sample # 1525424 (~1x106 genome copies/mL of B. anthracis strain “Pasteur”) was correctly reported. This particular strain is positive for the virulence plasmid pXO2 and the B. anthracis-specific markers rpoB and dhp61, but does not harbor “lethal and edema factor” encoding plasmid pXO1 and is therefore also negative for the commonly used pathogenicity marker pagA.

After this successful round of external quality assessment, “standardized samples” are again available for colleagues who are interested in obtaining B. anthracis DNA-positive material for assay validation purposes. Requests for backup samples should be addressed to the EQAS coordinator (U. Reischl).

RV 543: Francisella tularensis

General note to our participants: the concept of this proficiency testing series is designed to determine the analytical sensitivity and specificity of NAT-based assays for the direct detection of F. tularensis DNA in typical sample material. With the development and composition of the corresponding sample materials we want to mimic the situation of processing typical clinical samples. So the lyophilized samples may contain low amounts of target organisms in a natural background of human cells and other components typically present in patient specimens.

The current set of QC samples contained three positive samples: a high amount of Francisella tularensis spp novicida (~1x105 CFU/mL) was present in sample # 1525431, an approximately hundred lower amount (~1x103 CFU/mL) was present in sample # 1525434, and a relative high amount of Francisella tularensis spp holarctica (~1x104 CFU/mL) was present in sample # 1525432.

Similar to QC samples from past distributions, the positive sample # 1525431 (~1x105 CFU/mL of Francisella tularensis novicida) and # 1525432 (~1x104 CFU/mL of Francisella tularensis holarctica) were correctly tested positive by all of the 17 participating laboratories. Even with pathogen amounts of ~1x103 CFU/mL (sample #1525434) 15 out of 17 labs were able to detect Francisella DNA. As no false-positive result was observed for the “negative” sample # 1525433 – it seems that the participating laboratories have implemented functional precautions measures to prevent deleterious contamination events. Overall, these results corroborate the lower limits of detection observed in our previous EQAS distributions. Although the number of participating laboratories is still not very high, the results of the present distribution indicate that the lower limit of detection is about or slightly below 104 organisms/mL when using currently employed and well evaluated PCR/NAT-based assay concepts for the detection of F. tularensis DNA.

RV 544: Carbapenemase genes

The concept of this novel EQAS-panel for the detection of carbapenemase genes is designed exclusively for the testing of NAT-based methods and protocols for molecular resistance testing or the direct detection of carbapenemase genes from DNA preparations of Enterobacteriaceae culture isolates. Because of the methodologically challenging design of EQAs for the molecular resistance testing of the wide range of known carbapenemase coding genes in different bacteria, the panel is narrowed down to a small selection of the currently most common carbapenemase genes in Enterobacteriaceae: KPC, VIM, OXA-48 like genes, GES carbapenemases, NDM, IMP, and GIM.

As shown in Tab. 1 (Attachment 1 [Attach. 1], p. 18), the current set contained four samples with different carbapenem-resistant Enterobacteriaceae: sample # 1525441 contained Serratia marcescens with OXA-48 gene (~1x106 genome copies/mL), sample # 1525442 contained an Escherichia coli isolate with a NDM-5 and OXA-181 gene (~1x106 genome copies/mL), sample # 1525443 contained an Klebsiella pneumoniae isolate with a KPC-2 and VIM-1 gene (~1x106 genome copies/mL) and sample # 1525444 contained an Enterobacter cloacae-Complex strain harbouring a IMP-14 gene (~1x106 genome copies/mL).

All participants detected a carbapenemase gene in the OXA-48-positive S. marcescens sample (# 1525441) and with the exception of one participant classified the sample # 1525443 (Klebsiella pneumoniae with KPC-2 and VIM1 genes) correctly as positive for the presence of both carbapenemase genes. The participant with the false-negative result detected KPC-2 but missed the VIM-1 gene. For sample # 1525442 (NDM-5 and OXA-181 positive Escherichia coli), 39 of the 50 participants correctly reported presence of both carbapenemase genes. However, 11 participants detected only NDM-5 (10x) or only OXA-181 (1x). It is known that some OXA-48 like genes such as OXA-181 and OXA-232 are missed in certain commercial test systems. As these OXA-48 variants are increasingly imported into Europe any user should be fully aware if the molecular tests used in their laboratory is able to detect such OXA-48 variants. For sample # 1525444 (Enterobacter cloacae-Complex strain with an IMP-14 gene), only 14 participants detected this carbapenemase. Detection of beta-lactamase genes of the IMP family by molecular assays is technically demanding because of the heterogeneity of the many IMP variants. Currently IMP carbapenemases, including IMP-14, are exceedingly rare among carbapenemase-producing Enterobacteriaceae in Germany.

In-house NAT assays were used for the detection of carbapenemase coding genes by 17 of the 50 participating laboratories, while all others quoted the use of commercial test systems or kits on the result form. As these commercial test systems were not specified by all of the participants, a detailed comparisons between commercial kits and the heterogeneous group of proprietary (in-house) test systems with respect to sensitivity, specificity or susceptibility to contamination events is not yet possible.

RV 545: Clostridium difficile

General note to our participants: the concept of this proficiency testing series is designed to determine the analytical sensitivity and specificity of NAT-based assays for the direct detection of C. difficile DNA in typical sample material. With the development and composition of the corresponding sample materials we want to mimic the situation of processing typical clinical samples. The lyophilized samples may contain low amounts of target organisms in a natural background of human cells and other components typically present in patient specimens.

The current set of QC samples contained three Clostridium difficile-positive samples: sample # 1525453 with ~5x104 CFU/mL, sample # 1525451 with ~1x104 CFU/mL, and sample # 1525452 with ~5x103 CFU/mL. Sample # 1525454 contained only human cells and a considerable amount of E. coli organisms. The samples # 1525451 and # 1525453 containing relatively high amounts of C. difficile (1x104 CFU/mL and ~5x104 CFU/mL) were correctly reported as “positive” by 82 and 83 of the 84 participating laboratories, respectively.

False-negative results should prompt a thorough evaluation of the test system and the workflow. The latter is definitely warranted for the participant reporting a false-positive result for sample # 1525454, containing only E. coli, but no target organism. As cross-reaction of the applied test system with E. coli DNA is unlikely, probably cross-contamination during the process of sample preparation and analysis is causative.

Sample # 1525452 with the lowest amount of target organism (~5x103 CFU/mL) was correctly identified as positive by 79 participants. Again a false-negative result should prompt re-assessment of the sensitivity of the used test system. All participants included controls to detect inhibitions of the PCR reaction. Significant inhibitory events were not reported.

RV 546: VRE

General note to our participants: the concept of this proficiency testing series is designed to determine the analytical sensitivity and specificity of NAT-based assays for the direct detection of vancomycin-resistant enterococci DNA in typical sample material. With the development and composition of the corresponding sample materials we want to mimic the situation of processing typical clinical samples. So the lyophilized samples may contain low amounts of target organisms in a natural background of human cells and other components typically present in patient specimens.

Sample # 1525462 of the current set contained a relatively high amount of Enterococcus faecium vanB (~1x105 CFU/mL) and sample # 1525463 contained a similar amount of Enterococcus faecium vanA (~1x105 CFU/mL). Sample # 1525461 contained Enterococcus faecalis (~1x105 CFU/mL) and sample # 1525464 contained no target organisms but only human cells and E. coli cells. Of the 31 participating laboratories, 30 and 31 correctly reported positive results for the samples # 1525462 and # 1525463, respectively.

Of note, the reported dedicated vanA/vanB identifications for these two samples were all correct. We were pleased to see that also for the “negative” samples #1525461 and # 1525464, all participants reported correct “negative” results. This is especially important when considering the impact of molecular VRE detection on the clinical management of a patient. All participants included controls to detect inhibitions of the PCR reaction. Significant inhibitory events were not reported.

RV 560: Pneumocystis jirovecii

General note to our participants: the concept of this proficiency testing series, which was started in 2013, is designed to determine the analytical sensitivity and specificity of NAT-based assays for the direct detection of P. jirovecii DNA in typical sample material. With the development and composition of the corresponding sample materials we want to mimic the situation of processing typical clinical samples. So the lyophilized samples may contain low amounts of target organisms in a natural background of human cells and other components typically present in patient specimens.

The latest set of QC samples contained three positive specimens (see Tab. 1, Attachment 1 [Attach. 1], p. 21). A relatively high amount of Pneumocystis jirovecii (~5x105 organisms/mL) was present in sample # 1525601, an approximately tenfold lower amount of Pneumocystis jirovecii (~5x104 organisms/mL) was present in sample # 1525604 and an approximately hundredfold lower amount (~5x103 organisms/mL) was present in sample # 1525602. The set was completed by sample # 1525603 which contained only human cells and a considerable amount of E. coli organisms.

Sample # 1525601, which contained the highest amount of P. jirovecii target organisms (~5x105 organisms/mL) and sample # 1525604 with a ten-fold lower concentration of P. jirovecii, were reported “positive” by 90 and 86 of the 91 participating laboratories, respectively. Although this could be due to a loss of template DNA during pre-analytical sample preparation procedures or other reasons, observation of false-negative results should certainly trigger reassessment of the diagnostic workflow, sensitivity and/or specificity of the individual assay concept.

The sample containing the lowest amount of the target organism (# 1525602, ~5x103 organisms/mL) was correctly identified as positive by 58 participants, 29 false-negative results were recorded. The “negative sample” (# 1525603, containing only E. coli) was correctly classified “negative” by 89 participants. In case of false-positive or questionable results, this should definitely prompt investigations regarding all processes involved in sample preparation and analysis in order to optimize the NAT assay used.


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