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 2014 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
  • Thomas Holzmann - 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
  • 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
  • Wolf Splettstößer - 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
  • 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 2014;5:Doc03

doi: 10.3205/lab000013, urn:nbn:de:0183-lab0000133

This is the English version of the article.
The German version can be found at: http://www.egms.de/de/journals/lab/2014-5/lab000013.shtml

Published: July 1, 2014

© 2014 Reischl et al.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License. You are free: to Share - to copy, distribute and transmit the work, provided the original author and source are credited. See license information at http://creativecommons.org/licenses/by-nc-nd/3.0/.


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 ”Directive of the German Medical Association for quality assurance of medical laboratory examinations“ (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 quality control (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.instandev.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 2014

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 (~1x104 IFU/mL; sample # 1415301 and sample # 1415304), two samples with various amounts of N. gonorrhoeae organisms (~104 CFU/mL in sample # 1415304 and ~105 CFU/mL in sample # 1415303) and one sample without target organisms (# 1415302).

Despite relatively low amounts of C. trachomatis target cells in the positive samples # 1415301 and # 1415304, only 2 false-positive and 5 false-negative results were observed among the Chlamydia trachomatis-specific results, reported by the 194 participants. Among the N. gonorrhoeae-specific results, false-negative results were reported by only 2 of the 192 participants for samples # 1415303 and # 1415304, which contained N. gonorrhoeae target organisms in an amount of 1x105 CFU/mL and 1x104 CFU/mL respectively. Also false-positive results for the two GO-negative samples were reported by 2 participants.

Since the amount of target organisms in samples # 1415301, # 1415303 and # 1415304 (1x104 – 1x105 CFU/mL) could not be considered as „extremely low“, false negative results should encourage the participants to review and optimize their CT- and GO-specific NAT-based assays.

Inhibition controls were included by all of the 194 participants and no inhibitory events were reported.

Tab. 4 to 7 (see 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 (see Attachment 1 [Attach. 1], p. 2) only the C. trachomatis (CT) specific results and in the Tab. 6 and 7 (see Attachment 1 [Attach. 1], p. 3) only the Neisseria gonorrhoeae (GO) specific results are presented and evaluated statistically.

RV 531: Chlamydia trachomatis

The current set of QC samples contained two positive samples: # 1415312 with ~5x103 IFU/mL of C. trachomatis target organisms and sample # 1415311 with ~1x104 IFU/mL of C. trachomatis target organisms. Samples # 1415313 and # 1415314 contained no target organisms but only human cells and E. coli cells.

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

For the C. trachomatis-negative samples # 1415313 and # 1415314 containing only non-infectious human cells and E.coli, 2 false-positive results were observed of the 125 participants.

False positive results should encourage the participants to review and optimize their DNA extraction procedure and their CT-specific NAT-based test system. For both negative samples # 1415313 and # 1415314, results were classified as „questionable“ from one participant each. For questionable results, certificates are only issued when correct results are reported by the participant for the remaining 3 samples of RV 531.

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 125 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 125 participants.

RV 532: Bordetella pertussis

The current set of QC samples contained one sample with a relatively high amount of Bordetella pertussis (# 1415321; 5x105 CFU/mL), one sample with an approximately tenfold lower number of Bordetella pertussis (# 1415323; 1x104 CFU/mL), one negative sample containing Bordetella holmesii (# 1415324 with ~5x105 CFU/mL), as well as one sample containing only non-infected human cells and Escherichia coli (# 1415322).

The availability of well-established commercial or in-house NAT-assays has led to a high portion of correct results. Only two of the 152 participants reported a false-negative result for the sample # 1415321 (B. pertussis, 5x105 CFU/mL). Sample # 1415323 contained ~1x104 CFU/mL of Bordetella pertussis and was correctly reported by 142 of the 152 participants. The amount of 104 CFU/mL of B. pertussis target organisms is significantly above the previously observed lower limit of detection for the corresponding PCR assays or test systems. False-negative or questionable results should therefore lead to re-evaluations of the assay sensitivity. Sample # 1415322 contained only E. coli. All participants correctly reported this sample as negative for Bordetella pertussis. Sample # 1415324 contained the related Bordetella species Bordetella holmesii. As this strain also contains the IS481 insertion sequence which is a common target sequence to identify Bordetella pertussis, participants with IS481-based assays reported „false”-positive results. 39 laboratories identified this sample as negative for Bordetella pertussis, whereas 113 laboratories reported positive results. We included the Bordetella holmesii-containing sample to raise awareness for the issue of cross-reaction between these species. Consequently, false-positive results of participants had no negative effects on issuing the QC certificate. Of the 125 participants, 124 performed inhibition controls. Inhibition of the PCR-NAT-reaction was not reported. For the detection of B. pertussis, most participants used in-house test concepts.

RV 533: Helicobacter pylori

The current set of QC samples contained two samples with a clarithromycin-susceptible Helicobacter pylori patient strain. Sample # 1415331 contained approximately 1x105 CFU/mL and sample # 1415332 approximately 1x104 CFU/mL of the respective target organisms. Sample # 1415333 contained culture suspensions of the related species Helicobacter mustelae (~1x105 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 (# 1415331: ~1x105 CFU/mL and # 1415332: ~1x104 CFU/mL) led to positive predictive values of 100%.

One false-positive results was observed among the 48 participants for sample # 1415334, which contained only a significant number of E. coli cells within our proprietary sample matrix. Of note, four false-positive results and one questionable result were reported for sample # 1415333, containing ~1x105 CFU/mL Helicobacter mustelae. This may be indicative of lacking specificity of the applied test system and should prompt re-evaluation

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 37 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: # 1415344 (E. coli, 1x105 CFU/mL, clinical isolate, stx1-positive, stx2-positive, eae-positive and hlyA-positive) and # 1415342 (E. coli, 1x105 CFU/mL, clinical isolate, stx1-positive, stx2-negative, eae-positive and hlyA-negative). The other two EHEC-negative samples contained a Salmonella enterica ser. Typhi strain (sample # 1415341; 1x105 CFU/mL) and an eae- and hlyA-negative E. coli K12 strain (# 1415343).

All participants correctly reported negative results for sample #1415341, containing only Salmonella enterica ser. Typhi. The second „negative” sample (# 1415343), containing only E. coli K12 was with one exception also correctly commented as negative. For the EHEC/STEC positive samples # 1415342 and 1415344, the availability of well-established NAT-based assays and strategies for molecular differentiation resulted in consistently high accuracy rates. Sample # 1415342 was correctly reported positive by 130 of the 133 participants, while even 132 participants identified sample # 1415344 as positive.

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 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 (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. bissetii, B.lusitaniae and B. valaisiana are considered as potential human pathogens. Regarding OspA, especially B. garinii showed 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 # 1415353 (1x106 CFU/mL), sample # 1415354 (1x105 CFU/mL) and sample # 1415351 (1x104 CFU/mL). Sample # 1415352 contained no Borrelia organisms but a strain of Treponema phagedenis.

With the exception of 6 false-negative results for sample # 1415351 (containing the lowest amount of the target organism), all other B. garinii-containing samples were correctly identified by the participants. The false-negative results should prompt re-evaluation of the assay sensitivity.

The „negative” sample # 1415352 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 two positive samples with Legionella pneumophila serogroup 5 (# 1415362; ~1x106 CFU/mL and # 1415363; ~1x105 CFU/mL), as well as one sample containing Legionella dumoffii (# 1415364; ~1x105 CFU/mL). Sample # 1415361 contained no target organisms but only human cells and E. coli cells.

The L. pneumophila-positive (~1x106 and ~1x105 CFU/mL) samples # 1415362 and #1415363 were correctly tested positive by 107 and 106 of the 108 participating laboratories, respectively. Sample # 1415364, which contained ~1x105 CFU/mL was classified as false-positive by 9 laboratories. This should prompt investigations regarding the species-specificity of the applied test system. Additionally, three participants did not report a negative result for sample # 1415361, which contained only E. coli. As cross reaction is unlikely, accidental contamination during the process of sample preparation and analysis is most likely to be causative. Therefore, the workflow during the process should be re-checked. All but one participant included inhibition controls in their test systems. One laboratory reported significant inhibition of the PCR-/NAT-reaction for sample #14151361, whereas all other samples showed no inhibition.

RV 537: Salmonella enterica

The current set of QC samples contained two samples with Salmonella enterica serovar Enteritidis (sample # 1415374 with 1x105 CFU/mL, and sample # 1415371 with 1x104 CFU/mL). Sample # 1415372 and sample # 1415373 contained no target organisms but only human cells and E. coli cells. All participants reported correct results for all samples. 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. 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 (# 1415382; only E. coli cells), two samples positive for L. monocytogenes (# 1415384 and # 1415381) and one sample with Listeria innocua (# 1415383) as Listeria species other than L. monocytogenes. The Listeria monocytogenes-containing samples (# 1415381 and # 1415384) were correctly reported positive by all participants. In addition, the „negative” E. coli containing sample # 1415382 was also identified as negative by all laboratories. Most of the participants used Listeria monocytogenes-specific assays, which is reflected by the high number of „false-negative” results for sample # 1415383, containing 1x105 CFU/mL L. innocua. Only one participant reported a positive result for this sample. 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. Despite of one sample containing an MSSA isolate together with a methicillin resistant coagulase negative Staphylococcus species and one sample containing a mecC positive MRSA isolate, no „difficult“ or „interesting” sample was included into the current panel. All 298 participants consistently reported correct results for 3 of 4 samples this time.

Sample # 1415392 of the current set contained a mixture of S. aureus (MSSA, PVL-negative, ~1x103 CFU/mL) and a CoNS strain (S. epidermidis; mecA-positive, ~1x103 CFU/mL). One sample of the current set (# 1415393) contained no target organisms but only E. coli cells. Sample # 1415391 contained a relatively high number of a mecC positive and methicillin resistant S. aureus isolate (MRSA, PVL-negative, spa:t10009; ~1x105 CFU/mL) and sample # 1415394 contained a typical MRSA isolate (MRSA, PVL-negative; ~1x104 CFU/mL).

The MRSA negative sample # 1415393 was tested by 296 of 298 participants with their PCR-based MRSA-specific assays as „negative“, so only two participant observed a false positive result, which may have probably been caused by contamination with MRSA DNA during the sample preparation, amplification or detection. Fortunately, for the positive MRSA sample # 1415394, positive results were reported by nearly all of the 298 participants. Two fals-negative results were reported and two participants interpreted their results as „questionable”. Affected participants are encouraged to analyse and optimize their NAT-based assays, because the amount of MRSA target organisms (1x104 CFU/mL) was not abnormally low.

For the sample # 1415392, which contained a MSSA isolate together with a Methicillin resistant coagulase-negative Staphylococcus species, 278 of all 298 participants reported their results correctly as „MRSA-negative” and 9 participants classified the results as „questionable”. Five of these 9 participants indicated the use of test systems, which are based on a separated detection of the mecA gene and S.aureus specific target genes. In this case, 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 for these assays. The remainder 11 participants reported false-positive results for MRSA for sample # 1415392, containing a mixture of a MSSA isolate and a methicillin-resistant coagulase negative Staphylococcus species. These participants are encouraged to analyse the suitability of their test systems, as the described constellation is a relatively common scenario for microbiological routine diagnostic of MRSA. As Tab. 3 (see Attachment 1 [Attach. 1], p. 12) shows, all of participants, who the established SCCmec based NAT-assays for the detection of MRSA reported consistently correct (MRSA-negative) results.

The overall poor assay performance for MRSA positive sample # 1415391 is quickly explained after taking a closer look on the isolate included in this sample, which was a S. aureus patient isolate, where the phenotypic resistance for Oxacillin is not encoded by the „usual“ mecA gene, but the genetically distinct mecC gene.

These sporadically observed Oxacillin resistant S. aureus strains that include all commonly used genetic markers on the genome level inclusive of the SCCmec-orfX region, but on the sequence level, these MRSA strains clearly differ from isolates harboring the „popular“ mecA gene in their genome and are carrying a resistance mediating mecC gene instead with a strongly divergent gene sequence. This significant differing nucleotide sequence inevitably lead to false-negative results for MRSA in all commercial or in-house SCCmec-based PCR test systems, which were not yet optimized in the specific gene sequences of the mecC gene. As expected, the MRSA isolate in sample # 1415391 could only be detected by participants, who were using specially optimized assays for the detection of this „new“ Methicillin resistance gene. Even if such mecC positive MRSA isolates are rarely detected in our latitudes, this QC-panel aims to at least draw attention to the possible occurrence of such mecC positive MRSA isolates. Although mecC positive MRSAs are currently rarely detected (caused by an analytical gap or by a low prevalence?), the necessity of optimizing available NAT assays for the detection of mecC can only be clarified by further scientifical studies on the mecC prevalence. To avoid biasing the results in Tab. 3 (see Attachment 1 [Attach. 1], p. 12) by the mecC issue, we have included an additional Tab. 4 (see Attachment 1 [Attach. 1], p. 13) this time. Here, we listed the accuracy rates of each assay without taking the results for sample # 1415391 into account.

Overall, it should be noted that a pleasingly large proportion of participants reported a correct result, predominantly correctly positive results for one positive sample and correctly negative findings for the 2 MRSA negative samples. 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 65 of the total 298 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 [2] or [3]. A well evaluated protocol for the detection of PVL-positive PVL isolate can be found at [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 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 # 1415401 contained about 5x105 IFU/mL, sample # 1415403 about 5x104 IFU/mL and sample # 1415402 about 1x103 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 # 1415404 of the current set.

As depicted in Tab. 2 (see Attachment 1 [Attach. 1], p. 14), all participants reported correct results for the positive sample # 1415401. 128 of the 129 participants also reported correct positive results for sample # 1415403, and also for the sample with the lowest amount of C. pneumonia (# 1415402; 1x103 IFU/mL) 125 correct results were reported. Only two participants reported false-positive results for the „negative” sample # 1415404 (E. coli). Overall, there were no noticeable problems with the current set of QC samples and a good overall correlation with the expected results was observed.

RV 541: Mycoplasma pneumoniae

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

The current set of QC samples contained two positive samples. A relatively high amount of M. pneumoniae (~1x106 genome copies/mL) was present in sample # 1415412 and an approximately tenfold lower amount of M. pneumoniae (~1x105 genome copies/mL) was present in sample # 1415414. Sample # 1415413 was designed to monitor assay specificity: it contained a considerable amount of M. genitalium (~105 genome copies/mL) as a related species to the target organism. The set was completed by sample # 1415411, which contained only human cells and a considerable amount of E. coli organisms.

Similar to the result constellations observed with past distributions of our external quality assessment schemes for Mycoplasma pneumoniae PCR/NAT detection, the availability of well-established commercial or in-house PCR/NAT-assays has led to a high percentage of correct results. With the exception of one laboratory, all 135 participants correctly reported sample # 1415411 as negative. The Mycoplasma pneumoniae containing samples (#1415412, ~106 genome copies/mL and # 1415414 ~105 genome copies/mL) were correctly reported by all and all but two participants, respectively. Sample # 1415413 contained M. genitalium (~105 genome copies/mL), and was erroneously reported positive by nine laboratories. This may indicate lacking species-specificity of the test systems and trigger investigations.

RV 542: Coxiella burnetii & Bacillus anthracis

General note to our participants: the concept of this proficiency testing series is designed to determine the analytical sensitivity and specificity of NAT-based assays for the direct detection of C. burnetii DNA and/or Bacillus anthracis DNA in typical sample material. With the development and composition of the corresponding sample materials we 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 samples with different amounts of Coxiella burnetii organisms (~1x103 genome copies/mL in sample # 1415424 and ~1x104 genome copies/mL in sample # 1415423), two samples with fivefold different amounts of Bacillus anthracis (sample # 1415422 with ~1x104 genome copies/mL and sample # 1415423 with ~5x104 genome copies/mL) Sample # 1415421 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 (see Attachment 1 [Attach. 1], p. 16) for the Coxiella burnetii-specific results and Tab. 4 and 5 (see Attachment 1 [Attach. 1], p. 17) for the Bacillus anthracis-specific results.

Coxiella burnetii: The relatively high amount (~104 genome copies/mL) of C. burnetii organisms in sample # 1415423 (together with 5x104 genome copies/mL B. anthracis) was correctly reported by all participants, as well as the tenfold lower concentration of the pathogen in sample #1415424. The two „negative” samples (#1415421 contained only E. coli and #1415422 contained only B. anthracis) were with one exception correctly reported negative. Overall, there were no noticeable problems with the current set of QC samples and a good correlation with the expected results was observed.

Bacillus anthracis: The results for this newly introduced EQAS scheme are easily discussed. All of the 16 participants correctly reported positive results for both positive samples # 1415422 (~1x104 genome copies/mL) and # 1415423 (~5x104 genome copies/mL). In addition, all participants correctly reported negative results for the two „negative” samples # 1415421 (containing E. coli and human cells) and # 1415424 (containing ~103 genome copies of Coxiella burnetii in a suspension of human cells).

After this very successful round of external quality assessment, „standardized samples“ are now 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 (Prof. 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 holarctica (~1x105 CFU/mL) was present in sample # 1415432, an approximately tenfold lower amount (~1x104 CFU/mL) was present in sample # 1415431, and an approximately hundred fold lower amount (1x103 CFU/mL) was present in sample # 1415433.

Similar to QC samples from past distributions, the positive samples # 1415431 and # 1415432 (~1x104 CFU/mL and ~1x105 CFU/mL of Francisella tularensis holarctica, respectively) was correctly tested positive by all of the 16 participating laboratories. Even with pathogen amounts of ~1x103 CFU/mL (sample #1415433), 13 out of 16 labs were able to detect Francisella DNA. As no false-positive result was observed for the „negative“ sample # 1415434 – it seems that the participating laboratories have implemented functional precautions 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 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 probational EQA-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 carbapenemase, NDM, IMP, and GIM.

As shown in Tab. 1 (see Attachment 1 [Attach. 1], p. 19), the current set contained three samples with carbapenem-resistant Enterobacteriaceae: sample # 1415441 contained Escherichia coli with NDM-1 gene (approx. 1x107 genome copies/mL), sample # 1415442 contained an Enterobacter cloacae isolate with a VIM-1 gene (approx. 1x107 genome copies/mL) and sample # 1415444 contained a Klebsiella pneumoniae strain harbouring a KPC-3 gene (approx. 1x107 genome copies/mL). The fourth sample # 1415443 was designed negative control – it contained only E. coli without carbapenemase genes.

Results for the NDM-1 positive E. coli sample (# 1415441) as well as the VIM-1 positive E. cloacae sample (# 1415442) were reported correctly positive by all of the 26 participants.

For the sample # 1415444 containing a carbapenem-resistant K. pneumoniae only 23 of the 26 participants were able to clearly detect the resistance mediating KPC-3 gene. In case of false-negative results intensive trouble-shooting is necessary because it is not acceptable for a molecular test to miss the important carbapenemase KPC. It might be possible that the DNA yield after preparation from lyophilised EQA-samples is lower than from bacterial colonies. No false positive results were observed in the sample without target organisms (# 1415443), containing only human cell material and E. coli, so it seems that the participating laboratories have implemented efficient precautions to prevent contamination events.

In-house NAT assays were used for the detection of carbapenemase coding genes by 13 of the 26 participating laboratories, while the other half quoted the use of commercial test systems or kits on the result form. As these commercial test system were not specified by all of the participants a detailed comparisons between commercial kits and the heterogeneous group of proprietary (in-house) test systems with respect to sensitivity, specificity or susceptibility to contamination events is not yet possible. Overall, a very good diagnostic performance and no noticeable issues regarding sensitivity and specificity, as shown in Tab. 3 (see Attachment 1 [Attach. 1], p. 19), were observed for the carbapenemase gene-specific NAT assays used by the 26 participants.

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 actual set of QC samples was designed as a kind of dilution series of Pneumocystis jirovecii organisms in the sample matrix: sample # 1415602 contained about 1x105 genome copies/mL, sample # 1415604 about 1x104 genome copies/mL and sample # 1415601 about 2x103 genome copies/mL of Pneumocystis jirovecii. Sample # 1415603 of the current set contained no target organisms but a mixture of human cells and E. coli.

The promising results observed in the previous rounds of this external quality assessment scheme were confirmed in the current distribution. Sample # 1415602, which contained the highest amount of P. jirovecii target organisms (~1x105 genome copies/mL) and sample # 1415604 with a slightly lower concentration of P. jirovecii, were reported „positive“ by nearly all of the 84 participating laboratories. Only two laboratories reported a false-negative result for samples # 1415602 and # 1415602, each. 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 give reason to check the diagnostic workflow, consider improving the sensitivity and/or analyzing the species coverage of the individual assay concept.

For sample # 1515601 – containing a relatively low number of target organisms (2x103 genome copies/mL) – 75 out of 84 participants reported a positive result. The sample was, due to its low concentration of target organisms, this time excluded from issuing the certificates. However, participants with false-negative results should be encouraged to optimize their NAT assays.

Only one false-positive result was reported for sample # 1455603, which contained no target organisms but human cells and E. coli. The 83 reported negative results nicely demonstrate the efficiency of laboratory specific strategies for avoiding contamination events.

The yet limited number of participants in the INSTAND e.V. EQAS schemes RV 542, RV 543, RV 560, together with an incomplete reporting of the assays and manufacturers applied, do not allow a serious evaluation of the quality of either commercial tests or the very heterogeneous in-house PCR/NAT assays in regard to analytical sensitivity, analytical specificity, susceptibility to contamination, or simply the „overall performance“. In this regard we would like to encourage the participants to complete the protocol as good as possible.


References

1.
Reischl U, Lehn N, Wolf H, Straube E. „Bakteriengenom-Nachweis PCR/NAT“: Eine neue Ringversuchsreihe von INSTAND e.V. zur externen Qualitätskontrolle molekularbiologischer Nachweisverfahren in der bakteriologischen Diagnostik. Mikrobiologe. 2003 Aug;13(4):149-56.
2.
Linde HJ, Lehn N. Infektionen mit Methicillin-resistentem Staphylococcus aureus: Bedeutung des Pathogenitätsfaktors Panton-Valentine Leukozidin [Infections with methicillin-resistant Staphylococcus aureus: impact of Panton-Valentine leukocidin]. Dtsch Med Wochenschr. 2005 Oct 21;130(42):2397-401. DOI: 10.1055/s-2005-918583 External link
3.
Witte W, Braulke C, Cuny C, Strommenger B, Werner G, Heuck D, Jappe U, Wendt C, Linde HJ, Harmsen D. Emergence of methicillin-resistant Staphylococcus aureus with Panton-Valentine leukocidin genes in central Europe. Eur J Clin Microbiol Infect Dis. 2005 Jan;24(1):1-5. DOI: 10.1007/s10096-004-1262-x External link
4.
Reischl U, Tuohy MJ, Hall GS, Procop GW, Lehn N, Linde H. Rapid detection of Panton-Valentine leukocidin-positive Staphylococcus aureus by real-time PCR targeting the lukS-PV gene. Eur J Clin Microbiol Infect Dis. 2007 Feb;26(2):131-5. DOI: 10.1007/s10096-007-0254-z External link