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 2018 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 2019;10:Doc01

doi: 10.3205/lab000032, urn:nbn:de:0183-lab0000323

This is the English version of the article.
The German version can be found at: http://www.egms.de/de/journals/lab/2019-10/lab000032.shtml

Published: February 15, 2019

© 2019 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 June 2018

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 (~5x104 IFU/mL; # 1815302 and # 1815303), one sample with ~5x105 IFU/mL of C. trachomatis (# 1815304) and three samples with various amounts of N. gonorrhoeae organisms (~5x105 CFU/mL in sample # 1815302, ~5x104 CFU/mL in sample # 1815304, and ~5x102 CFU/mL in sample # 1815303). As depicted in tab. 2 (Attachment 1 [Attach. 1], p. 1) , the reported results were almost correct for the three C. trachomatis-positive samples and the one C. trachomatis-negative sample among the current set – only one false-positive and one false-negative result was observed among the datasets submitted by the 258 participating laboratories.

Among the N. gonorrhoeae-specific results, false-negative results were reported by 14 of the 256 participants for sample # 1815303, which contained a very low number of N. gonorrhoeae target organisms (5x102 CFU/mL), and by one participant for sample # 1815304. Also false-positive results for the GO-negative sample # 1815301 were reported by 4 participants.

Since the amount of target organisms in the CT- and GO-positive sample # 1815304 could not be considered as “extremely low”, false negative results should encourage the participant to review and optimize their CT- and GO specific NAT-based assays. Inhibition controls were included by 257 of the 258 participants and no inhibitory events were reported.

Tables 4 to 7 (Attachment 1 [Attach. 1], p. 2–4) 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 only the C. trachomatis (CT) specific results and in the tables 6 and 7 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: # 1815312 and # 1815314 with ~5x105 IFU/mL of C. trachomatis target organisms and sample # 1815311 with ~5x104 IFU/mL of C. trachomatis target organisms. Sample # 1815313 contained no target organisms but only human cells and E. coli cells.

As depicted in tab. 2 (Attachment 1 [Attach. 1], p. 5) 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, 6 false-positive results were observed among the 83 participants.

Assuming a sequential processing of the 4 individual samples of the current set, a contamination event of the “negative” samples “3” by target organism or PCR product carry-over from the positive samples “1” and “2” might have occurred 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 83 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 83 participants.

RV 532: Bordetella pertussis

The current set of QC samples contained one sample with a relatively high amount of Bordetella pertussis (# 1815321; 1x106 CFU/mL), one sample with an approximately hundredfold lower number of Bordetella pertussis (# 1815324; 1x104 CFU/mL), one negative sample containing Bordetella parapertussis (# 1815322 with ~1x105 CFU/mL), as well as one sample containing only non-infected human cells and Escherichia coli (# 1815323).

The availability of well-established commercial or in-house PCR/NAT-assays has led to a high portion of correct results. All of the 167 participating laboratories reported correct results for the very strongly B. pertussis positive sample # 1815321 (1x106 CFU/mL). Only 9 of the 167 participants reported false-positive results for the B. parapertussis sample # 1815322. The false-positivity issue with sample 2 of the current set is probably due to cross-reations of the applied PCR/NAT test systems (insufficient analytical specificity) or just due to simple contamination events in the course of sample preparation. For sample # 1815324 (104 CFU/mL of B. pertussis) 38 false-negative and 2 questionable 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 reached and so the results for the latter sample were not considered in the course of issuing the certificates (indicated by the three gray-shaded boxes in tab. 2 (Attachment 1 [Attach. 1], p. 6).

For the detection of B. pertussis, most participants used self-developed (in house) test systems with inhibition and/or positive controls. Therefor, 53 participating laboratories used IS481 insertion sequence, 9 the pertussis toxin coding gene and 2 ribosomal genes. Run controls were performed by 166 of 167 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 relatively high amount of target organisms. Sample # 1815331 contained approximately 5x104 CFU/ml of a Clarithromycin-susceptible Helicobacter pylori patient strain, and sample # 1815333 contained approximately 5x104 CFU/ml of a Clarithromycin-resistant Helicobacter pylori.

The availability of well evaluated NAT-based assays and the relatively high amount of target organisms in the two Helicobacter pylori-positive samples (# 1815331: ~5x104 CFU/mL and # 1815333: ~5x104 CFU/mL) led to positive predictive values of nearly 100%. Also for the Helicobacter pylori-negative sample # 1815332 correctly negative PCR/NAT-results were reported by nearly all the participating laboratories. Sample # 1815334 of the current distribution contained a culture suspension of Campylobacter jejuni (~5x104 CFU/ml), which was correctly reported “negative” by 51 of the 53 participants. Except the two false-positive results, this indicates an overall good analytical specificity of the used PCR test systems.

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 47 of the 53 participants. With three exceptions, the molecular antibiotic 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: # 1815343 (E. coli, 1x106 CFU/mL, clinical isolate, stx1-positive, stx2-positive, eae-positive and hlyA-positive) and # 1815344 (E. coli, 1x105 CFU/mL, clinical isolate, stx1-positive, stx2-positive, eae-positive and hlyA-positive). The other two EHEC-negative samples contained an eae-positive EPEC strain (sample # 1815341; 1x104 CFU/mL) and an eae- and hlyA-negative E. coli K12 strain (# 1815342). All of the 140 participants correctly reported negative results for samples # 1815342, containing only E. coli K12, and correctly detected the relatively high numbers of EHEC target organisms (1x106 and 1x105 CFU/mL) in samples # 1815343 and # 1815344. The other “negative” sample (# 1815341), containing an eae-positive but stx-negative EPEC isolate, was reported EHEC-negative by all but one of the participating diagnostic laboratories. The other participants, who reported a (false) positive result for sample # 1815341, indicated the molecular detection of the eae/intimin gene only. For an EPEC strain, the reported “eae-positive” result is theoretically correct. So we re-assigned the solely positive eae detection as EPEC and modified them as “negative” for EHEC target organisms.

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 120 of the 140 participating laboratories. With the exception of two laboratories, all 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 bissettiae and Borrelia garinii was included in the current EQAS distribution.

While B. garinii is a well-known human pathogenic species present in Europe and Asia, Borrelia bisettiae was isolated from ticks in the United States but never found in patient specimen. In Europe this species is still extremely rare in ticks (detected by PCR only) and patients, but one patient isolate (neuroborreliosis, Germany) is available.

The current distribution of QC samples contained one sample with Borrelia burgdorferi sensu stricto (# 1815353; ~5x105 organisms/mL), one sample with Borrelia garinii OspA type 3 (sample # 1815352; ~5x105 organisms/mL) and one sample with Borrelia bissettiae (sample # 1815354; ~5x105 organisms/mL). Sample # 1815351 contained no target organisms but only human cells and E. coli cells. With the exception of three false-negative results for sample # 1815352 (containing a high number of B. garinii target organisms), two false-negative result for sample # 1815354 (B. bissettiae) and two results classified as “questionable”, all participants reported correct results for the three positive samples and the negative sample. 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 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 scheme.

The current set of QC samples contained only one positive sample with Legionella pneumophila serogroup 2 (# 1815364; ~1x104 CFU/mL) next to two samples containing Legionella hackeliae (# 1815362; ~1x104 CFU/mL und # 1815363; ~1x103 CFU/mL). Sample # 1815361 contained no target organisms but only human cells and E. coli cells. The L. pneumophila-positive sample # 1815364 was correctly tested positive by 120 of the 126 participating laboratories. The negative sample # 1815361 was also correctly tested negative nearly all of the participants. Samples # 1815362 and # 1815363, which contained ~1x104 and ~1x103 CFU/mL of Legionella hackeliae, were classified false-positive by 6 and 5 of the participating laboratories, respectively. Observing false-positive L. pneumophila PCR-results for non-pneumophila Legionella spp. should encourage the corresponding participants to review and optimize the analytical specificity of their “L. pneumophila-specific” assays or PCR/NAT-protocols.

All but one of the 126 participants indicated the use of internal or external inhibition controls in their assay concepts and none of the investigated samples showed inhibition.

RV 537: Salmonella enterica

The current set of QC samples contained a kind of dilution series of Salmonella enterica serovar Typhi: sample # 1815371 contained 5x104 CFU/ml, sample # 1815374 contained 5x103 CFU/ml and sample # 1815373 contained 5x102 CFU/ml. Sample # 1815372 contained no target organisms but only human cells and E. coli cells.

Almost all participants reported correct results for the negative sample # 1815372, and for the positive samples # 1815371 and # 1815374. Although spiked with relatively low amounts of S. enterica target organisms (~5x103 CFU/mL), reporting a false-negative PCR/NAT result for sample # 1815374 should prompt a thorough re-evaluation of the performance of the test system.

Sample # 1815373, containing a very low amount of Salmonella enterica serovar Typhi target organisms (~5x102 CFU/mL) was correctly identified as “positive” by 17 of the 30 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 two samples without the corresponding target organisms (# 1815382 und # 1815383; only E. coli cells), one sample positive for L. monocytogenes (# 1815384 with ~5x103 CFU/mL) and one sample with Listeria ivanovii (# 1815381) as a Listeria species other than L. monocytogenes. The Listeria monocytogenes-containing sample (# 1815384) was correctly reported positive by all of the 42 participating diagnostic laboratories. In addition, the “negative” E. coli containing samples # 1815382 and # 1815383 were correctly identified as negative by all laboratories. Thirty-eight of the 42 participants indicated the use of Listeria monocytogenes-specific PCR/NAT assays, which is reflected by the high number of “false-negative” results for sample # 1815381, containing 5x104 CFU/mL of Listeria ivanovii organisms. 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 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 # 1815394 of the current distribution contained a mixture of a S. aureus strain (cMSSA, PVL-positive, ~1x104 CFU/mL) and a CoNS strain (S. epidermidis; mecA-positive, ~1x104 CFU/mL). Correct (negative) results were reported by 285 of the 307 participating laboratories. Most of the 8 participants who reported “questionable” for sample # 1815394 indicated the use of assay concepts for the independent detection of the mecA gene and a S. aureus species marker gene (where “questionable” is the expected and correct classification for this mixed sample). Some of the 14 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.

Sample # 1815391 contained a typical cMRSA or CA-MRSA isolate (MRSA, PVL-positive, spa:t 008; ~1x104 CFU/mL) which tested positive with the MRSA-specific assays in 304 participating laboratories. One sample of the current set (# 1815392) contained a relatively high number of a typical clinical MRSA isolate (MRSA, PVL-negative, ~5x104 CFU/mL) which was also found correctly MRSA positive by 304 of the participants. The last sample of the current set (# 1815393) contained no target organisms but only E. coli cells. Again, correct (negative) results were reported by 304 of our 307 participating laboratories. Assuming a sequential processing of the 4 individual samples of the current set, contamination events of the “negative” sample 3 by target organisms or PCR products originating from the positive samples “1” or “2” can not be ruled out. The false positive results, however, should encourage the affected participant to review and optimize their DNA extraction procedures and/or the MRSA specific NAT-based test systems.

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 50 of the 307 participating laboratories and within the current distribution, the results for the molecular PVL testing were correct in all but one case. Additional information can be found at: Linde et al. [2]; or Witte, W. et al. (2005) [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 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 sample material. With the development and composition of the corresponding sample materials we want to mimic the situation of processing typical clinical samples like BAL or other respiratory materials. 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 # 1815402 was spiked with ~5x105 IFU/ml of C. pneumoniae whereas sample # 1815401 contained an approximately ten fold lower number of C. pneumoniae (~5x104 IFU/ml). Sample # 1815404 contained significant numbers of Legionella pneumophila organisms to assess analytical specificity. Only E. coli and non-infected human cells but no C. pneumoniae target organisms were present in sample # 1815403. As depicted in tab. 2 (Attachment 1 [Attach. 1], p. 15), all participants reported correct results for the positive sample # 1815402. For the ten-fold low concentrated C. pneumoniae positive sample # 1815401 (~5x104 IFU/ml), still 138 from the 140 participants observed correct positive results. No false-positive results were observed for the “negative” sample # 1815403. Only three of the 140 participants reported false-positive results for sample # 1815404 (Legionella pneumoniae). As significant cross-reactions of C. pneumoniae-specific primer and probe sequences are not expected on molecular level with the use of well-evaluated PCR assays, these false-positives are probably due to cross-contamination events in the course of sample preparation, amplification or amplicon detection steps. 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 three positive samples. A relatively high amount of M. pneumoniae (~1x105 genome copies/mL) was present in sample # 1815413. An approximately tenfold lower amount of M. pneumoniae (~1x104 genome copies/mL) was present in sample # 1815411 and sample # 1815414 contained an approximately hundred fold lower amount of M. pneumoniae (~1x103 genome copies/mL). The set was completed by sample # 1815412, which contained only human cells and a considerable amount of E. coli. With the exception of two laboratories, which classified their results as “questionable” in the report forms, all of the 159 participants reported correct positive PCR/NAT results for samples # 1815411 and # 1815413.

The negative sample # 1815412 (containing only E. coli and uninfected human cells), was correctly reported “negative” by all of the 159 participants.

However, 28 laboratories reported false-negative results for the very weak positive sample # 1815414, containing the lowest amount of target organism (~1x103 genome copies/mL). Due to the relatively low amount of target organisms, results were not involved in the review for the certificates, but the 28 affected laboratories are encouraged to improve their diagnostic workflow or to check the analytical sensitivity of their PCR/NAT assays. Comparable results from some of our previous EQAS distributions (November 2016 and November 2011) let assume, that the lower detection limit of current M. pneumoniae-specific PCR/NAT assays is in the range between 5x102 to 102 M. pneumoniae genome copies/mL in appropriate sample material.

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: Attachment 1 [Attach. 1], p. 18) contained two samples with different amounts of C. burnetii DNA (~1x103 genome copies/mL in sample # 1815423 and ~1x105 genome copies/mL in sample # 1815424) and two samples with different amounts of B. anthracis strain UR-1 DNA (~1x104 genome copies/mL in sample # 1815423 and ~1x105 genome copies/mL in sample # 1815421). Sample # 1815422 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 [Attach. 1], p. 18) for the C. burnetii-specific results and tables 4 and 5 (Attachment 1 [Attach. 1], p. 19) for the B. anthracis-specific results.

Coxiella burnetii: The relatively high amount (1x105 genome copies/mL) of C. burnetii organisms in sample # 1815424 was correctly reported by all participants. The hundred-fold lower concentration of the pathogen in sample # 1815423 was correctly identified by 42 of the 44 participating laboratories. The two “negative” samples (# 1815421 containing only B. anthracis and # 1815422 containing only E. coli) 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: With the exception of one “questionable” result, all participants correctly reported negative results for the samples # 1815422 and # 1815424 which did not contain the target organism. The “positive” sample # 1815421 containing ~1x105 genome copies/mL B. anthracis strain “UR-1” was correctly reported by all 25 participants. The second positive sample # 1815423 (~1x104 genome copies/mL of B. anthracis strain “UR-1”) was correctly reported by 23 of the 25 participants. With the completion of 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 (udo.reischl@ukr.de).

RV 543: Francisella tularensis & Brucella spp.

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. 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, Attachment 1 [Attach. 1], p. 20) contained two samples with different amounts of Francisella tularensis subsp. tularensis DNA (~1x105 CFU/mL in sample # 1815432 and ~1x104 CFU/mL in sample # 1815434) two samples with different amounts of Brucella melitensis DNA (~1x105 CFU/mL in sample # 1815431 and ~1x104 CFU/mL in sample # 1815434). Sample # 1815433 contained only human cells and a considerable amount of E. coli organisms.

Francisella tularensis: Similar to QC samples from past distributions, the positive samples # 1815432 (~1x105 CFU/mL of Francisella tularensis spp. tularensis) and # 1815434 (~1x104 CFU/mL of Francisella tularensis spp. tularensis) were correctly tested positive by 25 and 26 of the 27 participating laboratories, respectively. Notably, one laboratory reported a ‘questionable’ result for the ‘negative’ samples # 1815431 and # 1815433. This should prompt an assessment of the performance of the test system used and the process of sample preparation.

Brucella spp.: The ‘positive’ samples # 1815431 and # 1815434 were correctly reported by 23 and 22 laboratories, respectively. In both samples without target organism (# 1815432 and #1815433), a total of 3 ‘questionable’ results were reported. 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 tab. 1 (Attachment 1 [Attach. 1], p. 22), the current set contained four samples with different carbapenem-resistant Enterobacteriaceae: sample # 1815441 contained a Klebsiella pneumoniae with a OXA-181 gene (~1x107 genome copies/mL), sample # 1815442 contained an Escherichia coli with a NDM-5 gene (~1x107 genome copies/mL), sample # 1815443 contained Klebsiella pneumoniae with a IMP-1 gene (~1x107 genome copies/mL) and sample # 1815444 contained a Klebsiella pneumoniae isolate with a KPC-2 gene (~1x107 genome copies/mL).

85 of the 88 participating laboratories reported sample # 1815441 (K. pneumoniae carrying a OXA-181 carbapenemase) as “carbapenemase positive”. Notably, all but 2 participants were able to detect carbapenemase genes in sample # 1815442 (E. coli carrying NDM-5). The third “positive” sample # 1815444 (containing K. pneumoniae with a KPC-2 gene) was correctly reported by 87 of the 88 participants. Apparently, not all test systems used were able to detect IMP-1, as 17 participants incorrectly reported ‘negative’ results for the IMP-1-carrying K. pneumoniae (sample # 1815443).

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 # 1815451 with ~1x105 CFU/mL, and sample # 1815452 with ~1x104 CFU/mL. Samples # 1815453 and # 1815454 contained only human cells and a considerable amount of E. coli organisms. The “positive” samples # 1815451 and # 1815452 were correctly reported as “positive” all participating laboratories. For the two ‘negative’ samples # 1815453 and # 1815454, 2 and 1 ‘questionable’ results were reported, respectively. 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 # 1815463 of the current set contained a relatively high amount of Enterococcus faecium vanA (~1x105 CFU/mL) and sample # 1815462 contained an approximately ten fold lower amount of Enterococcus faecium vanB (~1x104 CFU/mL). Sample # 1815461 contained Enterococcus faecalis (~1x105 CFU/mL) and sample # 1815464 contained no target organisms but only human cells and E. coli cells. Of the 54 participating laboratories, 51 and 54 correctly reported positive results for the samples # 1815462 and 1815463, 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 #1815461 and # 1815464 in total only two incorrect results were reported. 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. As the current and the forthcoming distribution (November 2018) are classified as “pilot studies”, 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 19 registered participants are depicted in tab. 2 (Attachment 1 [Attach. 1], p. 25) and a good overall correlation between the expected results (tab. 1, Attachment 1 [Attach. 1], p. 25) and the reported results was observed. The report forms of forthcoming RV 547 distributions will contain an extra field for a simple 7-digit code, where participants can specify the theoretical pathogen spectrum of their individual assay concepts. This will help to fairly consider the broad spectrum of different commercial and in-house PCR/NAT assays regarding species coverage, differentiation and multiplex capabilities.

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. 26). A relatively high amount of Pneumocystis jirovecii (~1x105 organisms/mL) was present in sample # 1815601, an approximately tenfold lower amount of Pneumocystis jirovecii (~1x104 organisms/mL) was present in sample # 1815602 and an approximately hundredfold lower amount (~1x103 organisms/mL) was present in sample # 1815603. The set was completed by sample # 1815604 which contained only human cells and a considerable amount of E. coli organisms.

Sample # 1815601, which contained the highest amount of P. jirovecii target organisms (~1x105 organisms/mL) and sample # 1815602 with a ten-fold lower concentration of P. jirovecii, were both reported “positive” by 104 and 103 of the 105 participating laboratories, respectively. The sample containing the lowest amount of target organism (# 1815603) was correctly reported by 82 participants, 23 laboratories reported false-negative results. Admittedly, the amount of P. jirovecii was really low and we thus decided not to rate a negative result as ‘false negative’. Nevertheless, participants should be aware of the potential limitations of the test system used. The “negative sample” (# 1815604, containing only E. coli) was correctly classified “negative” by all but two participants. In case of false-positive results, this should definitely prompt investigations to control all processes involved in sample preparation and analysis in order to optimize the NAT assay used.


References

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