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: comprehensive discussion of the November 2016 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
  • Michael Baier - 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
  • 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
  • Agnes Anders - National Reference Laboratory for multidrug-resistant gram-negative bacteria, Department for Medical Microbiology, Ruhr-University Bochum, Germany

GMS Z Forder Qualitatssich Med Lab 2017;8:Doc01

doi: 10.3205/lab000024, urn:nbn:de:0183-lab0000248

This is the English version of the article.
The German version can be found at: http://www.egms.de/de/journals/lab/2017-8/lab000024.shtml

Published: January 9, 2017

© 2017 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 an analysis report of 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.

A highly desired scheme for external quality assessment (EQAS) of molecular diagnostic methods in the field of medical microbiology was activated 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 German 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 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.instand-ev.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 style.


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 2016

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

Despite the mixed presence in different proportions and/or relatively low amounts of C. trachomatis and N. gonorrhoeae target organisms in some QC samples of the current set, 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 almost identical amounts of C. trachomatis (~1x105 IFU/mL; sample # 1625301 and sample # 1625302), one sample with ~1x104 IFU/mL of C. trachomatis (# 1625304) and three samples with various amounts of N. gonorrhoeae organisms (~5x106 CFU/mL in sample # 1625303, ~5x105 CFU/mL in sample # 1625304 and ~1x103 CFU/mL in sample # 1625302).

Despite relatively low amounts of C. trachomatis target cells and the simultaneous presence of N. gonorrhoeae organisms in the positive sample # 1625304, no false-negative results were observed among the Chlamydia trachomatis-specific results, reported by the 208 participants. Among the N. gonorrhoeae-specific results, 12 false-negative GO results were observed for sample # 1625302, which contained N. gonorrhoeae target organisms in a relatively low amount of 1x103 CFU/mL with C. trachomatis infected cells simultaneously present in high amounts. For sample # 1625303, results were classified as “questionable” by 7 participants. For questionable results, certificates are only issued when correct results are reported by the participant for the remaining samples of RV 531.

Since the amount of target organisms in CT-positive samples # 1625301, # 1625302, and NG-positive samples # 1625302, # 1625303 and # 1625304 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 207 participants and no inhibitory events were reported.

Tables 4 to 7 (Attachment 1 [Attach. 1], p. 2-3) were included this time to enable a detailed evaluation of the C. trachomatis- and GO-specific NAT components of combined GO/CT test systems. In the 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: # 1625313 with ~1x103 IFU/mL of C. trachomatis target organisms and sample # 1625311 with ~1x104 IFU/mL of C. trachomatis target organisms. Samples # 1625312 and # 1625314 contained no target organisms but only human cells and E. coli bacterial background.

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

Only two false-negative results were reported for C. trachomatis-positive sample # 1625313 containing a relatively low number of C. trachomatis-infected cells.

For the two C. trachomatis-negative samples # 1625312 and # 1625314 containing only human cells and E. coli, 3 false-positive results were observed among the 79 participants.

Assuming a sequential processing of the 4 individual samples of the current set, a contamination event of the “negative” samples 2 and 4 by target organism or PCR product carry-over from the positive samples 1 and/or 3 might have occured within the sample prep and amplification workflow of these laboratories. 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 79 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 79 participants.

RV 532: Bordetella pertussis

The current set of QC samples contained one sample with a very high amount of Bordetella pertussis (# 1625324; 1x106 CFU/mL), one sample with an approximately hundredfold lower number of Bordetella pertussis (# 1625322; 1x104 CFU/mL), as well as two samples containing only non-infected human cells and Escherichia coli (# 1625321 and # 1625323).

The availability of well-established commercial or in-house NAT-assays has led to a high portion of correct results. Only 1 of the 133 participants reported false-positive results for the B. pertussis-negative sample # 1625321 and three false-positives were observed with B. pertussis-negative sample # 1625323. The false-positivity issue is probably due to contamination events in the course of sample preparation or PCR/NAT amplification.

A cross reaction due to a possible low specificity of the used PCR/NAT test system is unlikely, because the negative samples contained only E. coli cells in the sample matrix as a kind of bacterial background. For sample # 1625322, 10 false-negative results were observed. With an amount of 104 CFU/mL of B. pertussis target organisms, the lower limit of detection of appropriate test systems is obviously “slightly touched” but not reached – as 123 participants were able to clearly detect the B. pertussis organisms in this sample.

For the detection of B. pertussis, most participants used self-developed (in house) test systems with inhibition and/or positive controls. Therefore, 84 participating laboratories used IS481 insertion sequence, 15 the B. pertussis toxin coding gene and 2 ribosomal genes.

RV 533: Helicobacter pylori

The current set of QC samples contained two samples with Clarithromycin-resistant Helicobacter pylori (sample # 1625333 with 1x104 CFU/ml and sample # 1625332 with 1x103 CFU/ml). Sample # 1625334 contained of a Clarithromycin-susceptible Helicobacter pylori patient strain (with 1x104 CFU/ml) and sample # 1625331 contained no target organisms but only human cells and E. coli cells.

The availability of well evaluated NAT-based assays and the relatively high amount of target organisms in two of the three Helicobacter pylori-positive samples (# 1625333 and # 1625334: ~1x104 CFU/mL) led to positive predictive values of 100%. Also for the Helicobacter pylori-negative sample # 1625331 correctly negative PCR/NAT-results were reported by all the 38 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 38 participants. With one exception, the molecular clarithromycin resistance testing 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: # 1625343 (E. coli, 1x105 CFU/mL, clinical isolate, stx1-positive, stx2-positive, eae-positive and hlyA-positive) and # 1625341 (E. coli, 1x105 CFU/mL, clinical isolate, stx1-negative, stx2c-positive, eae-positive and hlyA-positive). The other two EHEC-negative samples contained a Salmonella enterica ser. Enteritidis strain (sample # 1625344; 1x104 CFU/mL) and an eae- and hlyA-negative E. coli K12 strain (# 1625342).

Almost all of the 123 participants correctly reported negative results for sample # 1625344, containing only Salmonella enterica ser. Enteritidis. The second “negative” sample (# 1625342), containing only E. coli K12 was with one exception also correctly commented as EHEC/STEC-negative. For the EHEC/STEC-positive samples # 1625341 and # 1625343, the availability of well-established NAT-based assays and strategies for molecular differentiation (and relatively high numbers of target organisms present in the respective samples) resulted in consistently high accuracy rates. Sample # 1625341 was correctly reported positive by 122 of the 123 participants, while even 121 participants identified sample # 1625343 as positive and one participant classified his result as “questionable”.

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 106 of the 123 participating laboratories. All of 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.

Short recapitulation: So far 21 different species belonging to the B. burgdorferi sensu lato complex were described, that naturally present genetic differences in commonly used target genes. To further address this heterogeneity and to monitor the analytical sensitivity and specificity of the PCR/NAT assays applied by the diverse group of international participants, Borrelia valaisiana as well as Borrelia bissettiae were included in the actual panel.

While B. garinii is a well known human pathogenic species present in Europe and Asia, B. valaisiana – though frequently present in ticks in Europe and Asia – was rarely found in patient specimen and only by PCR, questioning a relevant human pathogenic potential. In the USA, Borrelia bisettiae was isolated from ticks but never found in patient specimen, while in Europe this species is extremely rare in ticks (1 study, by PCR only) and patients, but one patient isolate (neuroborreliosis, Germany) is available.

The current distribution of QC samples contained one sample with Borrelia valaisiana (# 1625353; ~1x105 organisms/mL), one sample with Borrelia garinii OspA type 3 (sample # 1625351; ~1x104 organisms/mL) and one sample with Borrelia bissettiae (sample # 1625354; ~1x104 organisms/mL). Sample # 1625352 contained no target organisms but only human cells and E. coli cells. With the exception of six false-negative results for sample # 1625353 (containing a high number of B. valaisiana target organisms), four false-negative result for sample # 1625354 (B. bissettiae) and two false-negative result for sample # 1625351 (B. garinii) all participants reported correct results for the three positive samples. The false-negative results should prompt re-evaluation of the assay specificity and/or 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. Looking at the species composition of the current panel, slight differences in test performance are getting apparent between commercially available kits and in-house assays for the diagnostic detection of Borrelia burgdorferi by PCR/NAT techniques.

RV 536: Legionella pneumophila

Referring to some recent requests of candidate participants: this EQAS panel is designed exclusively for assessment of PCR/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 newly established methods or protocols with the help of an external quality control.

In order to assess the analytical sensitivity of certain Legionella pneumophila-specific PCR assays, the current set of QC samples contained a kind of dilution series of Legionella pneumophila serogroup 2: sample # 1625364 (1x105 CFU/ml), sample # 1625361 (1x104 CFU/ml) and sample # 1625362 (1x103 CFU/ml). Sample # 1625363 contained no target organisms but only human cells and E. coli cells.

The L. pneumophila-positive samples # 1625361 (~1x104 CFU/mL) and # 1625364 (~1x105 CFU/mL) were correctly tested positive by 97 and 101 of the 103 participating laboratories, respectively. For the third positive sample within the current distribution, # 1625362, which containd a relatively low amount of L. pneumophila target organisms (~1x103 CFU/mL), only 67 of the 103 participants reported a correctly positive result.

With an amount of 103 CFU/mL of L. pneumophila, the lower limit of detection of appropriate test systems is obviously reached and so the results for the latter sample were not considered in the course of issuing the certificates.

Since the amount of target organisms in L. pneumophila-positive samples # 1625361 and # 1625364 could not be considered as “extremely low”, false-negative results should encourage the participants to review and optimize the workflow and concept of their individual L. pneumophila-specific PCR/NAT assays.

Sample # 1625363, which contained only E. coli, was classified as false-positive by one laboratory. This is probably due to contamination events in the course of sample preparation or PCR/NAT amplification. All participants have included inhibition controls in their test systems and no significant inhibitions of the PCR/NAT reactions were observed or reported.

RV 537: Salmonella enterica

The current set of QC samples contained a kind of dilution series of Salmonella enterica serovar Enteritidis: sample # 1625373 contained ~5x105 CFU/mL, sample # 1625372 contained ~5x104 CFU/mL and sample # 1625371 contained ~5x103 CFU/mL. Sample # 1625374 contained no target organisms but only human cells and E. coli cells. Almost all participants reported correct results for the negative sample # 1625374, and for the positive samples # 1625372 and # 1625373.

Reporting a false-negative result for sample # 1625372 should prompt a thorough re-evaluation of the performance of the test system.

Sample # 1625371, containing a relatively low amount of Salmonella enterica serovar Enteritidis target organisms (~5x103 CFU/mL) was correctly identified as “positive” by 18 of the 21 participants. Inhibitoric components in the sample matrix or events during 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 (# 1625382; only E. coli cells), and three samples positive for L. monocytogenes (# 1625381, # 1625384 and # 1625383). The Listeria monocytogenes-containing samples (# 1625381 with 5x105 CFU/mL of L. monocytogenes and # 1625384 with 5x104 CFU/mL of L. monocytogenes) were correctly reported positive by all participants. In addition, the “negative” E. coli containing sample # 1625382 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 # 1625383, containing only 5x103 CFU/mL of L. monocytogenes. Only one of the 36 participants has observed a false-negative result for this weak positive sample.

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 # 1625393 of the current distribution contained a S. aureus (MSSA, PVL-negative, ~1x104 CFU/mL). Correct (negative) results were reported by 297 of the 305 participating laboratories. The participant who reported “questionable” for sample # 1625393 indicated the use of an assay concept for the independent detection of the mecA gene and a S. aureus species marker gene. Some of the 7 participants who reported (false-) positive MRSA PCR results listed the use of in-house or commercial assay concepts relying on the quantitative detection of the mecA and S. aureus target genes.

One sample of the current set (# 1625391) contained an oxacillin-sensible CoNS strain (S. epidermidis; mecA-positive, ~1x105 CFU/mL). Correct (negative) results were reported by 299 of the 305 participating laboratories. Assuming a simultaneous processing of the 4 individual samples of the current set, a contamination event of the “negative” sample by target organisms or PCR products of the positive samples 2 or 4 is obvious.

False positive results for samples # 1625391 and/or # 1625393 should encourage the affected participants to review and optimize his DNA extraction procedure and/or the MRSA specific NAT-based test system.

Sample # 1625394 contained a typical cMRSA or CA-MRSA isolate (MRSA, PVL-positive, spa:t 310; ~1x105 CFU/mL) which tested positive with the MRSA-specific assays in 303 of 305 participating laboratories.

One sample of the current set (# 1625392) contained a relatively high number of a yet atypical methicillin resistant S. aureus isolate (clinical MRSA isolate with an “exotic” SCCmec cassette type IV, PVL-negative, ~1x105 CFU/mL). As expected, the latter organisms were not reliably detected by a number of in-house SCCmec-based assay concepts and they were also missed by some of the current commercial tests. MRSA strains containing weird MREJ variants are admittedly rare and hence false-negative results were not counted in the course of issuing the certificates. By the way, MREJ refers to the staphylococcal cassette chromosome mec (SCCmec) right-extremity junction. MREJ comprises the right extremity of SCCmec, the SCCmec integration site, and the staphylococcal orfX gene.

Overall, it should be noted that a pleasingly large proportion of participants reported correct PCR/NAT results for MRSA. This indicates the implementation of efficient sample workup and DNA extraction procedures, functioning of individual prevention measures to avoid the risk of contamination and carry-over events, as well as sensitive and specific PCR/NAT assay concepts for the detection of MRSA among the broad spectrum of our international EQAS participants.

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 124 of the 305 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 et al. (2005) [2] or Witte et al. (2005) [3]. A well evaluated protocol for the detection of PVL-positive PVL isolate can be found at: Reischl et al. (2007) [4]. In addition, commercial real-time PCR assays reliably targeting PVL-genes in MRSA and MSSA isolates are available from a number if diagnostic companies in the meantime (for example: r-biopharm or 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.

To assess the analytical sensitivity of the NAT-assays used by the individual participating laboratories, the current set of QC samples contained a kind of dilution series of C. pneumoniae organisms in the sample matrix: sample # 1625402 contained about 5x105 IFU/mL, sample # 1625401 about 5x104 IFU/mL and sample # 1625403 about 5x103 IFU/mL of C. pneumoniae-positive human cells. Only E. coli and non-infected human cells but no C. pneumoniae target organisms were present in sample # 1625404 of the current set.

As depicted in Tab. 2 (Attachment 1 [Attach. 1], p. 13), all but one participant reported correct results for the positive sample # 1625402. 106 of the 108 participants also reported correct positive results for sample # 1625401, and also for the sample with the lowest amount of C. pneumonia (# 1625403; 5x103 IFU/mL) 100 correct results were reported. Only two participants reported false-positive results for the “negative” sample # 1625404 (E. coli). Overall, there were no noticeable problems with the current set of QC samples and a good overall correlation with the expected results was observed.

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 two positive samples. A relatively high amount of M. pneumoniae (~1x105 genome copies/mL) was present in sample # 1625414 and a lower amount of M. pneumoniae (~5x103 genome copies/mL) was present in sample # 1625411. Sample # 1625412 was designed to monitor assay specificity: it contained a considerable amount of M. genitalium (~104 genome copies/mL) as a related species to the target organism. The set was completed by sample # 1625413, which contained only human cells and a considerable amount of E. coli organisms. Similar to the result constellations observed with past distributions of our external quality assessment schemes for Mycoplasma pneumoniae PCR/NAT detection, the availability of well-established commercial or in-house PCR/NAT-assays has led to a high percentage of correct results. With the exception of one laboratory, all 139 participants correctly reported sample # 1625413 as negative. The Mycoplasma pneumoniae-containing samples (#1625411 and # 1625414) were correctly reported by 118 and 122 of the 123 participants, respectively. Sample # 1625412 contained M. genitalium (~104 genome copies/mL), and was erroneously reported positive by five laboratories. This may indicate lacking species-specificity of the test systems and trigger further investigations.

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 aimed 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 (Tab. 1 in (Attachment 1 [Attach. 1], p. 15) contained two samples with different amounts of C. burnetii organisms (~1x103 genome copies/mL in sample # 1625424 and ~1x104 genome copies/mL in sample # 1625421), one sample with ~1x104 genome copies/mL of B. anthracis (sample # 1625421) and one sample with ~5x104 genome copies/mL of a B. anthracis Pasteur Strain (sample # 1625423). Sample # 1625422 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 Tab. 2 and 3 (Attachment 1 [Attach. 1], p. 15) for the Coxiella burnetii-specific results and Tab. 4 and 5 (Attachment 1 [Attach. 1], p. 16) for the Bacillus anthracis-specific results.

Coxiella burnetii: The relatively high amount (1x104 genome copies/mL) of C. burnetii organisms in sample # 1625421 was correctly reported by all participants. The ten-fold lower concentration of the pathogen in sample # 1625424 was correctly identified as “positive” by 34 of the 36 participating laboratories. False-negative or questionable results should prompt a reassessment of the performance of the test system used and evaluation of processes regarding sample workup and analysis. The two “negative” samples (# 1625422 contained only E. coli and # 1625423 contained only B. anthracis) were correctly reported as 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: All participants correctly reported negative results for the samples # 1625422 and # 1625424 which did not contain the target organism. The “positive” sample # 1625421 containing ~1x104 genome copies/mL B. anthracis strain “UR-1” was correctly reported by 16 of the 17 participants. The questionable result we received for this sample should definitely prompt investigations regarding sample workup and performance of the test system, as the amount of the target organism in the sample is still above the lower limit of detection.

With the exception of two participants, the second positive sample # 16225423 (~5x104 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 (coding for the "Protective Antigen") After this 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 (Tab. 1 in Attachment 1 [Attach. 1], p. 17) contained three positive samples: a high amount of Francisella tularensis spp holarctica (~1x105 CFU/mL) was present in sample # 1625432, an approximately ten lower amount (~1x104 CFU/mL) was present in sample # 1625431, and a relative high amount of Francisella tularensis spp. tularensis (~1x105 CFU/mL) was present in sample # 1625434. Both samples with the highest amount of target organism (# 1625432 and # 1625434) were correctly identified as “positive” by all but one participant (Tab. 2 and 3 in Attachment 1 [Attach. 1], p. 17). Even with pathogen amounts of ~1x104 CFU/mL (sample #1625431) 19 out of 21 labs were able to detect Francisella DNA. Two false-positive results were observed for the “negative” sample # 1625433 – this should be thoroughly evaluated, given the clinical consequences of a false-positive result! 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 in a sample harbouring even non-target DNA as it is given in clinical specimens.

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 three samples with different carbapenem-resistant Enterobacteriaceae: sample # 1625441 contained Klebsiella oxytoca with a KPC-3 and VIM-1 genes (~1x107 genome copies/mL), sample # 1625442 contained an Klebsiella pneumoniae isolate with a NDM-1 and OXA-48 genes (~1x107 genome copies/mL), and sample # 1625443 contained an Klebsiella pneumoniae isolate with a OXA-232 gene, which belongs to the OXA-48-like gene family (~1x107 genome copies/mL). The fourth sample # 1625444 was designed as negative control and contained only E. coli without carbapenemase genes.

All participants reported sample # 1625441 (K. oxytoca carrying KPC-3 and VIM-1 carbapenemase) as “carbapenemase positive”, although one participant did not detect VIM-1 and another reported the isolate OXA-positive. Notably, all participants were able to detect carbapenemase genes in sample # 1625442 (K. pneumoniae carrying NDM-1 and OXA-48), but again three laboratories missed the NDM-1 gene and one laboratory reported presence of VIM-1. The third “positive” sample # 1625443 (containing Klebsiella pneumoniae with a OXA-232 gene) was correctly reported by 58 of the 63 participants. A false-negative result for this sample should prompt investigations regarding the coverage of carbapenemase genes by the test system used.

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 two Clostridium difficile-positive samples: sample # 1625453 with ~1x105 CFU/mL, and sample # 1625452 with ~1x104 CFU/mL. Samples # 1625451 and # 1625454 contained only human cells and a considerable amount of E. coli organisms. The “positive” samples # 1625452 and # 1625453 were correctly reported as “positive” by 102 of the 103 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 # 1625451, 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 # 1625454 with no target organism was correctly identified as negative by all participants. 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.

The current set of QC samples contained this time two vancomycin-resistant Enterococcus strains: Enterococcus faecium vanA (# 1625463, ~1x105 CFU/mL) and an Enterococcus gallinarum vanA and vanC positive strain (# 1625461, ~1x105 CFU/mL). Sample # 1625464 contained Enterococcus faecium (~1x104 CFU/mL) and sample # 1625462 contained no target organisms but human cells and E. coli cells. All of the 39 participating laboratories reported correct results for the “positive” sample 1625463. The vanA and vanC-positive E. gallinarum strain was also correctly reported as “vancomycin-resistant” by 37 of the 39 participants. 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 #1625462 and # 1625464, 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 suitable clinical sample material. With the development of diagnostic material similar to clinical samples 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 two positive specimens (see Tab. 1 in Attachment 1 [Attach. 1], p. 22). A relatively high amount of Pneumocystis jirovecii (~5x105 genome copies/mL) was present in sample # 1625602, and an approximately hundredfold lower amount of Pneumocystis jirovecii (~5x103 genome copies /mL) was present in sample # 1625603. The set was completed by samples # 1625601 and # 1625604 which contained only human cells and a considerable amount of E. coli organisms.

Sample # 1625602, which contained the highest amount of P. jirovecii target organisms (~5x105 genome copies/mL) and sample # 1625603 with a hundered-fold lower concentration of P. jirovecii were reported "positive" by 96 and 83 of the 96 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 “negative samples” (# 1625601 and # 1625604, containing only E. coli) were correctly classified “negative” by 94 and 95 participants, respectively. 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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