gms | German Medical Science

GMS Zeitschrift zur Förderung der Qualitätssicherung in medizinischen Laboratorien

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

ISSN 1869-4241

Bacterial and fungal genome detection PCR/NAT: discussion of the May 2016 distribution for external quality assessment of nucleic acid-based protocols in diagnostic medical microbiology by INSTAND e.V.

Report

  • corresponding author Udo Reischl - Institute for Clinical Microbiology and Hygiene, University Hospital Regensburg, Germany
  • Wulf Schneider - Institute for Clinical Microbiology and Hygiene, University Hospital Regensburg, Germany
  • Martin Ehrenschwender - Institute for Clinical Microbiology and Hygiene, University Hospital Regensburg, Germany
  • Andreas Hiergeist - Institute for Clinical Microbiology and Hygiene, University Hospital Regensburg, Germany
  • Matthias Maaß - Labor Dr. Heidrich und Kollegen MVZ GmbH, Hamburg, Germany
  • Michael Baier - Institute of Medical Microbiology, University Hospital of the Friedrich Schiller University of Jena, Germany
  • Eberhard Straube - Institute of Medical Microbiology, University Hospital of the Friedrich Schiller University of Jena, Germany
  • Dimitrios Frangoulidis - Bundeswehr Institute of Microbiology, Munich, Germany
  • Gregor Grass - Bundeswehr Institute of Microbiology, Munich, Germany
  • Heiner von Buttlar - Bundeswehr Institute of Microbiology, Munich, Germany
  • 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
  • Martin Kaase - National Reference Laboratory for multidrug-resistant gram-negative bacteria, Department for Medical Microbiology, Ruhr-University Bochum, Germany

GMS Z Forder Qualitatssich Med Lab 2016;7:Doc03

doi: 10.3205/lab000023, urn:nbn:de:0183-lab0000232

This is the English version of the article.
The German version can be found at: http://www.egms.de/de/journals/lab/2016-7/lab000023.shtml

Published: July 5, 2016

© 2016 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 May 2016

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 (~1x105 IFU/mL in samples # 1615303 and # 1615304, and ~1x104 IFU/mL in sample # 1615301) and two samples with different amounts of N. gonorrhoeae target organisms (~106 CFU/mL in sample # 1615301 and ~103 CFU/mL in sample # 1615303).

Despite relatively low amounts of C. trachomatis target organisms in the positive sample # 1615301, all of the 216 participants reported correct positive CT results. For the two samples with a ten-fold higher amount of (# 1615303 and # 1615304), only 1 false-negative result was observed in the current distribution. Among the N. gonorrhoeae-specific results, false-negative results were reported by only 9 of the 216 participants for sample # 1615303, which contained a low number of N. gonorrhoeae target organisms (1x103 CFU/mL) next to a high amount of C. trachomatis (1x105 IU/mL). Also 10 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” samples “2” and “4” by target organism or PCR products of the positive samples “1” 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 sample # 1615303 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 216 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 Tab. 4 and 5 (Attachment 1 [Attach. 1], p. 2) only the C. trachomatis (CT) specific results and in Tab. 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: # 1615311 and # 1615314 with ~1x105 IFU/mL of C. trachomatis target organisms, and sample # 1615312 with ~1x104 IFU/mL of C. trachomatis target organisms. Sample # 1615313 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 # 1615313 and two of the positive samples # 1615311 and # 1615314 were correct.

For the C. trachomatis-positive sample # 1615312, containing a slightly weaker amount of target organisms, one false-negative result was observed among the 95 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 95 participants.

RV 532: Bordetella pertussis

The current set of QC samples contained two identical samples with a relatively high amount of Bordetella pertussis (# 1615321 and # 1615324; 1x105 CFU/mL), one negative sample containing Bordetella holmesii (# 1615322; 1x106 CFU/mL, IS481-positive strain !), as well as one sample containing only non-infected human cells and Escherichia coli (# 1615323).

The availability of well-established commercial tests or in-house PCR/NAT-assays has led to a high portion of correct results. Although none of the 152 participants reported false-negative results for the sample # 1615321 (B. pertussis, 1x105 CFU/mL), very surprisingly two participants observed negative results for the almost identical sample # 1615324 of the current distribution. Analytical sensitivity issues can be ruled out since 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.

Only one false-positive result was observed for the negative sample # 1615323 (E. coli) whereas the B. holmesii sample was tested false-positive by 101 of the 152 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 samples # 1615321 and # 1615324 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. Colleagues who are interested in the IS481 topic may read the following article: Njamkepo et al. (2011) [2].

However, for participants who have observed false-positive B. pertussis results with sample # 1615323 or false-negatives with sample # 1615324, it is strongly recommended to initiate appropriate measures to improve the analytical specificity and/or sensitivity of their assay concepts. All of the remaining results reported by the 152 participants were correct. Run controls were performed by 150 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 two samples with a clarithromycin-susceptible Helicobacter pylori patient strain. Sample # 1615331 contained approximately 1x105 CFU/mL and sample # 1615333 approximately 1x103 CFU/mL of the respective target organisms. Sample # 1615333 contained culture suspensions of the related species Helicobacter felis (~ 1x103 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 # 1615331 and # 1615333 led to very high positive predictive values.

Two false-negative results were observed among the 48 participants for sample # 1615333, which contained only a very low amount of H. pylori target organisms (1x103 CFU/mL) next to a significant number of E. coli cells within our proprietary sample matrix. Of note, five false-positive results and one result classified as “questionable” by the participant were reported for sample 1515332, containing ~1x103 CFU/mL Helicobacter felis. This may be indicative of “simple” DNA or sample contamination events originating from the strong positive sample # 1615333 or an insufficiently low level of analytic specificity of the applied PCR/NAT assays. Both possible reasons should prompt re-evaluation in the laboratories of the affected participants.

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 38 of the 48 participants. With one exception, the results were correct.

RV 534: EHEC/STEC

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

The current set of QC samples contained two samples positive for EHEC: # 1615342 (E. coli, 5x105 CFU/mL, clinical isolate, stx1-, stx2-, eae-, hlyA- and O157-positive) and # 1615344 (E. coli, 5x104 CFU/mL, clinical isolate, stx2c- and eae-positive). The other two EHEC-negative samples contained an EPEC strain (sample # 1615341; 5x104 CFU/mL) and an EIEC strain (sample # 1615343; 5x104 CFU/mL).

All but 2 participants correctly reported negative results for sample # 1615341, containing no EHEC target organisms but only relatively high numbers of an eae-positive EPEC strain. The other “EHEC-negative” sample (#1615343), containing a significant amount of EIEC isolate was also reported PCR-negative by all but two participants. For the EHEC/STEC positive samples # 1615342 and # 1615344, the availability of well-established NAT-based assays and strategies for molecular differentiation resulted in consistently high accuracy rates. Sample # 1615342 was correctly reported positive by 124 of the 125 participants and, again, all but one of the 125 participants detected the target organisms in the EHEC/STEC-positive sample # 1615344 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 125 participating laboratories. With one exception, the reported results were correct. None of the participants observed significant inhibition of the NAT-reaction.

RV 535: Borrelia burgdorferi

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

The current set of QC samples contained a kind of dilution series of B. afzelii organisms in our proprietary matrix: sample # 1615353 (1x105 B. afzelii organisms/mL), sample # 1615351 (1x104 organisms/mL) and sample # 1615354 (1x103 organisms/mL). Sample # 1615352 contained no Borrelia organisms but a strain of Treponema phagedenis.

With the exception of 3 false-negative results and one result classified as “questionable” for sample # 1615354 (containing the lowest amount of target organisms), and one false-negative result for sample # 1615353 (containing the highest amount of target organisms) all other B. afzelii-containing samples were correctly identified by the 121 participating laboratories. The false-negative result for sample # 1615353 should prompt re-evaluation of the assay sensitivity.

The “negative” sample # 1615352 was classified false-positive by three laboratories. Potentially, this is either due to cross-reactivity with this closely related spirochete or due to a contamination during sample preparation or 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

Due to numerous requests: this test is designed exclusively for the testing of NAT-based methods and protocols for direct detection of low amounts of Legionella pneumophila from appropriate clinical specimen (such as respiratory specimens for example). Individual samples may contain relatively small amounts of the corresponding target organism. For this reason, participation is promising only for diagnostic laboratories, which have established a highly sensitive and specific PCR-/NAT-based method for the detection of L. pneumophila DNA or who want to evaluate their method with the help of an external quality control.

The current set of QC samples contained only one positive sample with Legionella pneumophila serogroup 1 (# 1615361; ~1x105 CFU/mL) next to two samples containing Legionella longbeachae (# 1615364; ~1x105 CFU/mL and # 1615363; ~1x104 CFU/mL). Sample # 1615362 contained no target organisms but only human cells and E. coli cells.

The L. pneumophila-positive (~1x105 CFU/mL) sample # 1615361 was correctly tested positive by all of the 117 participating laboratories. Both of the L. longbeachae-positive samples (# 1615363 and # 1615364) were correctly tested negative by 109 participants. Sample # 1615362, which contained only E. coli, was tested false-positive by 3 participants whereas the remaining 114 participants reported correct negative PCR/NAT results for L. pneumophila DNA. The overall result constellation indicates that at least 3 participants may have contamination problems (false-positive results for sample # 1615361) and some more participants may have specifictity problems (false-positive results for samples # 1615363 and # 1615364) with their PCR/NAT-assays. 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 two samples with Salmonella enterica serovar enteritidis (sample # 1615371 with 1x106 CFU/ml, and sample # 1615372 with 5x104 CFU/ml). Sample # 1615373 contained Salmonella enterica serovar tennessee (with 1x105 CFU/ml) and sample # 1615374 contained no target organisms but only human cells and E. coli cells.

All of the 25 participants reported correct Salmonella enterica positive PCR/NAT-results for sample #1615371 and correct results for the negative sample # 1615374. Sample #1615373, containing ~5x105 CFU/mL Salmonella enterica serovar tennessee, was correctly identified as “positive” by 24 of the 25 participants. Reporting a false-negative result for this sample should prompt a thorough re-evaluation of the performance of the test system. The weak-positive sample #1615372, containing only ~5x104 CFU/mL of Salmonella enterica serovar enteritidis, was correctly reported “positive” by 17 of the 25 participants. Affected participants may consider to improve the analytical sensitivity of their PCR/NAT assay concepts.

Inhibitory events in the PCR-/NAT-reaction were not detected by any of the participants.

RV 538: Listeria spp.

The current set of QC samples contained a sample without the corresponding target organisms (# 1615383; only E. coli cells), two samples positive for L. monocytogenes (# 1615382 and # 1615384 with ~1x105 CFU/mL and one sample with Listeria innocua (# 1615381) as Listeria species other than L. monocytogenes. The results discussion of the current distribution is easy: all of the 39 participating laboratories detected the Listeria monocytogenes target organisms in the two (relatively strong) positive samples # 1615382 and # 1615384. In addition, the “negative” E. coli containing sample # 1615383 and the sample containing only Listeria innocua (# 1615381) were identified as negative by all laboratories. All of the participants used Listeria monocytogenes-specific assays, which is reflected by the high number of “false-negative” results for sample # 1615381. 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. Except of one sample containing an MSSA isolate together with almost equal amounts of a methicillin-resistant mecA-positive coagulase negative Staphylococcus species, no “difficult” or “interesting” Staphylococcal strains were included into the current panel.

Sample # 1615391 of the current distribution contained a mixture of S. aureus (MSSA, PVL-negative, ~1x105 CFU/mL) and a CoNS strain (S. epidermidis; mecA-positive, ~1x105 CFU/mL).

Sample # 1615394 contained a cMRSA or CA-MRSA isolate (MRSA, PVL-positive, spa:t 008; ~1x105 CFU/mL) which tested positive with the MRSA-specific assays in 313 participating laboratories.

Sample # 1615392 contained a cMRSA isolate (MRSA, PVL-positive, spa:t 310; ~1x104 CFU/mL) which tested positive with the MRSA-specific assays in 313 participating laboratories. One sample of the current set (# 1615393) contained no MRSA target organisms but only a clinical isolate of an oxacillin-sensible CoNS strain (S. epidermidis; mecA-negative, ~1x103 CFU/mL).

The MRSA negative sample # 1615393 was tested by 307 of 310 participants with their PCR-based MRSA-specific test systems as “negative”, so only three participants observed a false positive result for sample # 1615393, which may have probably been caused by contamination with MRSA DNA during the sample preparation, amplification or detection. Fortunately, for the relatively strong positive cMRSA sample # 1615394, positive results were reported by almost all participants. Even if sample # 1615392 contained a slightly lower amount of MRSA target organisms (1x104 CFU/mL), correct positive results were still reported by 307 participants, leading to positive predictive values of above 99%. The 2 false negative results and one result classified as “questionable” by the participating laboratory are probably due to an insufficient analytical specificity of the used in-house or commercial PCR/NAT test systems.

For the sample # 1615391, which contained a MSSA isolate together with a methicillin-resistant coagulase-negative Staphylococcus species, 286 of 310 participants reported their results correctly as “MRSA-negative” and 7 participants classified the results as “questionable”. Five of these 7 participants indicated the use of test systems, which are based on a separate detection of the mecA gene and S.aureus specific target genes. With this separate detection assays, the origin of the mecA target gene cannot definitively be correlated with the S. aureus or the coagulase-negative Staphylococcus species. Regarding this aspect, “questionable” is the scientifically correct result in the case when using duplex PCR/NAT assays, which are correlating quantitative results and using a kind of software algorithm for confirming or ruling out the presence of mecA-positive S. aureus organisms in the investigated sample. The remaining 17 participants reported false-positive results for MRSA for sample # 1615391, containing a mixture of a MSSA isolate and a methicillin-resistant coagulase-negative Staphylococcus species. These participants are encouraged to analyse the appropriateness of PCR/NAT their test systems or assay concepts since the simultaneous presence of MSSA ond mecA-positive CoNS organisms is a relatively common scenario for microbiological routine diagnostics of nasal swabs. On the other hand, all participants, who used SCCmec based test systems, reported correct MRSA-negative results for sample # 1615391.

Overall, it should be noted that a pleasingly large proportion of participants reported correct results for all 4 samples of the current EQAS distribution. 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 63 of the total 310 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. (2005) [5] or Witte et al. (2005) [6]. A well evaluated protocol for the detection of PVL-positive PVL isolate can be found at: Reischl et al. (2007) [7].

In addition, commercial real-time PCR assays reliably targeting PVL-genes in MRSA and MSSA isolates are available from a number if diagnostic companies in the meantime (e.g., r-biopharm or TIB Molbiol).

RV 540: Chlamydia pneumoniae

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

The current set of QC samples contained two samples positive for C. pneumoniae. Sample # 1615404 was spiked with ~5x105 IFU/ml of C. pneumoniae, whereas sample # 1615401 contained an approximately ten fold lower amount of C. pneumoniae (~5x104 IFU/ml). Sample # 1615403 contained significant numbers of C. trachomatis (~1x104 IFU/ml). Only E. coli and human cells were present in sample # 1615402.

As depicted in Tab. 2 (Attachment 1 [Attach. 1], p. 14), with two exceptions all participants reported correct results for the positive sample # 11615404. However, for the second sample containing a tenfold lower amount of C. pneumoniae (# 1615401) 10 false-negative results were reported. For the “negative” samples # 1615403 and # 1615402 (containing Chlamydia trachomatis and E. coli, respectively), 129 and 130 of 131 laboratories reports correct negative results. False-positive results are most probably due to (cross-)contamination during sample processing and extraction, as cross-reactivity of NAT/PCR-based assays specific for Chlamydia pneumoniae with Escherichia coli or Chlamydia trachomatis is unlikely. Certainly, a false-positive result should prompt investigations and improvement of the diagnostic workflow.

RV 541: Mycoplasma pneumoniae

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

The current set of QC samples contained three positive samples. A relatively high amount of M. pneumoniae (~1x106 genome copies/mL) was present in sample # 1615412. An approximately tenfold lower amount of M. pneumoniae (~1x105 genome copies/mL) was present in sample # 1615414 and sample # 1615413 contained an approximately hundred fold lower amount of M. pneumoniae (~1x104 genome copies/mL). The set was completed by sample # 1615411, which contained only human cells and a considerable amount of E. coli. With the exception of one laboratory, all participants correctly reported samples # 1615412 and # 1615414 as positive. However, 7 laboratories reported false-negative results for sample # 1615413, containing the lowest amount of target organism (~1x104 genome copies/mL). The E. coli (# 1525413) containing sample was correctly reported “negative” by all 143 participants.

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 contained two samples with different amounts of Coxiella burnetii organisms (~5x103 genome copies/mL in sample # 1615423 and ~5x104 genome copies/mL in sample # 1615421), one sample with ~1x103 genome copies/mL of Bacillus anthracis (sample # 1615422) and one sample with ~1x104 genome copies/mL of a Bacillus anthracis Pasteur Strain (sample # 1615423). Sample # 1615424 contained only human cells and a considerable amount of E. coli organisms.

For convenient data presentation and analysis, we decided to depict the PCR/NAT-results for each target organisms within this combined EQAS scheme in two separate tables: please see Tab. 2 and 3 (Attachment 1 [Attach. 1], p. 16 ) for the Coxiella burnetii-specific results and Tab. 4 and 5 (Attachment 1 [Attach. 1], p. 17) for the Bacillus anthracis-specific results.

Coxiella burnetii: The relatively high amount (5x104 genome copies/mL) of C. burnetii organisms in sample # 1615421 was correctly reported by all participants. The ten-fold lower concentration of the pathogen in sample # 1615423 was correctly identified as “positive” by 32 of the 34 participating laboratories. False-negative results should prompt a reassessment of the performance of the test system used and evaluation of processes regarding sample workup and analysis. The two “negative” samples (# 1615424 contained only E. coli and # 1615422 contained only B. anthracis) were correctly reported negative by 34 and 32 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.

Bacillus anthracis: All participants correctly reported negative results for the sample # 1615424 which did not contain the target organism. For the second “negative” sample (# 1615421), we received one false-positive result. The “positive” sample # 1615422 containing ~1x103 genome copies/mL B. anthracis strain “UR-1” was correctly reported by 17 of the 20 participants. Additionally, we received one questionable result and two false-negative results, which should definitely prompt investigations regarding sample workup and performance of the test system, as the amount of the target organism in the sample is still above the lower limit of detection.

With the exception of three participants, the second positive sample # 1645423 (~1x106 genome copies/mL of B. anthracis strain “Pasteur”) was correctly reported. This particular strain is positive for the virulence plasmid pXO2 and the B. anthracis-specific markers rpoB and dhp61, but does not harbor “lethal and edema factor” encoding plasmid pXO1 and is therefore also negative for the commonly used pathogenicity marker pagA (coding for the “Protective Antigen”) After this round of external quality assessment, “standardized samples” are again available for colleagues who are interested in obtaining B. anthracis DNA positive material for assay validation purposes. Requests for backup samples should be addressed to the EQAS coordinator (U. Reischl).

RV 543: Francisella tularensis

General note to our participants: the concept of this proficiency testing series is 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 two positive samples: a high amount of Francisella tularensis spp. tularensis (~1x105 CFU/mL) was present in sample # 1615431 and an approximately ten-fold lower amount (~1x104 CFU/mL) was present in sample # 1615433.

Similar to QC samples from past distributions, the positive samples # 1615431 and # 1615433 were correctly tested positive by all of the 22 participating laboratories. The “negative” samples # 1615432 and # 1615434, both containing only E. coli, were correctly reported by 21 and 22 of the 22 participating laboratories. Overall, these results corroborate the lower limits of detection observed in our previous EQAS distributions. Although the number of participating laboratories is still not very high, the results of the present distribution indicate that the lower limit of detection is about or slightly below 104 organisms/mL when using currently employed and well evaluated PCR/NAT-based assay concepts for the detection of F. tularensis DNA.

RV 544: Carbapenemase genes

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

As shown in Tab. 1 (Attachment 1 [Attach. 1], p. 19), the current set contained three samples with different carbapenem-resistant Enterobacteriaceae: sample # 1615441 contained Klebsiella pneumoniae with a KPC-3 gene (~1x106 genome copies/mL), sample # 1615442 contained an Serratia marcescens isolate with a NDM-1 gene (~1x106 genome copies/mL), and sample # 1615444 contained an Enterobacter cloacae-Complex with a VIM-1 and OXA-48 genes (~1x106 genome copies/mL). The fourth sample # 1615443 was designed as negative control and contained only E. coli without carbapenemase genes.

All participants detected a carbapenemase gene in the NDM-1 positive S. marcescens sample (# 1615422). Additionally, all participants classified the sample # 1615444 (E. cloacae-complex with VIM-1 and OXA-48 genes) correctly as positive for the presence of carbapenemase genes. The third “positive” sample # 1615441 (containing Klebsiella pneumoniae with a KPC-3 gene) was correctly reported by 59 participants. A false-negative result for this sample should prompt investigations regarding the coverage of carbapenemase genes by the test system used.

RV 545: Clostridium difficile

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

The current set of QC samples contained two Clostridium difficile positive samples: sample # 1615451 with ~5x104 CFU/mL and sample # 1615453 with ~5x103 CFU/mL. Samples # 1615452 and # 1615454 contained only human cells and a considerable amount of E. coli organisms.

The samples # 1615451 and # 11615453 containing relatively high amounts of C. difficile (1x104 CFU/mL and ~5x103 CFU/mL) were correctly reported as “positive” by 109 and 108 of the 110 participating laboratories, respectively. False-negative results should prompt a thorough evaluation of the test system and the workflow. The latter is definitely warranted for the participants reporting false-positive results for samples # 1615452 and # 1615454, 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. 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 # 1615461 of the current set contained a relatively high amount of Enterococcus faecium vanB (~1x105 CFU/mL) and sample # 1615464 contained a similar amount of Enterococcus faecium vanA (~1x105 CFU/mL). Sample # 1615462 contained a tenfold lower amount of Enterococcus faecium vanA (~1x104 CFU/mL) and sample # 1615463 contained no target organisms but only human cells and E. coli. Of the 41 participating laboratories, 41 and 40 correctly reported positive results for the samples # 1615461 and 1615463, respectively. Of note, the reported dedicated vanA/vanB identifications for these two samples were all correct. Sample # 1615462 contained the lowest amount of target organism (Enterococcus faecium vanA, ~1x104 CFU/mL) and only 36 laboratories identified the sample as “VRE-positive”. This should prompt investigations regarding the sensitivity of the used test system as well as re-evaluation of sample processing and -analysis. Importantly, this should also be considered by the two participants reporting false-positive results for sample # 1615463. All participants included controls to detect inhibitions of the PCR reaction. Significant inhibitory events were not reported.

RV 560: Pneumocystis jirovecii

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

The latest set of QC samples contained three positive specimens (see Table 1: Attachment 1 [Attach. 1], p. 22). A relatively high amount of Pneumocystis jirovecii (~1x106 organisms/mL) was present in sample # 1615601, an approximately tenfold lower amount of Pneumocystis jirovecii (~1x105 organisms/mL) was present in sample # 1615603 and an approximately hundredfold lower amount (~1x104 organisms/mL) was present in sample # 1615604. The set was completed by sample # 1615602 which contained only human cells and a considerable amount of E. coli organisms.

Sample # 1615601, which contained the highest amount of P. jirovecii target organisms (~1x106 organisms/mL) and sample # 1615603 with a ten-fold lower concentration of P. jirovecii, were both reported “positive” by 89 out of the 90 participating laboratories. Although this could be due to a loss of template DNA during pre-analytical sample preparation procedures or other reasons, observation of false-negative results should certainly trigger reassessment of the diagnostic workflow, sensitivity and/or specificity of the individual assay concept. The sample containing the lowest amount of the target organism (# 1615604, ~1x104 organisms/mL) was correctly identified as positive by 84 participants, 6 false-negative results were recorded. The “negative sample” (# 1615602, containing only E. coli) was correctly classified “negative” by 89 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.


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