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 June 2019 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
  • 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 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
  • Holger Scholz - 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
  • Roger Dumke - Institute for Medical Microbiology and Hygiene, Technical University 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 2020;11:Doc01

doi: 10.3205/lab000036, urn:nbn:de:0183-lab0000361

This is the English version of the article.
The German version can be found at: http://www.egms.de/de/journals/lab/2020-11/lab000036.shtml

Published: March 16, 2020

© 2020 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 schemes (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 INSTAND e.V. website (https://www.instand-ev.de). Although the preferred language of these documents is German, we aim 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 June 2019

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 almost identical amounts of C. trachomatis (~5x105 IFU/mL; # 1915301 and # 1915302), and two samples with different amounts of N. gonorrhoeae organisms (~5x105 CFU/mL in sample # 1915303 and ~5x104 CFU/mL in sample # 1915304).

As depicted in Table 2 (Attachment 1 [Attach. 1], p. 1), the reported results were almost correct for the two relatively strong C. trachomatis-positive samples and the two C. trachomatis-negative sample among the current set – only 8 false-positive and two false-negative results were observed among the 1056 results submitted by the 264 participating laboratories.

Among the N. gonorrhoeae-specific results, false-negative results were reported by one of the 264 participants for sample # 1915303, which contained a relatively high number of N. gonorrhoeae target organisms (5x105 CFU/mL), and by 3 participants for sample # 1915304 (5x104 CFU/mL). Fortunately, no false-positive results were reported for the GO-negative samples # 1915301 and # 1915302 of the current distribution.

Since the amount of target organisms in the CT- and GO-positive samples of the current distribution could not be considered as “extremely low”, false-negative results and also false-positive results for either of the two target organisms should encourage the participant to review and optimize their CT- and GO-specific NAT-based assays. Inhibition controls were included by 263 of the 266 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 Tables 4 and 5 (Attachment 1 [Attach. 1], p. 2), only the C. trachomatis (CT)-specific results, and in 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 three positive samples: # 1915312 and # 1915314 with ~5x105 IFU/mL of C. trachomatis target organisms and sample # 1915313 with ~5x104 IFU/mL of C. trachomatis target organisms. Sample # 1915311 contained no target organisms but only human cells and E. coli cells.

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

For the C. trachomatis-negative sample # 1815313 containing only non-infectious human cells and E.coli, only one false-positive result was observed among the 66 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 product carry-over from the positive samples “2”, “3” and/or “4” might have occurred within the sample prep and amplification workflow of the affected laboratory. In general, the observation of false-positive and/or false-negative results should encourage the affected participants to review and optimize their DNA extraction procedure and their CT-specific NAT-based test system.

However, 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 66 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 66 participants.

RV 532: Bordetella pertussis

The current set of QC samples contained one sample with a relatively high amount of Bordetella pertussis (# 1915322; 1x104 CFU/mL), and three samples negative for the respective target organism: one negative sample containing Bordetella parapertussis (# 1915321; 1x105 CFU/mL), one negative sample containing Bordetella bronchiseptica (# 1915324 with ~1x105 CFU/mL), as well as one sample containing only human cells and Escherichia coli (# 1915323).

The availability of well-established commercial or in-house NAT assays has led to a high portion of correct results. Nearly all of the 169 participants reported correct-positive results for the sample # 1915322 (B. pertussis, 1x104 CFU/mL). Three of the participating laboratories observed false-negative results for B. pertussis DNA in sample # 1915322. 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 # 1915324 and # 1915321 which contained ~1x105 CFU/mL of Bordetella bronciseptica and Bordetella parapertussis, respectively, were correctly tested negative by the majority of the 169 participants, but 3 (for sample # 1915324) and 2 (for sample # 1915321) of the participating laboratories observed false-positive results for B. pertussis DNA. Sample # 1915323 contained only E. coli. All but three 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 # 1915322, one result was classified as “questionable" by one participant. For questionable results, it should be noted that 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 DNA, most participants used self-developed (in-house) test systems with inhibition and/or positive controls or “other” commercial tests. Therefore, 42 participating laboratories indicated the use of the IS481 insertion sequence, 7 the pertussis toxin coding gene, and 3 ribosomal genes as target. Run controls were performed by 168 of 169 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 two samples with a Clarithromycin-resistant Helicobacter pylori strain isolated from a patient in the course of an antibiotic therapy failure study. Sample # 1915331 contained approximately 5x105 CFU/mL and sample # 1915333 approximately 5x104 CFU/mL of the respective target organisms. Sample # 1915334 contained culture suspensions of the related species Helicobacter mustelae (~5x104 CFU/mL).

The availability of well-evaluated NAT-based assays and the relatively high amount of target organisms in the two Helicobacter pylori-positive samples (# 1915331: ~5x105 CFU/mL and # 1915333: ~5x104 CFU/mL) led to positive predictive values of 100%.

One false positive result was observed among the 52 participants for sample # 1915332, which contained only a significant number of E. coli cells within our proprietary sample matrix. Of note, seven false-positive results were reported for sample # 1915334, containing ~5x104 CFU/mL Helicobacter mustelae.

Assuming a sequential processing of the 4 individual samples of the current set, a contamination event of the “negative” sample “4” by target organism or PCR product carry-over from the positive sample “1” and/or “3” might have occurred within the sample prep and amplification workflow of the affected laboratories. In the current distribution, false-positive H. pylori results for sample # 1915334 could also be due to a lacking specificity of the applied test system. 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 45 of the 56 participants. All of the reported 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: # 1915341 (E. coli, 1x104 CFU/mL, clinical isolate, stx1-, stx2-, eae-, hlyA- and O157-positive) and # 1915344 (E. coli, 1x105 CFU/mL, clinical isolate, stx2c- and eae-positive). The other two EHEC-negative samples contained an EPEC strain (sample # 1915342; 1x104 CFU/mL) and an EIEC strain (sample # 1915344; 1x104 CFU/mL).

All but 3 participants correctly reported negative results for sample # 1915342, containing no EHEC target organisms but only relatively high numbers of an eae-positive EPEC strain. The other “EHEC-negative” sample # 1915344, containing a significant amount of EIEC isolate was also reported PCR-negative by all but one of the participants. For the EHEC/STEC-positive samples # 1915341 and # 1915343, the availability of well-established NAT-based assays and strategies for molecular differentiation resulted in consistently high accuracy rates. Sample # 1915341 was correctly reported positive by all of the 139 participants, and all but two of the 139 participants detected the target organisms in the EHEC/STEC-positive sample # 1915343 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 126 of the 139 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.

Short recapitulation: So far, more than 20 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 lusitaniae and Borrelia garinii were included in the current EQAS distribution.

While B. garinii is a well-known human pathogenic species present in Europe and Asia, Borrelia lusitaniae is found in Europe – mainly western Mediterranean – in Ixodes ticks and in lizzards as host. Though having been known for nearly 30 years, only two patient isolates exist so far – both from atypical skin diseases. Therefore, the human pathogenicity is not well assured. In Europe, this species is still extremely rare in ticks (detected by PCR only) and patients, but one isolate from a German patient with neuroborreliosis is available.

The current distribution of QC samples contained one sample with Borrelia recurrentis (# 1915351; ~5x105 organisms/mL), one sample with Borrelia garinii OspA type 7 (sample # 1915352; ~5x105 organisms/mL) and one sample with Borrelia lusitaniae (sample # 1915354; ~5x105 organisms/mL). Sample # 1915353 contained no Borrelia organisms but a strain of Treponema phagedenis. With the exception of one false-negative result, all participants reported correct results for sample # 1915352 containing a high number of B. garinii target organisms. For the Borrelia spp.-negative sample # 1915353 (containing a high number of Treponema phagedenis organisms) of the current distribution, 118 correct-negative and 3 false-positive results for B. burgdorferi DNA were observed. About two thirds of all participants missed the detection of B. lusitaniae target organisms in sample # 1915354 and one third of all participants reported a false-positive PCR/NAT B. burgdorferi DNA result for the B. recurrentis organisms present in sample # 1915351 of the current distribution.

Admittedly, the current set of 4 samples contained some analytical challenges in good faith and with an educative background. But, as always, obtaining false-negative results should prompt thorough re-evaluation of the assay’s 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

Due to numerous requests: this EQA scheme is designed exclusively for the testing of NAT-based methods and protocols for direct detection of low amounts of Legionella pneumophila from appropriate clinical specimens. Individual samples may contain relatively small amounts of the corresponding target organism. For this reason, participation is promising only for those diagnostic laboratories that 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 scheme.

The current set of QC samples contained only one positive sample with Legionella pneumophila serogroup 1 (# 1915361; ~1x105 CFU/mL) next to two samples containing Legionella gormanii (# 1915364; ~1x105 CFU/mL and # 1915363; ~1x104 CFU/mL). Sample # 1915362 contained no target organisms but only human cells and E. coli cells.

The L. pneumophila-positive (~1x105 CFU/mL) sample # 1915361 was correctly tested positive by all but one of the 123 participating laboratories. Both of the L. gormanii-positive samples (# 1915363 and # 1915364) were correctly tested negative by 114 and 108 of the participants, respectively. Sample # 1915362, which contained only E. coli, was tested false-positive by 2 participants, whereas the remaining 121 participants reported correct-negative PCR/NAT results for L. pneumophila DNA. The overall result constellation with the sporadically observed false-positive results indicates that target DNA or amplicon contaminations may have occurred in the analytical workflow of some participants, or that some of the applied L. pneumophila-specific PCR/NAT assay concepts may show analytical specificity problems. In general, the observation of false-positive or false-negative results should encourage the affected laboratories to review and optimize their DNA extraction procedures and/or their L. pneumophila-specific PCR/NAT test systems. All but two participants have implemented inhibition controls in their test systems and no inhibition events were observed among the samples of the current distribution.

RV 537: Salmonella enterica

The current set of QC samples contained three positive samples with Salmonella enterica serovar enteritidis: sample # 1915373 contained ~1x104 CFU/mL, sample # 1915371 contained ~5x103 CFU/mL and sample # 1915372 contained ~1x103 CFU/mL. Sample # 1915374 contained no target organisms but only human cells and E. coli cells. All of the participants reported correct results for the negative sample # 1915374, and all but three participants reported correct-positive results for the Salmonella enterica-positive sample # 1915373 (1x104 CFU/mL). Due to the relatively low numbers of Salmonella enterica target organisms in samples # 1915371 (5x103 CFU/mL) and # 1915372 (1x103 CFU/mL) of the current distribution, about one half of the 28 participants reported false-negative results for the latter two samples. With an amount of 5x103 CFU/mL or less of S. enterica target organisms, the lower limit of detection of appropriate test systems is obviously reached and so the results for sample # 1915372 were not considered in the course of issuing the certificates (indicated by the gray-shaded box in Table 2, Attachment 1 [Attach. 1], p. 10).

However, a negative PCR/NAT result for sample # 1915373 should prompt a thorough re-evaluation of the performance of the S. enterica-specific PCR/NAT test systems. Inhibitoric components in the sample matrix or other inhibition 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 (# 1915382; only E. coli cells), two samples positive for L. monocytogenes (# 1915381 with ~1x106 CFU/mL and # 1915384 with ~5x104 CFU/mL) and one sample with Listeria innocua (# 1915383) as Listeria species other than L. monocytogenes. The results discussion of the current distribution is easy: all of the 43 participating laboratories detected the Listeria monocytogenes target organisms in the two (relatively strong) positive samples # 1915381 and # 1915384. In addition, the “negative” E. coli-containing sample # 1915382 was identified as negative by all laboratories. The majority of participants used Listeria monocytogenes-specific PCR/NAT assays, which is reflected by the high number of “false-negative” results for sample # 1915383. However, as noted in the report form, 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 (like L. innocua in sample # 1915383 of the present distribution) 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 # 1915392 of the current distribution contained a mixture of S. aureus (MSSA, PVL-negative, ~5x104 CFU/mL) and a CoNS strain (S. haemolyticus; mecA-positive, ~1x105 CFU/mL).

Correct-(negative) results were reported by 270 of the 293 participating laboratories. The two participants who reported “questionable” for sample # 1915392 indicated the use of assay concepts for the independent detection of the mecA gene and an S. aureus species marker gene (where “questionable” is the expected and correct classification for this mixed sample). Some of the 21 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 (# 1915391) contained an oxacillin-sensible CoNS strain (S. epidermidis; mecA-negative, ~1x103 CFU/mL). Correct-(negative) results were reported by 291 of the 293 participating laboratories. Assuming a sequential processing of the 4 individual samples of the current set, a contamination event of the “negative” sample 1 by target organisms or PCR products of the positive samples “3” and “4” is not really obvious, but cannot be ruled out. Such false-positive results should encourage the affected participants to review and optimize their DNA extraction procedures and/or the MRSA-specific NAT-based test systems.

Sample # 1915394 contained a typical cMRSA or CA-MRSA isolate (MRSA, PVL-positive, spa: t310; ~1x105 CFU/mL) which tested positive with the MRSA-specific assays in 290 participating laboratories.

One sample of the current set (# 1915393) contained a relatively high number of an “atypical” methicillin-resistant S. aureus SCC mec Type V isolate (MRSA, 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. Such isolates are admittedly rare and hence false-negative results were not counted in the course of issuing the certificates.

Overall, it should be noted that a pleasingly large proportion of participants reported correct PCR/NAT results for MRSA. This indicates excellent sample workup functioning of laboratory-specific prevention measures to avoid the risk of contamination and carry-over events.

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 81 of the 293 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 in Linde et al. [2] and Witte et al. [3]. A well-evaluated protocol for the detection of PVL-positive PVL isolate can be found in Reischl et al. [4].

In addition, commercial real-time PCR assays reliably targeting PVL genes in MRSA and MSSA isolates are now available.

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. Consequently, the lyophilized samples usually contain low amounts of target organisms in a natural background of human cells and other components. As a result, 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 three different amounts of C. pneumoniae organisms in the sample matrix: sample # 1915403 contained about 5x106 IFU/mL, sample # 1915401 about 1x105 IFU/mL and sample # 1915404 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 # 1915402 of the current set.

As depicted in Table 2 (Attachment 1 [Attach. 1], p. 14), all participants reported correct results for two of the positive samples # 1915401 and # 1915403. All but one of the 139 participants also reported correct positive results for the slightly weaker C. pneumoniae-positive sample # 1915403. Only three of the 139 participating laboratories reported false-positive results for the C. pneumoniae-“negative” sample # 1915402 (E. coli). Again, such false-positive results should encourage the affected participants to review and optimize their DNA extraction procedures and/or the C. pneumoniae-specific NAT-based test systems. 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

A 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 (~1x106 genome copies/mL) was present in sample # 1915413 and a lower amount of M. pneumoniae (~1x104 genome copies/mL) was present in sample # 1915412. Sample # 1915411 was designed to monitor assay specificity: it contained a considerable amount of M. genitalium (~105 genome copies/mL) as a related species to the target organism. The set was completed by sample # 1915414, 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 two laboratories, all participants correctly reported sample # 1915414 as negative. The Mycoplasma pneumoniae-containing samples (#1915413 and # 1915412) were correctly reported by 158 and 153 of the 158 participants, respectively. Sample # 1915411 contained M. genitalium (~105 genome copies/mL), and was erroneously reported positive by four laboratories. This may indicate lacking species specificity of the test systems and trigger further investigations.

RV 542: Coxiella burnetii & Bacillus anthracis

A 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 B. 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. Consequently, 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 C. burnetii organisms (~1x104 genome copies/mL in sample # 1915421 and ~1x105 genome copies/mL in sample # 1915422), one sample with ~1x104 genome copies/mL of B. anthracis (sample # 1915421) and one sample with ~1x104 genome copies/mL of a B. anthracis Pasteur Strain (sample # 1915423). Sample # 1915424 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 organism within this combined EQAS scheme in two separate tables: please see Tables 2 and 3 (Attachment 1 [Attach. 1], p. 16) for the C. burnetii-specific results and Tables 4 and 5 (Attachment 1 [Attach. 1], p. 17) for the B. anthracis-specific results.

Coxiella burnetii: The relatively high amount (~105 genome copies/mL) of C. burnetii organisms in sample # 1915422 was correctly reported by all participants, as well as the ten-fold lower concentration of the pathogen in sample #1915421. The two “negative” samples (#1915424 contained only E. coli and #1915422 contained only B. anthracis) were correctly reported negative by all but two 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. 27 of the 28 participants correctly reported a positive result for sample # 1915421 (~1x105 genome copies/mL). The second “positive” sample # 1915423 contained ~1x104 genome copies/mL of B. anthracis strain “Pasteur”. 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. With one exception, all participants correctly reported negative results for the two “negative” samples # 1915424 (containing E. coli and human cells) and # 1915422 (containing ~105 genome copies of C. burnetii in a suspension of human cells).

After this very 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 & Brucella spp.

A 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 and Brucella spp. 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. Consequently, 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 (Table 1, Attachment 1 [Attach. 1], p. 18) contained two samples with similar amounts of Francisella tularensis subsp. holarctica DNA (~1x104 CFU/mL in sample # 1915431 and # 1915432), two samples with different amounts of Brucella melitensis DNA (~1x105 CFU/mL in sample # 1915434 and ~1x104 CFU/mL in sample # 1915432). Sample # 1915433 contained only human cells and a considerable amount of E. coli organisms.

Francisella tularensis: Similar to QC samples from past distributions, the positive samples # 1915431 (~1x104 CFU/mL of Francisella tularensis spp. holarctica) and # 1015432 (also ~1x104 CFU/mL of Francisella tularensis spp. holarctica) were correctly tested positive by 33 of the 33 participating laboratories, respectively. None of the participants observed inhibition of the nucleic acid amplification reactions with the samples of the current distribution.

Brucella spp.: The “positive“” samples # 1915432 and # 1915434 were correctly reported by all participating laboratories. Additionally, the two samples without target organism (# 1915431 and #1915433) were correctly classified as “negative”. None of the participants observed an inhibition of the nucleic acid amplification.

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 Table 1 (Attachment 1 [Attach. 1], p. 20), the current set contained three samples with different carbapenem-resistant Enterobacteriaceae: sample # 1915441 contained a Klebsiella pneumoniae with a KPC-3 gene (~1x106 genome copies/mL), sample # 1915442 contained a Citrobacter freundii with a GIM-1 gene (~1x107 genome copies/mL), sample # 1915444 contained Serratia marcescens with a VIM-1 gene (~1x107 genome copies/mL). The fourth sample # 1915443 was designed as negative control and contained only E. coli without carbapenemase genes.

88 of the 90 participating laboratories reported sample # 1915441 (K. pneumoniae carrying a KPC-3 carbapenemase) as “carbapenemase-positive”. Notably, only 18 of the 90 participants were able to detect carbapenemase genes in sample # 1915442 (C. freundii carrying GIM-1). The third “positive” sample # 1915444 (containing S. marcescens with a VIM-1 gene) was correctly reported by 89 of the 90 participants. Additionally, 2 false-positive results were submitted for sample # 1915443, which contained carbapenemase-negative E. coli K12.

RV 545: Clostridium difficile

A 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 aim 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 # 1915452 with ~1x105 CFU/mL, sample # 1915451 with ~1x104 CFU/mL and sample # 1915454 with ~1x103 CFU/mL. Sample # 1915453 contained only human cells and a considerable amount of E. coli organisms.

The samples # 1915451 and # 1915452 containing relatively high amounts of C. difficile (1x104 CFU/mL and ~5x105 CFU/mL) were correctly reported as “positive” by 168 and 167 of the 168 participating laboratories, respectively. The sample # 1915454 (~1x103 CFU/mL) was correctly reported by 145 participants. False-negative results should prompt a thorough evaluation of the test system and the workflow. The latter is definitely warranted for the participants reporting false-positive results for samples # 1615453, containing only E. coli, but no target organism. As a 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. All participants included controls to detect inhibitions of the PCR reaction. Significant inhibitory events were not reported.

RV 546: VRE

A 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. Consequently, 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 vancomycin-resistant Enterococcus strains this time: Enterococcus faecalis vanA (# 1915461, ~1x105 CFU/mL), an Enterococcus faecium vanB (# 1915462, ~1x105 CFU/mL) and an Enterococcus gallinarum vanA- and vanC-positive strain (# 1915463, ~1x105 CFU/mL). Sample # 1915464 contained no target organisms but human cells and E. coli cells. All but one of the 59 participating laboratories reported correct results for the “positive” samples # 1915461 and # 1915462. The vanA- and vanC-positive E. gallinarum strain was also correctly reported as “vancomycin-resistant” by all 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” sample #1915464, 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 547: Urogenital panel

The concept of this novel EQAS panel for the detection of the most prominent urogential pathogens was recently established to meet the demands of current and future multiplex PCR/NAT assay concepts. Making some helpful experiences during the pilot phase of two previous distributions, we are starting with our first “regular distribution” in the current round.

Regarding the statistical analysis, data presentation and results discussion, we are still in the learning phase to optimize the informative and intuitive depiction of the complex result constellations as well as developing a rational scheme for issuing individual certificates for the participants. The results reported by the 57 registered participants are depicted in Table 2 (Attachment 1 [Attach. 1], p. 23). A good overall correlation between the expected results (Table 1, Attachment 1 [Attach. 1], p. 23) and the reported results was observed. The report forms of RV 547 distributions now contain an extra field for a simple 7-digit code, where participants have to specify the theoretical pathogen spectrum of their individual assay concepts. This extra information will help to consider and fairly assess the broad spectrum of different commercial and in-house PCR/NAT assays regarding species coverage, differentiation and multiplex capabilities.

RV 560: Pneumocystis jirovecii

A 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 aim to mimic the situation of processing typical clinical samples. Consequently, 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 (Table 1, Attachment 1 [Attach. 1], p. 24). A relatively high concentration of Pneumocystis jirovecii (~1x105 CFU/mL) was present in sample # 1915602, whereas in sample # 1915603 Pneumocystis jirovecii (~1x104 CFU/mL) was present at an approximately ten-fold lower concentration. The set was completed by samples # 1915601 and # 1915604 which contained only human cells and a considerable amount of E. coli organisms.

For sample # 1915602, which contained P. jirovecii target organisms (~1x105 CFU/mL) at a relatively high concentration, all of the 117 participants reported correctly positive results.

Sample # 1915603 of the current distribution, with a ten-fold lower concentration of P. jirovecii, was tested “positive” by all but two of the 117 participating laboratories, and one laboratory reported a “questionable” result. 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 in clinical samples with target organism loads around 104 CFU/mL should certainly trigger reassessment of the diagnostic workflow, sensitivity and/or specificity of the individual assay concept. The “negative samples” within the current distribution (# 1915601 and # 1915604, containing only E. coli) were correctly classified “negative” by all of our 117 participants, respectively. Overall, there were no noticeable problems with the current set of QC samples, and a good correlation with the expected results was observed.


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