gms | German Medical Science

For the growing number of international participants, we provide a brief discussion of the current results in an English version.

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 similar amounts of C. trachomatis (~5x10⁵ IFU/mL in samples # 1925302 and # 1925303, ~1x10⁵ IFU/mL in sample # 1925301) and three samples with different amounts of N. gonorrhoeae target organisms: ~5x10⁵ CFU/mL in sample # 1925304, ~5x10⁴ CFU/mL in sample # 1925301, and ~1x10³ CFU/mL in sample # 1925302.

Due to the relatively high amounts of C. trachomatis target organisms in the three positive samples of the current distribution, all but four of the 256 participants reported correct-positive CT results for samples # 1925301, # 1925302 and # 1925303. For the CT-negative sample # 1925304, only 5 false-positive results were noted. Among the N. gonorrhoeae-specific results, false-negative results were reported by 15 of the 256 participants for sample # 1925302, which contained a relatively low number of N. gonorrhoeae target organisms (1x10³ CFU/mL) next to a high amount of C. trachomatis (5x10⁵ IU/mL). Also 4 false-positive results for the GO-negative sample # 1925303 were reported by the participants. Assuming a sequential processing of the 4 individual samples of the current set, contamination events of the GO-negative sample “3” by target organisms or PCR products of the positive samples “1” or “2” are by far not unlikely in the current sample constellation. As a consequence, 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 # 1925302 could not be considered as “extremely low”, false-negative results should also encourage the corresponding participants to carefully investigate 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 nearly all of the participants and no inhibitoric 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 256 participants.

The current set of QC samples contained three positive samples: # 1925311 and # 1925313 with ~5x10⁵ IFU/mL of C. trachomatis target organisms. Samples # 1925312 and # 1925314 contained no target organisms but only human cells and E. coli cells.

As depicted in Table 2 (Attachment 1 [Attach. 1], p. 4), the reported results were generally correct for the two C. trachomatis-positive samples. For the two C. trachomatis-negative samples # 1925312 and # 1925314, containing only non-infectious human cells and E. coli, only two false-positive results were observed by two laboratories among the 65 participants.

Assuming a sequential processing of the 4 individual samples of the current set, a contamination event of the “negative” sample “2” or “4” by target organism or PCR product carry-over from the positive samples “1” and “3” might have occurred within the sample prep and amplification workflow of the affected laboratory. In general, the observation of false-positive and/or false-negative results should encourage the affected participants to review and optimize their DNA extraction procedure and their CT-specific NAT-based test system. However, this striking match of the current results with observations and accuracy rates during the past distributions of our EQAS scheme can again be considered as evidence for the high reliability and consistency of the applied assays and overall sample processing.

Run controls were performed by nearly all of the 65 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 65 participants.

The current set of QC samples contained one sample with a relatively high amount of Bordetella pertussis (# 1925321; 5x10⁵ CFU/mL), one sample with an approximately ten-fold lower number of Bordetella pertussis (# 1925324; 5x10⁴ CFU/mL), one sample with an approximately hundred-fold lower number of Bordetella pertussis (# 1925323 with ~5x10³ CFU/mL), as well as one sample containing only non-infected human cells and Escherichia coli (# 1925322).

The availability of well-established commercial or in-house NAT assays has led to a high portion of correct results. All of the 155 participants reported correct-positive results for the relatively strong positive sample # 1925321 (B. pertussis, 5x10⁵ CFU/mL). Sample # 1925324, which contained ~5x10⁴ CFU/mL of Bordetella pertussis, was correctly tested by 153 of the 155 participants, whereas only 133 of the participating laboratories observed positive PCR/NAT results for B. pertussis DNA with the weak positive sample # 1925323. It should be noted that the amount of 5x10³ 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. However, given the relatively small amount of target organisms in the sample # 1925323, reported results were not included in the assessment for the EQAS certificates. This fact is characterized by the three gray shaded boxes in Table 2 (Attachment 1 [Attach. 1], p. 5).

Sample # 1925322 of the current distribution contained only E. coli. All participants correctly reported this sample as negative for Bordetella pertussis DNA.

For the detection of B. pertussis, most participants used self-developed (in-house) test systems with inhibition and/or positive controls. According to our report files, 54 participating laboratories indicated the use of the IS481 insertion sequence, 12 the pertussis toxin coding gene, and 5 participants mentioned ribosomal genes as the PCR/NAT target region. Run controls were performed by 154 of 155 participants, and no inhibition events were observed with the samples of the current distribution.

The current set of QC samples contained three samples with a Clarithromycin-susceptible Helicobacter pylori patient strain in a kind of dilution series. Sample # 1925333 contained approximately 5x10⁵ CFU/mL, sample # 1925331 approximately 5x10⁴ CFU/mL, and sample # 1925332 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 two Helicobacter pylori-positive samples (# 1925333: ~5x10⁵ CFU/mL and # 1925331: ~5x10⁴ CFU/mL) led to correctness values of 100% for the positive as well as for the negative result (sample # 1925334) reported by the 47 participants. For the weaker positive sample # 1925332, all but 2 of the participating laboratories observed correct-positive PCR/NAT results for Helicobacter pylori DNA.

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 probe-based qPCR assays. Results for clarithromycin resistance were reported by 41 of the 47 participants for the three H. pylori-positive samples of the current distribution. With the exception of one result, all of the reported molecular clarithromycin resistance testing results were correct.

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: # 1925342 (E. coli, 1x10⁵ CFU/mL, clinical isolate, stx₁-positive, stx₂-positive, eae-positive and hlyA-positive) and # 1925343 (E. coli, 1x10⁴ CFU/mL, clinical isolate, stx_1a-positive, stx₂-negative, eae-positive and hlyA-positive). The other two EHEC-negative samples contained a Salmonella enterica ser. Enteritidis strain (sample # 1925341; 5x10⁴ CFU/mL) and an eae- and hlyA-negative E. coli K12 strain (# 1925344).

Almost all of the 133 participants reported correct EHEC/STEC-negative results for sample # 1925341, containing only Salmonella enterica ser. Enteritidis. The second “negative” sample (# 1925344), containing only E. coli K12 was with one exception also correctly reported as EHEC/STEC-negative by all of our participants. For the EHEC/STEC-positive samples # 1925342 and # 1925343, the availability of well-established NAT-based assays and strategies for molecular differentiation (and relatively high numbers of target organisms present in the respective samples) resulted in consistently high accuracy rates. Sample # 1925343 was correctly reported EHEC/STEC-positive by 132 of the 133 participants, while all of the 133 participants observed correct-positive PCR/NAT results for sample # 1925342.

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 analytical sensitivity or lower limit of detection. Partial or complete shiga-toxin subtyping, eae-, and hlyA-detection techniques were performed by 113 of the 133 participating laboratories. Except of two molecular typing results, all of the reported results were correct. None of the participants observed significant inhibition of the PCR/NAT reaction with the samples of the current distribution.

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 # 1925352 (5x10⁵ B. afzelii organisms/mL), sample # 1925351 (5x10⁴ organisms/mL) and sample # 1925353 (5x10³ organisms/mL). Sample # 1925354 contained no target organisms but only human cells and E. coli cells.

With the exception of one false-negative result and two results classified as “questionable” for sample # 1925353 (containing the lowest amount of target organisms), one false-negative result for samples # 1925351 and # 1925352 (containing the highest amount of target organisms), the three B. afzelii-containing samples were correctly identified by the 100 participating laboratories. At least the three false-negative results for the positive samples should prompt re-evaluation of the assay’s sensitivity. The “negative” sample # 1925354 was classified false-positive by one laboratory and one participant reported a “questionable” PCR/NAT result for B. burgdorferi DNA. Potentially, the false-positive results are probably due to contamination events during sample preparation or analysis. Therefore, the workflow should be optimized to minimize clinically misleading false-positive results for the detection of B. burgdorferi.

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.

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 1: sample # 1925364 (5x10⁵ CFU/mL), sample # 1925361 (5x10⁴ CFU/mL), and sample # 1925362 (5x10³ CFU/mL). Sample # 1925363 contained no target organisms but only human cells and E. coli cells.

The L. pneumophila-positive samples # 1925361 (~5x10⁴ CFU/mL) and # 1925364 (~5x10⁵ CFU/mL) were correctly tested positive by 112 and 111 of the 114 participating laboratories, respectively. For the third positive sample within the current distribution, # 1925362, which contained a significantly lower amount of L. pneumophila target organisms (~5x10³ CFU/mL), only 92 of the 114 participants reported a correctly positive result. With an amount of 5x10³ CFU/mL of L. pneumophila, the lower limit of detection of several PCR/NAT test systems and analytical workflows is obviously reached, and 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 samples # 1925361 and # 1925364 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 # 1925363, which contained only E. coli, was classified as false-positive by two laboratories. This is probably due to contamination events in the course of sample preparation or PCR/NAT amplification. All participants have included inhibition controls in their test systems and no significant inhibitions of the PCR/NAT reactions were observed or reported.

The current set of QC samples contained one sample with Salmonella enterica serovar enteritidis (sample # 1925374 with 1x10⁵ CFU/mL. Sample # 1925372 contained Salmonella enterica serovar paratyphi (with 5x10⁴ CFU/mL), sample # 1925373 contained Salmonella enterica serovar tennesee (with 5x10⁵ CFU/mL) and sample # 1925371 contained no target organisms but only human cells and E. coli cells.

All of the 24 participants reported correct Salmonella enterica-positive PCR/NAT results for sample # 1925373 and correct results for the negative sample # 1925371. Sample # 1925372, containing ~5x10⁴ CFU/mL Salmonella enterica serovar paratyphi, and # 1925374, containing ~1x10⁵ CFU/mL Salmonella enterica serovar enteritidis, were correctly identified as “positive” by 23 of the 24 participants. Reporting a false-negative result for these samples should prompt a thorough re-evaluation of the performance of the test system.

Inhibitory events in the PCR/NAT reaction were not detected by any of the 24 participants.

The current set of QC samples contained a sample without the corresponding target organisms (# 1925384; only E. coli cells), and three samples positive for L. monocytogenes (# 1925381, # 1925382 and # 1925383). The Listeria monocytogenes-containing samples # 1925381 (with 5x10⁵ CFU/mL of L. monocytogenes) and # 1925382 (with 5x10⁴ CFU/mL of L. monocytogenes) were correctly reported positive by all of the 43 participants. In addition, the “negative” E. coli containing sample # 1925384 was also correctly identified as negative by all laboratories. Most of the participants used very sensitive Listeria monocytogenes-specific assays, which is reflected by the high number of correctly positive results for sample # 1925383, containing only 1x10³ CFU/mL of L. monocytogenes. Only 8 of the 43 participants observed a false-negative PCR/NAT result for this very weak positive sample. Due to the relatively small amount of target organisms in sample # 1925383, the respective results were not included in the assessment for the EQAS certificates.

It should be noted that 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.

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 aim to mimic the situation of processing clinical samples like wound or nasal swabs. Consequently, 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 # 1925391 and # 1925393 of the current set contained a relatively high number of methicillin-resistant S. aureus isolate (MRSA, PVL-negative; ~5x10⁵ CFU/mL and ~1x10⁵ CFU/mL respectively), sample # 1925394 of the current set contained relatively high amounts of a mecA dropout MSSA isolate, and only E. coli and human cells were present in sample # 1925392 of the current distribution.

The MRSA-negative sample # 1925392 was correctly reported negative by 283 of the 288 participants. Only two participants classified their results as “questionable” and three participants reported false-positive results, presumably due to intra-laboratory contamination events from the highly positive sample # 1925391 during the sample preparation, amplification or detection. The MRSA-positive samples # 1925391 and # 1925393 were correctly reported positive by 286 and 282 of the 288 participants, respectively. Participants with false-negative results are encouraged to analyse and optimize their PCR/NAT-based assays, because the amount of MRSA target organisms in samples # 1925391 and # 1925393 (1x10⁵ and 5x10⁵ CFU/mL) were not abnormally low.

Twenty-three false-positive MRSA results were reported for MSSA sample # 1925394 and four participants classified their results as “questionable” for MRSA. The apparently “bad” performances for this MRSA-negative sample are quickly explained on closer inspection: it contained one of the yet still relatively rare S. aureus strains that belongs to the group of so-called mecA dropout MSSA isolates: Oxacillin-sensitive S. aureus strains which contain the MRSA-typical SCCmec cassette, but significant parts or the entire mecA gene are deleted on the genomic level. Consequently, only 261 of the 288 participants reported correct-negative MRSA results for this tricky sample. Compared to the previous rounds of PCR/NAT external quality assessment, a much better diagnostic performance was observed for this variant S. aureus genetic constellation. The mecA dropout variant, sent out formerly in the November 2012 distribution, was detected by 30% of the participants. A similar mecA dropout variant MSSA strain, sent out in the November 2015 distribution, was detected by 53% of the participants, whereas around 90% (!) of the participants correctly reported MRSA-negative results in the current distribution. This situation nicely reflects the various (and obviously successful) efforts of diagnostic companies and in-house assay development teams to continuously improve and adapt 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 115 of the 288 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 in Linde et al. [2] and Witte et al. [3]. A well-evaluated protocol for the detection of PVL-positive PVL isolate can be found in Reischl et al. [4].

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

The concept of this proficiency testing series is designed to determine the analytical sensitivity and specificity of NAT-based assays for the direct detection of C. pneumoniae in typical sample material. With the development and composition of the corresponding sample materials, we intended to mimic the situation of processing typical clinical samples like BAL or other respiratory materials. Consequently, 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 # 1925402 was spiked with ~5x10⁵ IFU/mL of C. pneumoniae, whereas sample # 1925401 contained an approximately ten-fold lower number of C. pneumoniae (~1x10⁴ IFU/mL). Sample # 1925403 contained significant numbers (~1x10⁵ genome copies/mL) of Mycoplasma pneumoniae organisms to assess analytical specificity. Only E. coli and non-infected human cells but no C. pneumoniae target organisms were present in sample # 1925404.

As depicted in Table 2 (Attachment 1 [Attach. 1], p. 13), all of the 120 participants reported correct results for the strong-positive sample # 1925402, and all of the participants also reported correctly positive results for the slightly weaker positive sample # 1925401, which still contained a relatively high concentration of C. pneumoniae target organisms (1x10⁴ IFU/mL). Only one participant classified his result as “questionable” for the negative sample # 1925404 (E. coli and non-infected human cells) and one participant reported a false-positive result for the negative sample # 1925403 (containing a significant number of Mycoplasma pneumoniae organisms), which could probably be due to cross-contamination events in the course of sample preparation, amplification, or amplicon detection steps. Overall there were no noticeable problems with the current set of QC samples and a good overall correlation with the expected results.

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

The current set of QC samples contained three positive samples. A relatively high amount of M. pneumoniae (~5x10⁵ genome copies/mL) was present in sample # 1925413. An approximately ten-fold lower amount of M. pneumoniae (~5x10⁴ genome copies/mL) was present in sample # 1925411, and sample # 1925414 contained an approximately hundred-fold lower amount of M. pneumoniae (~5x10³ genome copies/mL). The set was completed by sample # 1925412, which contained only human cells and a considerable amount of E. coli.

With the exception of 2 laboratories, all of the 139 participants correctly reported samples # 1925411 and # 1925413 positive for M. pneumoniae DNA. The third positive sample with a relatively low amount of the target organisms (#1925414) was correctly identified by 133 participants. Sample # 1925412, which contained only human cells and E. coli, was correctly reported as negative for M. pneumoniae DNA by 137 laboratories. Only two participants observed false-positive results for the “negative” sample, which could be due to cross-contamination events in the course of sample preparation, amplification, or amplicon detection steps. Overall, there were no noticeable problems with the current set of QC samples and a good overall correlation with the expected results.

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

The current set of QC samples contained two samples with different amounts of Coxiella burnetii organisms (~5x10⁴ genome copies/mL in sample # 1925424 and ~1x10⁴ genome copies/mL in sample # 1925421), one sample with ~5x10⁴ genome copies/mL of Bacillus anthracis (sample # 1925424) and two samples with ~1x10⁶ and ~5x10³ genome copies/mL of a Bacillus anthracis Pasteur Strain (sample # 1925422 and sample # 1925421, respectively). Sample # 1925423 contained only human cells and a considerable amount of E. coli organisms.

For convenient data presentation and analysis, we decided to depict the PCR/NAT results for each target organism within this combined EQAS scheme in two separate tables: please see Tables 2 and 3 (Attachment 1 [Attach. 1], p. 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 (~5x10⁴ genome copies/mL) of C. burnetii organisms in sample # 1925424 was correctly reported by all participants, as well as the five-fold lower concentration of the pathogen in sample #1925421. The two “negative” samples (#1925423 contained only E. coli and #1925422 contained only B. anthracis) were correctly reported negative by all but three participants. Overall, there were no noticeable problems with the current set of QC samples and a good correlation with the expected results was observed.

Bacillus anthracis: The results for this newly introduced EQAS scheme are easily discussed. 21 of the 22 participants correctly reported positive results for sample # 1925422 (~1x10⁶ genome copies/mL) and # 1925424 (~5x10⁴ genome copies/mL). The third “positive” sample # 1925421 contained ~5x10³ genome copies/mL of B. anthracis strain “Pasteur”. Only 15 participants reported correct results for this sample. This particular strain is positive for the virulence plasmid pXO2 and the B. anthracis-specific chromosomal markers rpoB and dhp61, but does not harbor “protective antigen, lethal and edema factor” encoding plasmid pXO1, and is therefore also negative for the commonly used pathogenicity marker pagA. All participants correctly reported negative results for the “negative” sample # 1925423 (containing E. coli and human cells). After this very successful round of external quality assessment, “standardized samples” are again available for colleagues who are interested in obtaining B. anthracis DNA-positive material for assay validation purposes. Requests for backup samples should be addressed to the EQAS coordinator (U. Reischl).

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

The current set of QC samples (Table 1, Attachment 1 [Attach. 1], p. 17) contained two samples with different amounts of F. tularensis subsp. holarctica DNA (~1x10⁵ CFU/mL in sample # 1925432 and ~1x10⁴ CFU/mL in sample # 1925433), three samples with different amounts of Brucella melitensis DNA (~1x10⁵ CFU/mL in sample # 1925431, ~1x10³ CFU/mL in sample # 1925432, and ~1x10⁴ CFU/mL in sample # 1925433). Sample # 1925434 contained only human cells and a considerable amount of E. coli organisms.

Francisella tularensis: Similar to QC samples from past distributions, the positive samples # 1925432 (~1x10⁵ CFU/mL of F. tularensis spp. holarctica) and # 1925433 (~1x10⁴ CFU/mL of F. tularensis spp. holarctica) were correctly tested positive by 24 and 22 of the 25 participating laboratories, respectively.

Brucella spp.: The ‘positive’ samples # 1925431 and # 1925433 were correctly reported by 23 and 20 of the participating laboratories, respectively. The third positive sample #1925432 contained a relatively low amount of the target organism (~1x10³ CFU/mL), and only 8 participants correctly reported positive results. The sample without target organism (# 1925434) was correctly classified as ‘negative’ by all laboratories. None of the participants observed an inhibition of the nucleic acid amplification.

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

As shown in Table 1 (Attachment 1 [Attach. 1], p. 19), the current set contained three samples with different carbapenem-resistant Enterobacteriaceae: sample # 1925441 contained a Klebsiella oxytoca isolate with two carbapenemase genes: KPC-3 and VIM-1 (~1x10⁶ genome copies/mL), sample # 1925443 contained a Klebsiella pneumoniae with an OXA-245 gene (~1x10⁶ genome copies/mL), sample # 1925444 contained Klebsiella pneumoniae with a NDM-9 gene (~1x10⁶ genome copies/mL). The fourth sample # 1925442 was designed as negative control and contained only E. coli without carbapenemase genes. All participating laboratories reported sample # 1925441 (K. oxytoca carrying a KPC-3 & VIM-1 carbapenemase) as “carbapenemase-positive”. Notably, 8 of the 86 participants missed the carbapenemase gene in sample # 1925443 (K. pneumoniae carrying OXA-245). The third “positive” sample # 1925444 (containing K. pneumoniae with an NDM-9 gene) was correctly reported by 85 of the 86 participants. Additionally, no false-positive results were submitted for sample # 1925442, which contained carbapenemase-negative E. coli K12.

Interestingly, two of last year’s samples (November 2018 distribution) turned out to be candidates for two outbreaks this year in Germany (see [5], [6]).

Laboratories can support the outbreak investigation initiated by the Robert Koch Institute and German National Reference Laboratory if the lab is able to specify and report OXA-48-like carbapenemases as OXA-244 carbapenemases. If this is not the case, the German National Reference Lab may help you free of charge to identify OXA-48-like carbapenemases. The second outbreak strain requires the ability to detect two carbapenemases simultaneously (NDM-1 and OXA-48).

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

The current set of QC samples contained two Clostridium difficile-positive samples: sample # 1925451 with ~1x10⁶ CFU/mL, and sample # 1925454 with ~1x10⁵ CFU/mL. Samples # 1925452 and # 1925453 contained only human cells and a considerable amount of E. coli organisms. The “positive” samples # 1925451 and # 1925454 were correctly reported as “positive” by 148 and 146 of the 149 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 # 1925452 and # 1925453, 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 but two participants included controls to detect inhibitions of the PCR reaction. Significant inhibitory events were not reported.

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

Sample # 1925461 of the current set contained a relatively high amount of Enterococcus faecalis vanA (~1x10⁴ CFU/mL) and sample # 1925464 contained a similar amount of Enterococcus faecium vanA (~1x10⁴ CFU/mL). Sample # 1925463 contained Enterococcus faecalis (~1x10⁵ CFU/mL), and sample # 1925462 contained no target organisms but only human cells and E. coli cells.

Of the 56 participating laboratories, 56 and 54 correctly reported positive results for the samples # 1925461 and 1925464, respectively. Of note, 53 participants reported dedicated vanA/vanB identification for these two samples, 48 were correct. We were pleased to see that also for the “negative” samples #1925462 and # 1925463, all participants reported correct results. All but one participant included controls to detect inhibitions of the PCR reaction. Significant inhibitory events were not reported.

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

Regarding the statistical analysis, data presentation and results discussion, we are still in the learning phase to optimize the informative and intuitive depiction of the complex result constellations as well as developing a rational scheme for issuing individual certificates for the participants.

The results reported by the 68 registered participants are depicted in Tables 2 to 7 (Attachment 1 [Attach. 1], p. 23–25), and a good overall correlation between the expected results (Table 1, Attachment 1 [Attach. 1], p. 23) and the reported results was observed. Briefly, only sporadic false-negative or false-positive results were observed. For example, one false-positive Treponema pallidum DNA result was reported for sample # 1925474 of the current 4-sample set, which could probably be due to cross-contamination events in the course of sample preparation, amplification, or amplicon detection steps.

The online results input mask of RV 547 distributions now contains extra fields where participants are asked to specify the theoretical pathogen spectrum of their individual assay concepts. This extra information will help to consider and fairly assess the broad spectrum of different commercial and in-house PCR/NAT assays regarding species coverage, differentiation, and multiplex capabilities.

A general note to our participants: the concept of this external quality assessment scheme is designed to determine the analytical sensitivity and specificity of NAT-based assays for the direct detection of P. jirovecii DNA in suitable clinical sample material. With the development of diagnostic material similar to clinical samples, we aim to mimic the situation of processing typical clinical samples. Consequently, the lyophilized samples may contain low amounts of target organisms in a natural background of human cells and other components typically present in patient specimens.

The current set of QC samples contained three positive specimens (see Table 1, Attachment 1 [Attach. 1], p. 26). A relatively high concentration of Pneumocystis jirovecii (~1x10⁵ organisms/mL) was present in sample # 1925602, whereas in sample # 1925601 a ten-fold lower (~1x10⁴ organisms/mL), and in sample # 1925603 an approximately twenty-fold lower concentration of Pneumocystis jirovecii (~1x10⁴ organisms/mL) were present. The set was completed by sample # 1925604, which contained only human cells and a considerable amount of E. coli organisms.

For sample # 1925602, which contained P. jirovecii target organisms (~1x10⁵ CFU/mL) at a relatively high concentration, all of the 120 participants reported correctly positive results. Sample # 1925603 of the current distribution, with a ten-fold lower concentration of P. jirovecii, was tested “positive” by all but one of the 120 participating laboratories. Sample # 1925601, which contained a relatively low amount of P. jirovecii target organisms (~5x10³ organisms/mL) was still classified as “positive” by 105 of the 120 participants of the current distribution.

The negative sample within the current distribution (# 1925604, containing only E. coli) was correctly classified “negative” by all of our 120 participants.

Overall, there were no noticeable problems with the current set of QC samples and a good correlation with the expected results was observed.