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 different amounts of C. trachomatis (~5x10⁴ IFU/mL in samples # 2025302 and # 2025303, and ~5x10³ IFU/mL in sample # 2025304), as well as three samples with different amounts of N. gonorrhoeae target organisms: ~5x10⁴ CFU/mL in sample # 2025303, ~5x10³ CFU/mL in sample # 2025301 and ~5x10² CFU/mL in sample # 2025304.

Due to the relatively high amounts of C. trachomatis target organisms in the three positive samples of the current distribution, all but 2 of the 239 participants reported correct-positive CT results for samples # 2025302, # 2025303 and # 2025304. Three participants reported false-positive results for sample # 2025301. For the CT-negative sample # 2025301, only 3 false-positive results were noted. Among the N. gonorrhoeae-specific results, false-negative results were reported by 12 of the 239 participants for sample # 2025304, which contained a relatively low number of N. gonorrhoeae target organisms (5x10² CFU/mL) next to a significant amount of C. trachomatis (5x10³ IU/mL). Due to the relatively low amounts of GO target organisms (~5x10² CFU/mL) in sample # 2025304, we have not scored those (false-)negative results in the course of issuing the corresponding QC certificates. Also 3 false-positive results for the GO-negative sample # 2025302 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 “2” by target organisms or PCR products of the positive sample “1” 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 assay concepts. Since the amount of target organisms in the GO-positive sample # 2025303 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 239 participants.

The current set of QC samples contained two positive samples (# 2025311 and # 2025313) with ~5x10⁴ IFU/mL of C. trachomatis target organisms, and sample # 2025312 with ~5x10³ IFU/mL. Sample # 2025314 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 three C. trachomatis-positive samples # 2025311, # 2025312 and # 2025313, and no false-negative results were observed for the current set. For the C. trachomatis-negative sample # 2025314, containing only non-infectious human cells and E. coli, also no false-positive results were observed among the 51 participants. 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 an evidence for high reliability and consistency of the applied assays and overall sample processing.

Run controls were performed by all of the 51 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 participants.

The current set of QC samples contained two samples with a relatively high amount of Bordetella pertussis (# 2025321 with 5x10⁵ CFU/mL and # 2025324 with 5x10⁴ CFU/mL), one sample with the closely related species Bordetella holmesii (# 2025323, ~5x10⁴ CFU/mL; IS481-positive strain!), as well as one sample containing only non-infected human cells and Escherichia coli (# 2025322). The availability of well-established commercial or in-house NAT assays has led to a high portion of correct results. Only two of the 146 participants reported false-negative results for the samples # 2025321 and # 2025324 (B. pertussis, 5x10⁵ CFU/mL and 5x10⁴ CFU/mL, respectively) and one participant has classified his result as “questionable”. By the way, even an amount of 5x10⁴ CFU/mL of B. pertussis target organisms is significantly above the previously observed lower limits of detection for the currently used PCR/NAT assays. Sample # 2025322, which only contained E. coli, was falsely scored positive by 2 of the participating laboratories. These sporadically observed false-positive results are most likely caused by contamination events in the course of sample preparation or the PCR/NAT amplification and detection workflow.

The B. holmesii organisms in sample # 2025323 were tested false-negative by 54 of the 146 participants. Since it is well known that B. holmesii strains may contain copies of the most popular B. pertussis target gene IS481, the high rate of false-positives is not surprising for the latter sample. Considering that the detection rate of the B. pertussis samples 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 for detection of B. pertussis DNA in clinical samples, we have not scored those (false-)positive results for the B. holmesii samples in the course of issuing the corresponding QC certificates. For colleagues who are interested in the IS481 topic, there is an informative paper by Njamkepo et al. [1].

For the two participants who observed false-positive B. pertussis PCR/NAT results with sample # 2025322, it is strongly recommended to initiate appropriate measures to improve the analytical specificity of their assay concepts [2]. However, it is good to see that most of the remaining results reported by the 146 participants were correct. 145 participants performed run controls, and inhibition events were not observed among the samples of the current distribution.

The current set of QC samples contained only one sample with a Clarithromycin-resistant Helicobacter pylori strain isolated from a patient in the course of an antibiotic therapy failure study. Sample # 2025331 contained approximately 5x10⁵ CFU/mL of the target organisms. Sample # 2025333 contained culture suspension of the related species Helicobacter salomonis (~5x10⁴ CFU/mL), and sample # 2025334 contained culture suspension of Helicobacter acinonyx (~5x10⁴ CFU/mL).

The availability of well-evaluated NAT-based assays and the relatively high amount of target organisms in the Helicobacter pylori-positive sample (# 2025331: ~1x10⁵ CFU/mL) led to high proportions of correct-positive and correct-negative PCR/NAT results in samples # 2025331, # 2025332 and # 2025333 of the current distribution. One false-positive result was observed among the 45 participants for sample # 2025332, which contained only a significant number of E. coli cells within our proprietary sample matrix. Of note, seven false-positive results were reported for sample # 2025333, containing ~5x10⁴ CFU/mL of the related species Helicobacter salomonis. False-positive H. pylori results for sample # 2025333 could be due to contamination events or just lacking analytical specificity of the applied PCR/NAT assays. Sample # 2025334 of the current set contained a relatively high amount of Helicobacter acinonyx (~5x10⁴ CFU/mL) contaminated with traces of H. pylori organisms during bacterial culture out of our stock collection vials originating from the year 1998. When we noted the traces of H. pylori in the H. acinonyx culture (meanwhile also known as H. acinonychis), we decided to keep this suspension as starting material for the preparation of EQAS samples and were already keen to see how they would perform in the current distribution. As expected, most of the participating labs were able to even detect the relatively low amounts of H. pylori organisms in sample # 2025334 next to H. acinonyx. Of course, we have not scored the results from this sample in the course of issuing the corresponding QC certificates.

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 42 of the 45 participants. Except one result, all of the reported results were correct for both of the H. pylori-positive samples.

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: # 2025342 (E. coli, 5x10⁴ CFU/mL, clinical isolate, stx₁-, stx₂-, eae- and hlyA-positive) and # 2025343 (E. coli, 1x10⁴ CFU/mL, clinical isolate, stx₁-, stx₂-, eae- and hlyA-positive). The other two EHEC-negative samples contained a Shigella sonnei strain (sample # 2025341, 5x10⁴ CFU/mL) and an eae- and hlyA-negative E. coli K12 strain (# 2025344).

The majority of the 127 participants reported correct EHEC/STEC-negative results for sample # 2025341, which contained a significant number of Shigella sonnei organisms. Also the second “EHEC/STEC-negative” sample (# 2025344) of the current distribution, containing only E. coli K12, was correctly reported as EHEC/STEC-negative by all except two of the participating laboratories. Assuming a sequential processing of the 4 individual samples of the current set, contamination events of the negative sample “4” by target organisms or PCR products originating from positive samples “2” or “3” are by far not unlikely in the current sample constellation.

For the EHEC/STEC-positive samples # 2025342 and # 2025343, 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. Samples # 2025342 and # 2025343 were correctly reported EHEC/STEC-positive by 126 and 125 of the 127 participants, respectively.

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 108 of the 127 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 from outside Europe: as this proficiency testing panel is designed for a specific and sensitive detection of B. burgdorferi sensu lato DNA, the positive samples do not necessarily contain suspensions of “prototype” isolates of B. burgdorferi sensu stricto; and in many of the bi-annual rounds of our external quality assessment scheme (EQAS), also other B. burgdorferi genotypes or genospecies will be present in individual samples.

Short recapitulation: So far, more than 20 different species belonging to the B. burgdorferi sensu lato complex were described that naturally present genetic differences in commonly used target genes. To further address this heterogeneity and to monitor the analytical sensitivity and specificity of the PCR/NAT assays applied by the diverse group of international participants for members of the B. burgdorferi sensu lato complex, Borrelia valaisiana, Borrelia garinii and Borrelia hispanica were included in the current EQAS distribution.

While B. garinii is a well-known human pathogenic species present in Europe and Asia, pathogenicity of B. valaisiana for humans – though known for more than 15 years – is still questionable. Borrelia hispanica is not a member of the B. burgdorferi sensu lato complex, but like e.g. Borrelia duttonii, it is one of the causative agents for tick-borne relapsing fever, which is most common in Spain and Northern Africa. In Europe, this species is still extremely rare – and especially of differential diagnostic importance for travelers with febrile illnesses.

The current distribution of QC samples contained one sample with Borrelia valaisiana (# 2025351, ~5x10⁴ organisms/mL), one sample with Borrelia garinii OspA type 3 (sample # 2025353, ~5x10⁴ organisms/mL) and one sample with Borrelia hispanica (sample # 2025354, ~1x10⁵ organisms/mL). Sample # 2025352 contained no Borrelia organisms but a strain of Treponema phagedenis. With the exception of one false-negative result, all participants reported correct results for sample # 2025353 containing a high number of B. garinii target organisms. For the Borrelia spp.-negative sample # 2025352 (containing a high number of Treponema phagedenis organisms) of the current distribution, 98 correctly negative and no false-positive results for B. burgdorferi sensu lato DNA were observed. About 15% of all participants reported a false-positive result for the B. hispanica organisms in sample # 2025354, and 7 of the 98 participants reported a false-negative or questionable PCR/NAT B. burgdorferi DNA result for the B. valaisiana organisms present in sample # 2025351 of the current distribution.

Admittedly, the current set of 4 samples contained some analytical challenges with an educative background. But, as always, obtaining false-negative or false-positive results should prompt a thorough re-evaluation of the assay’s analytical specificity and/or sensitivity. Approximately half of the participating laboratories used self-developed (in-house) PCR/NAT assays with inhibition and/or positive controls. None of the participants noted significant inhibition of the NAT reaction. Looking at the species composition of the current panel, slight differences in test performance become apparent 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 the assessment of PCR/NAT-based methods and protocols for the 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 those diagnostic laboratories that have established a highly sensitive and specific PCR/NAT-based method for the detection of L. pneumophila DNA or who would like 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 three samples with Legionella pneumophila serogroup 1: samples # 2025363 and # 2025364 with 5x10⁵ CFU/mL and sample # 2025361 with 1x10⁴ CFU/mL. Sample # 2025362 contained no target organisms but only human cells and E. coli cells.

The relatively strong L. pneumophila-positive samples # 2025363 and # 2025364 (~5x10⁵ CFU/mL) were correctly tested positive by 107 of the 109 participating laboratories. For the third positive sample within the current distribution, # 2025361, which contained an about 50-fold lower amount of L. pneumophila target organisms (~1x10⁴ CFU/mL), only 106 of the 109 participants reported a correctly positive result. Since the amount of target organisms in L. pneumophila-positive samples # 2025363 and # 2025364 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 # 2025362, which contained only E. coli, was correctly classified as negative by all of the participating diagnostic laboratories. All but one of the participants have included inhibition controls in their test systems. No significant inhibitions of the PCR/NAT reactions were observed among the samples of the current distribution.

The current set of QC samples contained two positive samples with Salmonella enterica. A relatively high amount of the corresponding target organism was present in sample # 2025371 (Salmonella enterica serovar Typhimurium, ~5x10⁵ CFU/mL), and a similar amount was present in sample # 2025374 (Salmonella enterica serovar Typhi, ~1x10⁵ CFU/mL). Sample # 2025372 contained a Shigella sonnei strain (clinical isolate, 1x10⁵ CFU/mL). No target organisms but only E. coli cells were present in sample # 2025373.

The results reported by the 24 participants make the discussion of the current distribution very easy and also pleasing. Only correct-positive and correct-negative results were observed for all of the 4 samples. This again indicates a remarkably high analytical sensitivity of the current Salmonella enterica-specific PCR/NAT assays and improved procedures with respect to the effective prevention of contamination events during the individual sample preparation and PCR/NAT analytics in the participating diagnostic laboratories.

The current set of QC samples contained a sample without the corresponding target organisms (# 2025381; only E. coli cells), and three samples positive for L. monocytogenes (# 2025383, # 2025384 and # 2025382). The Listeria monocytogenes-containing samples # 2025383 with 5x10⁵ CFU/mL of L. monocytogenes, # 2025384 with 5x10⁴ CFU/mL of L. monocytogenes and # 2025382 with 5x10³ CFU/mL of L. monocytogenes were correctly reported positive by all of the 41 participants. In addition, the “Listeria-negative” E. coli containing sample # 2025381 was also correctly identified as negative by all but one of the participating 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 # 2025384, containing only 5x10³ CFU/mL of L. monocytogenes.

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. Except for one sample containing a rarely encountered MRSA isolate (missing the normally very reliable, very sensitive and robust pSA442 S. aureus species marker gene), no other difficult or interesting staphylococcal strains were included into the current panel.

Sample # 2025391 of the current distribution contained an MRSA patient isolate (MRSA, PVL-negative, pSA442-negative, ~1x10⁵ CFU/mL). Samples # 2025394 and # 2025393 contained different amounts of a CA-MRSA isolate (MRSA, PVL-positive; ~5x10⁴ CFU/mL and ~5x10³ CFU/mL respectively). One sample of the current set (# 2025392) contained no MRSA target organisms but only a clinical isolate of an oxacillin-susceptible CoNS strain (S. epidermidis, mecA-negative, ~1x10³ CFU/mL).

Fortunately, for the two relatively strong positive CA-MRSA samples # 2025393 and # 2025394, correct-positive results were reported by almost all participants. The 5 false-negative results are probably caused by problems in the course of the sample workup or experimental workflow of commercial PCR/NAT test systems. Considering the assay-specific percentage of true-positive and true-negative results given in Table 3 (Attachment 1 [Attach. 1], p. 13), no serious problems can be seen for individual commercial assay concepts. Consequently, any “problems” are obviously due to the technical or manual workflow of individual operators.

The MRSA-negative sample # 2025392 tested correctly negative by 273 of 277 participants with their PCR-based MRSA-specific test systems. Only 5 participants reported a false-positive result, which could be due to genomic DNA or amplicon contamination events during sample preparation, amplification or detection.

For the sample # 2025391, which contained an MRSA isolate lacking the pSA442 chromosomal S. aureus species marker gene, 250 of 277 participants reported correct MRSA-positive results, whereas 24 participants missed this MRSA strain with their individual PCR/NAT detection assay, and 3 participants classified their results as “questionable”. Five of these 24 participants indicated the use of test systems that are based on a separate detection of the mecA gene and S. aureus specific target genes. When they used the pSA442 target gene as S. aureus marker, it is likely that they have missed this particular isolate. For further information on pSA442, see Martineau et al. [3]. Due to the special genetic constellation of the MRSA strain in sample # 2025391, it was classified as “educative”, and the corresponding PCR/NAT results were not included into the assessment for the EQAS certificates.

Overall, it should be noted that a pleasingly large proportion of participants reported correct results for at least 3 samples of the current EQAS distribution. This indicates excellent sample workup that manages to avoid the risk of contamination and carry-over events through laboratory-specific prevention measures. Moreover, an optional molecular detection of putative pathogenicity factor PVL (Panton-Valentine leukocidine) or its coding gene lukF/S-PV is possible in this EQAS scheme. Corresponding results were reported by 78 of the 277 participating laboratories. Within the current distribution, the (positive) results for molecular PVL testing were correct in all but two cases.

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. Therefore, 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 # 2025403 was spiked with ~1x10⁶ IFU/mL of C. pneumoniae, whereas sample # 2025402 contained an approximately hundred-fold lower amount of C. pneumoniae (~5x10⁴ IFU/mL). Only E. coli and human cells were present in samples # 2025401 and # 2025404.

As depicted in Table 2 (Attachment 1 [Attach. 1], p. 14), all but one of the 118 participants reported correct-positive results for both the relatively strong positive C. pneumoniae sample # 2025403 (5x10⁵ IFU/mL) as well as for the ten-fold lower C. pneumoniae-positive sample # 2025402 (5x10⁵ IFU/mL). For both of the C. pneumoniae-negative samples # 2025401 and # 2025404 in the current distribution, all but 4 laboratories reported correct-negative results. The sporadically observed false-positive results could be due to simple cross-contamination events in the course of sample processing and extraction. Interestingly, all of the 4 affected participants indicated the use of LDT or in-house PCR/NAT assay concepts. From the methodical point of view, a severe cross-reactivity of C. pneumoniae-specific NAT/PCR assays with E. coli DNA seems to be unlikely. False-positive results for samples # 2025401 and # 2025404, however, may prompt investigations and improvement of the preanalytical workup, assay concepts and/or the diagnostic workflow.

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 # 2025414. An approximately ten-fold lower amount of M. pneumoniae (~5x10⁴ genome copies/mL) was present in sample # 2025412, and sample # 2025413 contained an approximately hundred-fold lower amount of M. pneumoniae target organisms (~5x10³ genome copies/mL). The set was completed by sample # 2025411, which contained only human cells and a considerable amount of E. coli.

With the exception of 3 laboratories, all of the 136 participants correctly reported samples # 2025414 and # 2025412 positive for M. pneumoniae DNA. The third positive sample with a relatively low amount of target organisms (# 2025413) was correctly identified by 128 participants, and 8 laboratories observed false-negative PCR/NAT results. Due to the relatively small amount of M. pneumoniae target organisms, sample # 2025413 was not included in the assessment for the EQAS certificates.

Sample # 2025411, which contained only human cells and E. coli, was correctly reported as negative for M. pneumoniae DNA by 133 laboratories. Only 3 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 was observed.

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 aimed 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 similar amounts of C. burnetii DNA (~5x10⁴ genome copies/mL in sample # 2025421 and # 2025424) and two samples with different amounts of B. anthracis strain UR-1 DNA (~1x10⁵ genome copies/mL in sample # 2025421 and ~5x10⁵ genome copies/mL in sample # 2025422). Sample # 2025423 contained only human cells and a considerable amount of E. coli organisms. For convenient data presentation and analysis within this combined EQAS scheme, we decided to depict the PCR/NAT results for each target organism in two separate tables: please see Tables 2 and 3 (Attachment 1 [Attach. 1], p. 17) for the C. burnetii-specific results and Tables 4 and 5 (Attachment 1 [Attach. 1], p. 18) for the B. anthracis-specific results.

Coxiella burnetii: With the exception of two laboratories, all participants correctly reported correct-positive results for the C. burnetii-containing samples # 2025421 (5x10⁴ genome copies/mL) and # 2025424 (5x10⁴ genome copies/mL). The two negative samples (# 2025422 contained only B. anthracis and # 2025423 contained only E. coli) were correctly reported as negative by 45 and 46 of the 46 participants, respectively. These single false-negative and false-positive results should be taken as an opportunity by the affected participants to investigate the efficiency of their individual sample preparation and PCR/NAT amplification/detection procedures.

Bacillus anthracis: All participants correctly reported negative results for samples # 2025423 and # 2025424, which did not contain B. anthracis target organisms. The positive samples # 2025421 containing ~1x10⁵ genome copies/mL and # 2025422 containing ~5x10⁵ genome copies/mL of B. anthracis strain “UR-1” were correctly reported by all of the 24 participants.

With the completion of this round of external quality assessment, standardized samples are again available for colleagues who are interested in obtaining B. anthracis DNA-positive material for assay validation purposes. Requests for backup samples should be addressed to the EQAS coordinator (udo.reischl@ukr.de).

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 EQAS samples (Table 1, Attachment 1 [Attach. 1], p. 19) contained two samples with different amounts of F. tularensis subsp. tularensis DNA (~1x10⁵ CFU/mL in sample # 2025433 and ~1x10⁴ CFU/mL in sample # 2025431), two samples with different amounts of B. melitensis DNA (~1x10⁵ CFU/mL in sample # 2025432 and ~1x10⁴ CFU/mL in sample # 2025431). Sample # 2025434 contained only human cells and a considerable amount of E. coli organisms.

Francisella tularensis: Similar to many of the past distributions, the F. tularensis-positive samples # 2025431 (~1x10⁴ CFU/mL of F. tularensis subsp. tularensis) and # 2025433 (~1x10⁵ CFU/mL of F. tularensis subsp. tularensis) were correctly tested positive by all but 2 of the 33 participating laboratories. As a notable improvement to some of our previous EQAS distributions, also the F. tularensis-negative samples # 2025432 and # 2025434 were consistently reported as negative by all of the current participants. This indicates a remarkably high analytical sensitivity of the current F. tularensis-specific PCR assays and an obvious improvement with regard to preventing carry-over or other contamination events during the individual sample preparation and PCR/NAT analyses in the participating diagnostic laboratories. All laboratories indicated the use of appropriate internal or external inhibition controls in their assay workflow, and none of the investigated samples showed inhibition.

Brucella spp.: All of the 25 participants correctly reported negative results for samples # 2025433 and # 2025434, which did not contain B. melitensis target organisms. The two positive samples of the current distribution, # 2025431 containing ~1x10⁴ CFU/mL and sample # 2025432 containing ~1x10⁵ CFUs/mL of B. melitensis, were also correctly reported by all of the 24 participants.

None of the participants observed any inhibition events in the course of PCR/NAT amplification and detection. Overall, there were no noticeable problems with the current set of EQAS samples, and a good correlation with the expected results was observed.

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 Enterobacterales culture isolates. As shown in Table 1 (Attachment 1 [Attach. 1], p. 21), the current set contained three samples with different carbapenem-resistant Enterobacterales: sample # 2025441 contained Serratia marcescens with a VIM-4 gene (~1x10⁷ genome copies/mL), sample # 2025443 contained Escherichia coli with an OXA-162 gene (~1x10⁷ genome copies/mL), sample # 2025444 contained a GES-5-positive Citrobacter freundii complex isolate (~1x10⁷ genome copies/mL). The fourth sample # 2025442 was designed as negative control and contained only E. coli without carbapenemase genes.

All of the 80 participating laboratories reported samples # 2025441 and # 2025443 correctly positive for the presence of a carbapenemase gene. The third positive sample # 2025444 (C. freundii complex with a GES-5 gene) was tested positive by only 21 of the 80 participants. Apparently, most of the PCR/NAT assays currently used for molecular detection of carbapenemase genes lack the GES-5 target sequence. This limitation should be kept in mind when discrepancies between phenotypic and molecular testing of carbapenem susceptibility arise. As GES-5 carbapenemases occur among Enterobacterales and Pseudomonas aeruginosa in increasing numbers in Germany, they represent an important target for this EQAS panel.

For sample # 2025442, which contained a carbapenemase-negative E. coli K12 strain, only one false-positive result was reported (probably due to sporadic cross-contamination issues).

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 # 2025453 with ~5x10⁵ CFU/mL, and sample # 2025452 with ~5x10⁴ CFU/mL. Samples # 2025451 and # 2025454 contained only human cells and a considerable amount of E. coli organisms. The samples # 2025452 and # 2025453 containing relatively high amounts of C. difficile (5x10⁵ CFU/mL and ~5x10⁴ CFU/mL) were correctly reported as “positive” by 164 and 162 of the 165 participating laboratories, respectively. False-negative results should prompt a thorough evaluation of the test system and the workflow, including a re-assessment of total analytical sensitivity. Re-assessment of the individual assay concept is also warranted for the two participants who reported a false-positive result for sample # 2025454, containing only E. coli but no C. difficile target organisms. 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. The second C. difficile-negative sample # 2025451 was correctly identified as negative by all but one of the 165 participants. All but 4 participants included appropriate controls to monitor DNA extraction and/or detect inhibition 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 # 2025461 of the current set contained a relatively high amount of Enterococcus faecium vanA (~1x10⁵ CFU/mL), and sample # 2025464 contained an approximately half amount of Enterococcus faecium vanB (~5x10⁴ CFU/mL). Sample # 2025462 contained Lactobacillus rhamnosus (~1x10⁵ CFU/mL), and sample # 2025463 contained no target organisms but only human cells and E. coli cells.

All of the 63 participating laboratories correctly reported positive results for samples # 2025461 and # 2025464. Also the negative samples # 2025462 and # 2025463 were correctly reported as negative for VRE by all but one participating laboratory. Of note, the reported vanA/vanB differentiation for the two VRE-positive samples were correct with two exceptions. All but one of the participants reported the use of mandatory 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 has recently been 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 71 participants are depicted in Tables 2 to 9 (Attachment 1 [Attach. 1], p. 25–29) and a good overall correlation between the expected results (Table 1, Attachment 1 [Attach. 1], p. 25) and the reported results was observed. Briefly, only sporadic false-negative or false-positive results were observed. In general, false-positive results for given species within the multiplex panel 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 contain extra fields where participants should 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 proficiency testing series 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 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 latest set of QC samples contained two positive specimens (Table 1, Attachment 1 [Attach. 1], p. 30). A relatively high amount of Pneumocystis jirovecii (~5x10⁴ organisms/mL) was present in sample # 2025603, and an approximately ten-fold lower amount of Pneumocystis jirovecii (~5x10³ organisms/mL) was present in sample # 2025601. The set was completed by P. jirovecii-negative samples # 2025602 and # 2025604, which contained only human cells and a considerable amount of E. coli organisms next to the lyophilization matrix.

Sample # 2025603 of the current distribution, which contained the highest amount of P. jirovecii target organisms (~1x10⁵ organisms/mL), and sample # 2025601 with a ten-fold lower concentration of P. jirovecii were classified as “positive” by 111 and 107 of the 111 participating laboratories, respectively. Observing false-negative results, which could be due to a loss of template DNA during pre-analytical sample preparation procedures or limited analytical sensitivity of the entire PCR/NAT workflow, should encourage the affected laboratories to check their individual procedures for overall diagnostic sensitivity. Two laboratories reported a false-positive result for samples # 2025602 and # 2025604 of the current distribution, both containing no target organisms but only E. coli cells and our sample matrix. All of the participating laboratories included appropriate DNA extraction and PCR inhibition controls. Significant inhibitory events were not reported.

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