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 2017 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 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
  • 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:Doc03

doi: 10.3205/lab000026, urn:nbn:de:0183-lab0000265

This is the English version of the article.
The German version can be found at: http://www.egms.de/de/journals/lab/2017-8/lab000026.shtml

Published: July 19, 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 June 2017

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

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

The current set of QC samples contained three samples with different amounts of C. trachomatis (~5x104 IFU/mL in sample # 1715303, ~1x104 IFU/mL in sample # 1715301 and 5x103 IFU/mL in sample # 1715302) and two samples with different amounts of N. gonorrhoeae target organisms (~5x105 CFU/mL in sample # 1715302 and ~5x102 CFU/mL in sample # 1715303).

Despite relatively low amounts of C. trachomatis target organisms in the positive sample # 1715302, all but one of the 237 participants reported correct positive CT results. For the two samples with a two- or ten-fold higher amount of C. trachomatis (#1715301 and # 1715303), only 1 false-negative result was observed in the current distribution. Among the N. gonorrhoeae-specific results, false-negative results were reported by only 15 of the 237 participants for sample # 1715303, which contained a very low number of N. gonorrhoeae target organisms (5x102 CFU/mL) next to a high amount of C. trachomatis (5x104 IU/mL). Also 7 false-positive results for the two GO-negative samples were reported by participants. Assuming a sequential processing of the 4 individual samples of the current set, contamination events of the “GO-negative” sample “4” by target organism or PCR products of the positive samples “2” and/or “3” is by far not unlikely in the current sample constellation. So the observation of false-positive results should encourage the affected participants to review and optimize their DNA extraction procedure and their GO-specific NAT-based test system.

Since the amount of target organisms in the GO-positive samples # 1715302 and # 1715303 could not be considered as “extremely low”, false negative results should also encourage the corresponding participants to review and optimize their GO specific NAT-based assays (or at least the GO-specific components if they are using multiplex assay concepts).

Inhibition controls were included by all but one of the participants and no inhibitory events were reported. Overall, a very good diagnostic performance and no noticeable issues regarding sensitivity and specificity were observed for the C. trachomatis- and N. gonorrhoeae-specific NAT assays used by the 237 participants.

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 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 in a kind of dilution series: # 1715313 with ~5x104 IFU/mL of C. trachomatis target organisms, # 1715311 with ~1x104 IFU/mL of C. trachomatis target organisms, and sample # 1715314 with ~5x103 IFU/mL of C. trachomatis target organisms. Sample # 1715312 of the current set contained no target organisms but only human cells and E. coli cells.

As depicted in Tab. 2 (Attachment 1 [Attach. 1], p. 4), all of the results reported for the negative sample # 1715312 and the three CT-positive samples # 1715311, # 1715313 and # 1715314 were correct.

Also for the C. trachomatis-positive sample # 1715314, containing a relatively weak amount of target organisms, no false-negative result was observed among the 97 participants.

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 implemented and performed by all but one of the 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 97 participants.

RV 532: Bordetella pertussis

The current set of QC samples contained one sample with a relatively high amount of Bordetella pertussis (# 1715321; 1x105 CFU/mL), and three negative samples containing Bordetella parapertussis (# 1715324; 1x105 CFU/mL), Bordetella holmesii (# 1715323 with ~1x105 CFU/mL; IS481-positive B. holmesii strain!), as well as one sample containing only non-infected human cells and Escherichia coli (# 1715322).

The availability of well-established commercial or in-house NAT-assays has led to a high portion of correct results. Surprisingly, seven of the 155 participants reported false-negative results for the sample # 1715321 (B. pertussis, 1x105 CFU/mL). The amount of 105 CFU/mL of B. pertussis target organisms is significantly above the previously observed lower limit of detection for the corresponding PCR assays or test systems.

The B. parapertussis sample # 1715324 was tested false-positive by 5 participants and (as expected somehow) the B. holmesii sample #1715323 was tested false-positive by 103 of the 155 participants. Since it is well known that B. holmesii strains my contain copies of the most popular B. pertussis-target gene IS 481, the high rate of false-positives is not really surprising for the latter sample. Considering that the detection rate of the B. pertussis sample # 1715321 was very high (indicating a good performance of the B. pertussis-specific PCR/NAT assays), and IS481 is still one of the most practical and sensitive target genes, we have not scored those (false) positive results for the B. holmesii samples in the course of issuing the corresponding QC certificates. For colleagues who are interested in the IS481 topic, there is a recent paper [2].

However, for participants who have observed false-negative B. pertussis results with sample # 1715321 or false-positive B. pertussis results with Bordetella parapertussis in sample # 1715324, it is strongly recommended to initiate appropriate measures to improve the analytical specificity of their assay concepts. Except two false-positive results with the “negative” sample # 1715322 (presumably due to carry-over from the strong positive sample 1715321), all of the remaining results reported by the 155 participants were correct. Run controls were performed by 154 participants and inhibition events were not observed among the samples of the current distribution.

RV 533: Helicobacter pylori

The current set of QC samples contained three samples with a Clarithromycin-resistant Helicobacter pylori isolated from a patient in the course of an antibiotic therapy failure study in a kind of dilution series. Sample # 1715331 contained approximately 5x105 CFU/mL, sample # 1715332 approximately 5x104 CFU/mL and sample # 1715334 approximately 5x103 CFU/mL of the respective target organisms.

The availability of well evaluated NAT-based assays and the relatively high amount of target organisms in the three Helicobacter pylori-positive samples (#1715331, # 17153322, and # 1715334) led to positive predictive values of nearly 100%. Only one participant observed a false-negative PCR result for sample # 1715334. Also for the Helicobacter pylori-negative sample # 1715333 correctly negative PCR/NAT results were reported by nearly all the participating laboratories. Only one participant observed a false-positive PCR result.

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 39 of the 50 participants and all reported results of molecular susceptibility testing 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: # 1715342 (E. coli, 1x104 CFU/mL, clinical isolate, stx1-positive, stx2-positive, eae-positive and hlyA-positive) and # 1715341 (E. coli, 1x105 CFU/mL, clinical isolate, stx1-negative, stx2d-positive, eae-negative and hlyA-negative). The other two EHEC-negative samples contained a Shigella sonnei strain (sample # 1715343; 1x105 CFU/mL) and an eae- and hlyA-negative E. coli K12 strain (# 1715344).

All but one participant correctly reported negative results for sample # 1715344, containing only an eae-negative and hlyA-negative E. coli K12 strain. The second EHEC/STEC-“negative” sample (#1715343), containing a significant amount of a clinical Shigella sonnei isolate was also reported PCR-negative by all but four participants. For the EHEC/STEC positive samples # 1715341 and # 1715342, the availability of well-established NAT-based assays and strategies for molecular differentiation resulted in consistently high accuracy rates. The stx2d-positive EHEC sample # 1715341 was correctly reported positive by 124 of the 133 participants and 125 of the 133 participants detected the “classical” EHEC target organisms in the positive sample # 1715342 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 112 of the 133 participating laboratories. With two exceptions, 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 (EQAS) scheme 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. Of special interest – since of proven human pathogenicity and widely distributed in Europe – are B. burgdorferi sensu stricto, B. afzelii, B. garinii and B. bavariensis. B. spielmanii, a further species with proven pathogenicity for humans, seems to be rare and was so far only recovered from skin manifestations of Lyme borreliosis. B. bissettiae, B. lusitaniae und B. valaisiana are considered as potential human pathogens. Regarding OspA, especially B. garinii shows a striking heterogeneity with at least 5 genetic distinguishable “genotypes” in Europe.

The current set of QC samples contained a kind of dilution series of B. garinii organisms in our proprietary matrix: sample # 1715353 (5x105 CFU/ml), sample # 1715351 (5x104 CFU/ml) and sample # 1715354 (5x103 CFU/ml). Sample # 1715352 contained no target organisms but only human cells and E. coli cells.

With the exception of 13 false-negative results for sample # 1715354 (containing the lowest amount of the target organism), 3 false-negative and one “questionable” result for sample # 1715351 and 2 false-negative results for sample # 1715353 (containing the highest amount of the target organism) all of the remaining participants reported correctly results for the B. garinii-containing samples. The false-negative results should prompt re-evaluation of the assay sensitivity.

The “negative” sample # 1715352 was classified false-positive by only one laboratory. Potentially, this may be due to a contamination event from the positive sample # 1715351 during sample preparation or PCR/NAT analysis. Therefore, the workflow should be optimized to minimize clinically misleading false-positive results.

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

RV 536: Legionella pneumophila

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 # 1715361 (5x104 CFU/ml), sample # 1715364 (5x103 CFU/ml) and sample # 1715363 (5x102 CFU/ml). Sample # 1715362 contained no target organisms but only human cells and E. coli cells.

The L. pneumophila-positive samples # 1715361 (~5x104 CFU/mL) and # 1715364 (~5x103 CFU/mL) were correctly tested positive by all and by 106 of the 119 participating laboratories, respectively. For the third positive sample within the current distribution, # 1715363, which contained a very low amount of L. pneumophila-target organisms (~5x102 CFU/mL), only 62 of the 119 participants reported a correctly positive result.

With a target organism load of below 103 CFU/mL of L. pneumophila, the lower limit of detection of appropriate test systems is obviously clearly 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 sample # 1715364 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 # 1715362 which contained only E. coli, was classified as false-positive by 2 of the participating laboratories. This is probably due to contamination events in the course of sample preparation or PCR/NAT amplification. All but one of the 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 Typhimurium: sample # 1715371 contained 5x104 CFU/ml, sample # 1715374 contained 5x103 CFU/ml and sample # 1715372 contained 5x102 CFU/ml. Sample # 1715373 contained no target organisms but only human cells and E. coli cells.

Sample # 1715371, containing the highest amounts of Salmonella enterica target organisms, was reported correctly by all of the 27 participants. Only two false-negative result was reported for the weak positive sample # 1715374, and two false-positive results were reported for the “negative” sample # 1715373. Even sample # 1715372, containing very low amounts of Salmonella enterica organisms (5x102 CFU/ml) was reported correctly positive by 21 of the 27 participants. This indicates a remarkably high analytical sensitivity of the current Salmonella enterica-specific PCR assays and an improved procedure with regard to the prevention of contamination events during the individual sample preparation and PCR/NAT analytics in the participating diagnostic laboratories.

Assuming a sequential processing of the 4 individual samples of the current set, contamination events of the negative sample “3” by target organism or PCR products of the positive samples “1” and/or “2” is by far not unlikely in the current sample constellation. So the observation of false-positive results for sample # 1715373 in the laboratory of 2 participants should encourage them to review and optimize their DNA extraction procedure and their specific PCR/NAT-based test system.

RV 538: Listeria spp.

The current set of QC samples contained a sample without the corresponding target organisms (# 1715382; only E. coli cells), two samples positive for L. monocytogenes (# 1715381 with ~5x104 CFU/mL and # 1715383 with ~5x105 CFU/mL) and one sample with Listeria ivanovii (# 1715384) as a Listeria species other than L. monocytogenes. The Listeria monocytogenes-containing samples (# 1715381 and # 1715383) were correctly reported positive by all but one participant. In addition, the “negative” E. coli-containing sample # 1715382 was correctly identified as negative by all laboratories. Thirty-five of the 46 participants indicated the use of Listeria monocytogenes-specific PCR/NAT assays, which is reflected by the high number of “false-negative” results for sample # 1715384, containing 1x105 CFU/mL L. ivanovii. 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.

Samples # 1715394 and # 1715391 of the current distribution contained a typical clinical MRSA isolate (MRSA, PVL-negative; ~1x105 CFU/mL and ~1x104 CFU/mL, respectively) whereas sample # 1715392 contained a significant amount of a so called “mecA dropout” MSSA isolate (~1x104 CFU/mL). Only E. coli and human cells were present in sample # 1715393 of the current distribution.

The MRSA-negative sample # 1715393 was correctly reported negative by 296 of the 302 participants. Only three participants reported false-positive results, presumably due to intra-laboratory contamination events from the positive sample # 17153931 during sample preparation, amplification or detection. The MRSA-positive samples # 1715391 and # 1715394 were correctly reported positive by almost all of the 302 participants. One false-negative result was observed for sample # 1715391. Three false-negative results were reported for MRSA sample # 1715394 and one participant classified his/her result as “questionable”. Affected participants are encouraged to analyse and optimize their NAT-based assays, because the amount of MRSA target organisms in the positive samples, especially # 1715394 (1x105 CFU/mL), was not abnormally low.

The apparently “bad” performances for the MRSA-negative but MSSA-positive sample # 1715392 are quickly explained on closer inspection. Sample # 1715392 contained one of the yet still relatively rare S. aureus strain, that belongs to the group of so-called mecA dropout MSSA isolate: Oxacillin sensitive S. aureus strains which contain the MRSA-typical SCCmec cassette, but significant parts or the entire mecA gene are deleted on genomic level. Consequently only 254 of the 302 participants reported correct negative MRSA results for this tricky sample.

Compared to the some previous rounds of PCR/NAT external quality assessment for MRSA, a much better diagnostic performance was observed for these variant S. aureus genetic constellations. Similar mecA dropout variants, which have been sent out formerly in the November 2012 and November 2015 distributions, were detected by 30% (2012) and by 53% (2015) of participants who reported correctly MRSA negative results in the previous distributions. In the current distribution, 84% of the participants reported correct MRSA-negative results. This situation nicely reflects the various (and obviously successful) efforts of diagnostic companies and in-house assay development teams to continuously improve and adopt their protocols to the current challenges of direct PCR/NAT testing for MRSA.

Also, an optional molecular detection of putative pathogenicity factor PVL (Panton-Valentine Leukocidin) or its coding gene lukF/S-PV was inquired. Corresponding results were reported by 92 of the 302 participating laboratories and within the current distribution, the results for the molecular PVL testing were correct in all but three cases. Additional information can be found at: Linde et al. [8] or Witte, W. et al. [9]. A well evaluated protocol for the detection of PVL-positive PVL isolate can be found at Reischl et al. [10].

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

RV 540: Chlamydia pneumoniae

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

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 # 1715402 contained about 5x105 IFU/mL, sample # 1715404 about 5x104 IFU/mL and sample # 1715403 about 1x104 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 # 1715401 of the current set.

As depicted in Tab. 2 (Attachment 1 [Attach. 1], p. 13), all participants reported correct results for the positive sample # 1715402. 132 of the 134 participants also reported correct positive results for sample # 1715404, and also for the sample with the lowest amount of C. pneumonia (# 1715403; 1x104 IFU/mL) 129 correct results were reported. Only one participant reported false-positive results for the “negative” sample # 1715401 (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 want to mimic the situation of processing typical clinical samples like BAL or other respiratory materials. 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. 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 # 1715412 and an approximately tenfold lower amount of M. pneumoniae (~1x104 genome copies/mL) was present in sample # 1715413. Sample # 1715414 was designed to monitor assay specificity: it contained a considerable amount of Haemophilus influenzae (~1x104 CFU/mL), a species of a genus related to Mycoplasma. The set was completed by sample # 1715411, which contained only human cells and a considerable amount of E. coli organisms.

As also observed during the past distributions of our EQAS scheme 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. Among the M. pneumoniae-specific results reported by the 149 participants, all laboratories reported correct positive results for the relatively high positive sample # 1715412, but 15 participants reported false-negative results for sample # 1715413, which contained 1x 104 genome copies/mL.

Sample # 1715414, which contained Haemophilus influenzae, was tested correctly negative by 147 of the 149 participants. Two participants reported false-positive results for the Haemophilus influenzae sample, which could be due to shortcomings in analytical specificity or just cross-contamination events in the course of sample preparation, amplification or amplicon detection steps. The affected laboratories are encouraged to improve their diagnostic workflow or to check the analytical specificity of their PCR/NAT assays.

Sample # 1715411 of the current set contained no target organisms but only non-infected human cells and E. coli cells. As no false-positive results were observed for the “negative” sample, it seems that the participating laboratories have implemented functional precautions to prevent deleterious contamination events. No inhibitory events or other noticeable problems with the current set of QC samples and a good overall correlation with the expected results were observed.

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. 15) contained two samples with different amounts of C. burnetii organisms (~1x104 genome copies/mL in sample # 1715423 and ~1x105 genome copies/mL in sample # 1715421), one sample with ~1x105 genome copies/mL of B. anthracis strain UR-1 (sample # 1715421) and one sample with ~1x106 genome copies/mL of a B. anthracis STI vaccine strain (sample # 1715424). Sample # 1715422 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. 15) for the C. burnetii-specific results and Tables 4 and 5 (Attachment 1 [Attach. 1], p. 16) for the B. anthracis-specific results.

Coxiella burnetii: The relatively high amount (1x105 genome copies/mL) of C. burnetii organisms in sample # 1715421 was correctly reported by all participants. The ten-fold lower concentration of the pathogen in sample # 1715423 was correctly identified as “positive” by 38 of the 39 participating laboratories. The participant reporting a questionable result should reassess the performance of the test system used and evaluate processes of sample workup and analysis. The two “negative” samples (# 1715422 contained only E. coli and # 1715424 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 # 1715422 and # 1715423 which did not contain the target organism. The “positive” sample # 1715421 containing ~1x105 genome copies/mL B. anthracis strain “UR-1” was correctly reported by all 22 participants. The second positive sample # 1715424 (~1x106 genome copies/mL of B. anthracis strain “STI”) was correctly reported. This particular strain is positive for the B. anthracis-specific markers rpoB (or dhp61) and pagA and also contains the “protective antigen, lethal and edema factor” encoding plasmid pXO1, but not the virulence plasmid pXO2. 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 (U. Reischl).

RV 543: Francisella tularensis

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

The current set of QC samples contained three positive samples: a high amount of Francisella tularensis spp novicida (~1x105 CFU/mL) was present in sample # 1715434, an approximately ten-fold lower amount of Francisella tularensis spp. holarctica (~1x104 CFU/mL) was present in sample # 1715432, and a relative high amount of Francisella tularensis spp. tularensis (~1x105 CFU/mL) was present in sample # 1715431.

Similar to QC samples from past distributions, the positive samples # 1715431 (~1x105 CFU/mL of Francisella tularensis spp. tularensis) and # 1715434 (~1x105 CFU/mL of Francisella tularensis spp. novicida) were correctly tested positive by 25 and 24 of the 26 participating laboratories, respectively. Even with pathogen amounts of ~1x104 CFU/mL (sample #1715432) 18 out of 26 labs were able to detect Francisella DNA. As no false-positive result was observed for the “negative” sample # 1715433, it seems that the participating laboratories have implemented functional precaution measures to prevent deleterious contamination events. Overall, these results corroborate the lower limits of detection observed in our previous EQAS distributions. Although the number of participating laboratories is still limited, the results of the present distribution indicate that the lower limit of detection is about or slightly below 104 organisms/mL when using currently employed and well evaluated PCR/NAT-based assay concepts for the detection of F. tularensis DNA.

RV 544: Carbapenemase genes

The concept of this novel EQAS-panel for the detection of carbapenemase genes is designed exclusively for the testing of NAT-based methods and protocols for molecular resistance testing or the direct detection of carbapenemase genes from DNA preparations of Enterobacteriaceae culture isolates. Because of the methodologically challenging design of EQAs for the molecular resistance testing of the wide range of known carbapenemase coding genes in different bacteria, the panel is narrowed down to a small selection of the currently most common carbapenemase genes in Enterobacteriaceae: KPC, VIM, OXA-48 like genes, GES carbapenemases, NDM, IMP, and GIM. As shown in Tab. 1 (Attachment 1 [Attach. 1], p. 18), the current set contained three samples with different carbapenem-resistant Enterobacteriaceae: sample # 1715441 contained Klebsiella pneumoniae with a VIM-2 gene (~1x107 genome copies/mL), sample # 1715442 contained an Klebsiella pneumoniae isolate with two carbapenemase genes: KPC-2 and OXA-48 (~1x107 genome copies/mL), and sample # 1715444 contained an Serratia marcescens isolate with a NDM-1 gene. The fourth sample # 1715443 was designed as negative control and contained only E. coli without carbapenemase genes.

74 of the 77 participating laboratories reported sample # 1715441 (K. pneumoniae carrying a VIM-2 carbapenemase) as “carbapenemase-positive”. Notably, all but 2 participants were able to detect carbapenemase genes in sample # 1715442 (K. pneumoniae carrying KPC-2 and OXA-48), but six laboratories missed the KPC-2 gene. The third “positive” sample # 1715444 (containing Serratia marcescens with a NDM-1 gene) was correctly reported by 76 of the 77 participants. A false-negative result for carbapenemase gene-positive samples 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 three Clostridium difficile positive samples: sample # 1715451 with ~1x105 CFU/mL and samples # 1715453 and # 1715454 with ~1x104 CFU/mL. Sample # 1715452 contained only human cells and a considerable amount of E. coli organisms.

The sample # 1715451 containing a relatively high amount of C. difficile (1x105 CFU/mL) was correctly reported as “positive” all of the 130 participating laboratories. Additionally, the two samples containing a ten-fold lower amount of the target organism (# 1715453 and # 1715454) were correctly identified by 128 and 129 participant, respectively. False-negative results should prompt a thorough evaluation of the test system and the workflow. The latter is definitely warranted for the participants reporting a false-positive result for sample # 1715452, 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. With one exception, 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 three vancomycin-resistant Enterococcus strains: an Enterococcus faecalis vanA positive strain (# 1715461, ~1x105 CFU/mL), an Enterococcus faecium vanB positive strain (# 1715462, ~1x105 CFU/mL), and an Enterococcus avium vanA positive strain (# 1715463, ~1x105 CFU/mL). Sample # 1715464 contained no target organisms but human cells and E. coli cells.

48 and 47 of the 50 participating laboratories reported correct results for the “positive” samples # 1715461 and #1715462, respectively. The vanA-positive E. avium strain was also correctly reported as “vancomycin-resistant” by 43 of the 50 participants. Of note, the reported dedicated vanA/vanB identifications for these three samples were all correct. We were pleased to see that also for the “negative” sample #1715464, 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. With one exception, 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: Attachment 1 [Attach. 1], p. 22). A relatively high concentration of Pneumocystis jirovecii (~1x105 CFU/mL) was present in sample # 1715601, whereas in sample # 1715603 Pneumocystis jirovecii (~1x104 CFU/mL) was present at an approximately ten-fold lower concentration. The set was completed by samples # 1715602 and # 1715604 which contained only human cells and a considerable amount of E. coli organisms.

Sample # 1715601, which contained P. jirovecii target organisms (~5x105 CFU/mL) at highest concentration and sample # 1715603 with a ten-fold lower concentration of P. jirovecii, were both reported “positive” by 102 and 100 of the 102 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” (# 1715602 and # 1715604, containing only E. coli) were correctly classified “negative” by 101 and 100 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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