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 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 three samples with different amounts of C. trachomatis (~5x10⁵ IFU/mL in sample # 1825303, ~1x10⁵ IFU/mL in sample # 1825304 and 5x10⁴ IFU/mL in sample # 1825301) and two samples with different amounts of N. gonorrhoeae target organisms: ~5x10⁵ CFU/mL in sample # 1825304 and ~5x10² CFU/mL in sample # 1825303. Sample # 1825302 contained no target organisms but only human cells and E. coli cells.

Despite the relatively low amounts of C. trachomatis target organisms in the positive sample # 1825301, all of the 250 participants reported correct positive CT results. For the two samples with a two- or tenfold higher amount of C. trachomatis (#1825304 and 1825303), only one false-negative results for each sample was observed in the current distribution. Among the N. gonorrhoeae-specific results, false-negative results were reported by only 1 of the 250 participants for sample # 1825304, and by 10 participants for sample # 1825303 which contained a relatively low number of N. gonorrhoeae target organisms (5x10² CFU/mL) next to a high amount of C. trachomatis (1x10⁵ IU/mL). Also 7 false-positive results for the GO-negative samples # 1825301 and # 1825302 were reported by the participants. Think that we are all aware of the fact that contamination issues are a permanent threat within the workup or the workflow of highly-sensitive PCR/NAT procedures. Assuming a sequential processing of the 4 individual samples of the current set, contamination events of the GO-negative samples “1” and “2” by target organism or PCR products of the positive samples “3” and/or the very high positive sample “4” is not really likely 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. With a target organism load around 5x10² CFU/mL of N. gonorrhoeae the lower limit of detection of appropriate test systems is obviously reached and so the results for the GO-positive sample # 1825303 were not considered in the course of issuing the certificates. Inhibition controls were included or at least indicated by 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 250 participants.

Tables 4 to 7 (Attachment 1 [Attach. 1], p. 2–4) were included this time to enable a detailed evaluation of the C. trachomatis- and GO-specific NAT components of combined GO/CT test systems. In tables 4 and 5 only the C. trachomatis (CT) specific results and in the tables 6 and 7 only the Neisseria gonorrhoeae (GO) specific results are presented and evaluated statistically.

The current set of QC samples contained three positive samples: # 1825313 with ~5x10⁵ IFU/mL of C. trachomatis target organisms, # 1825314 with ~1x10⁵ IFU/mL and # 1825311 with ~5x10⁴ IFU/mL. Sample # 1825312 contained no target organisms but only human cells and E. coli cells.

As depicted in table 2 (Attachment 1 [Attach. 1], p. 5), the reported results were generally correct for the three positive samples.

One false-negative result was reported for the C. trachomatis-positive sample # 1825314 and for the C. trachomatis-negative sample # 1825312, containing only non-infectious human cells and E.coli, one false-positive result was observed among the 70 participants. Assuming a sequential processing of the 4 individual samples of the current set, a contamination event of the “negative” sample “2” by target organism or PCR product carry-over from the positive sample “1” might have occurred within the sample prep and amplification workflow of these laboratories. So false positive results should encourage the affected participants to review and optimize their DNA extraction procedure and their CT-specific NAT-based test system.

This pleasant 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 70 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 70 participants.

The current set of QC samples contained one sample with a relatively high amount of Bordetella pertussis (# 1825322; 5x10⁴ CFU/mL), one negative sample containing Bordetella parapertussis (# 1825323; 1x10⁵ CFU/mL), one negative sample containing Bordetella holmesii (# 1825324 with ~1x10⁴ CFU/mL; IS481-positive strain!), as well as one sample containing only non-infected human cells and Escherichia coli (# 1825321).

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 sample # 1825322 (B. pertussis, 5x10⁴ CFU/mL) and one participant has classified his result as “questionable”. By the way, an amount of 5x10⁴ 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. Sample # 1825321 which contained only E. coli, were classified as false-positive by 2 of the participating laboratories. These sporadically observed false-positive results are most likely due to contamination events in the course of sample preparation or the PCR/NAT amplification and detection workflow.

The B. parapertussis sample was tested false-positive by 4 participants whereas the B. holmesii sample was tested false-positive by 52 of the 146 participants. Since it is well known that B. holmesii strains my contain copies of the most popular B. pertussis-target gene IS 481, the high rate of false-positives is not really surprising for the latter sample. Considering that the detection rate of the B. pertussis sample # 1825322 was very high (indicating a good performance of the B. pertussis-specific PCR/NAT assays), and IS481 is still one of the most practical and sensitive target genes, we have not scored those (false) positive results for the B. holmesii samples in the course of issuing the corresponding QC certificates. For colleagues who are interested in the IS481 topic, there is an informative paper [2].

However, for participants who have observed false-positive B. pertussis results with Bordetella parapertussis in sample # 1825323, it is strongly recommended to initiate appropriate measures to improve the analytical specificity of their assay concepts. However, it is good to see that most of the remaining results reported by the 146 participants were correct. Run controls were performed by 144 participants and inhibition events were not observed among 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 # 1825333 contained approximately 1x10⁶ CFU/mL, sample # 1825331 approximately 1x10⁵ CFU/mL and sample # 1825332 approximately 1x10⁴ CFU/mL of the respective target organisms.

The availability of well evaluated NAT-based assays and the relatively high amount of target organisms in two of three positive samples (#1825333: ~1x10⁶ CFU/mL and # 1825331: ~1x10⁵ CFU/mL) led to consistently correct results (positive predictive values of 100%) in the current distribution. Only one false-negative result was observed for the weaker positive sample #1825332 by one of the 47 participants.

As noted in the test 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 were communicated by 45 of the 47 participants and the results for molecular susceptibility testing were also correct (wild-tpye), apart from one report of a mutation in one of the H. pylori strains.

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: # 1825341 (E. coli, 1x10⁵ CFU/mL, clinical isolate, stx₁-, eae-, hlyA- and O157-positive) and # 1825342 (E. coli, 1x10⁵ CFU/mL, clinical isolate, stx₁-, stx₂-, eae-, hlyA- and O157-positive). The other two EHEC-negative samples contained an ETEC strain (sample # 1825344; 1x10⁴ CFU/mL) and an eae- and hlyA-negative E. coli K12 strain (# 1825343).

All of the 134 participants correctly reported EHEC-negative results for sample # 1825343, containing only E. coli K12. The other “EHEC-negative” sample (# 1825344), containing a significant amount of an LT- and ST-positive ETEC isolate was also reported PCR-negative by all but one of the participants. For the EHEC/STEC positive samples # 1825341 and 1825342, the availability of well-established NAT-based assays and strategies for molecular differentiation resulted in consistently high accuracy rates. Sample # 1825341 was correctly reported positive by 134 of the 135 participants and all of the participants detected the target organisms in the EHEC/STEC-positive sample # 1825343 correctly. Since the amount of target organisms in the EHEC-positive sample # 1825341 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 EHEC/STEC-specific PCR/NAT assays.

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 104 of the 134 participating laboratories. With one exception, the reported results were correct. None of the participants observed significant inhibition of the NAT-reaction.

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 more than 21 different species belonging to the B. burgdorferi sensu lato complex were described, that naturally present genetic differences in commonly used target genes. Of special interest – since of proven human pathogenicity and widely distributed in Europe – are B. burgdorferi sensu stricto, B. afzelii, B. garinii and B. bavariensis. B. spielmanii, a further species with proven pathogenicity for humans, seems to be rare and was so far only recovered from skin manifestations of Lyme borreliosis. B. bissettiae and B. lusitaniae are considered as potential human pathogens while for B. valaisiana evidence for human pathogenicity is missing. Regarding OspA, especially B. garinii shows a striking heterogeneity with at least 5 genetic distinguishable “genotypes” in Europe.

The current set of QC samples contained a kind of dilution series of B. bavariensis organisms in our proprietary matrix: sample # 1825354 (5x10⁵ CFU/ml), sample # 1825351 (5x10⁴ CFU/ml) and sample # 1825353 (5x10³ CFU/ml). Sample # 1825352 contained no Borrelia organisms but a strain of Treponema phagedenis.

With the exception of 3 false-negative results for sample # 1825353 (containing the lowest amount of the B. bavariensis target organism), one false-negative result for sample # 1825351 and only one false-negative results for sample # 1825354 (containing the highest amount of the target organism) all of the remaining participants reported correctly results for the B. bavariensis-containing samples. The couple of false-negative results should prompt re-evaluation of the assay sensitivity. The “negative” sample # 1825352 was classified false-positive by only one laboratory. Potentially, this may be due to a contamination event from the positive sample # 1825351 during sample preparation or PCR/NAT analysis. Therefore, the workflow should be optimized to minimize clinically misleading false-positive results.

Approximately half of the participating laboratories used self-developed (in-house) tests with inhibition and/or positive controls. None of the participants noted significant inhibition of the NAT-reaction. There were also no significant differences in test performance noticed 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.

The current set of QC samples contained two positive ones with Legionella pneumophila serogroup 2: sample # 1825361 (5x10⁵ CFU/ml) and sample # 1825364 (5x10³ CFU/ml). Samples # 1825362 and # 1825363 contained no target organisms but only human cells and E. coli cells.

The L. pneumophila-positive sample # 1825361 (~5x10⁵ CFU/mL) was correctly tested positive by all of the 120 participating laboratories. For the second positive sample within the current distribution, # 1825364 which contained a significantly lower amount of L. pneumophila-target organisms (~5x10³ CFU/mL), only 85 of the 120 participants reported a correctly positive result.

With a target organism load of around 5x10³ CFU/mL of L. pneumophila, the lower limit of detection of appropriate test systems is obviously reached and so the results for the latter sample were not considered in the course of issuing the certificates.

Samples # 1825362 and # 1825363 which contained only E. coli, were classified as false-positive by 2 and 1 of the participating laboratories, respectively. These sporadically observed false-positive results are probably due to contamination events in the course of sample preparation or the PCR/NAT amplification and detection workflow. All but one of the participants have included inhibition controls in their test systems and no significant inhibitions of the PCR/NAT-reactions were observed or reported this time.

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 # 1825372 (Salmonella enterica serovar Enteritidis, ~1x10⁴ CFU/ mL) and a similar amount was present in sample # 1825374 (Salmonella enterica serovar Typhi, ~1x10⁴ CFU/ mL). Sample # 1825371 contained an EHEC strain (clinical isolate, 1x10⁵ CFU/mL). No target organisms but only E. coli cells were present in sample # 1825373.

Only one false-negative result was reported for the positive sample # 1825374, which contained a significant number of Salmonella enterica serovar typhi target organisms. One false-positive result was reported for the Salmonella negative sample # 1825371, which contained an EHEC strain. One false-positive result was reported for the Salmonella negative sample # 1825373, which contained only the matrix and a significant number of E. coli K12 cells. For the remaining sample, only correct positive PCR/NAT results were reported among all of the 25 participating laboratories.

This indicates again a remarkably high analytical sensitivity of the current Salmonella enterica-specific PCR 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 two samples without the corresponding target organisms (# 1825381 and # 1825383; only E. coli cells), and two samples positive for L. monocytogenes (# 1825382, and # 1825384). The Listeria monocytogenes-containing sample # 1825382 with 5x10⁴ CFU/mL of L. monocytogenes was correctly reported positive by all of the 40 participants.

The Listeria monocytogenes weak-positive sample # 1825384, containing about 1x10³ CFU/mL of L. monocytogenes, was tested positive by only 35 of the 40 participating laboratories.

In addition, the two L. monocytogenes negative samples of the current distribution (samples # 1825381 and # 1825383) were reported correctly negative by all of our participants. Most of them have obviously used very sensitive Listeria monocytogenes-specific PCR/NAT assays, which is reflected by the high portion of correctly positive results for sample # 1825384, containing only 1x10³ 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 want to mimic the situation of processing clinical samples like wound or nasal swabs, so the lyophilized samples usually contain low amounts of target organisms in a background of human cells and other components. It is therefore important to note that NAT assays designed mainly for MRSA culture confirmation purposes may fail due to the low number of MRSA organisms in individual samples of the QC set. Except of one sample containing an MSSA isolate together with almost equal amounts of a methicillin-resistant mecA-positive coagulase negative Staphylococcus species, no “difficult” or “interesting” Staphylococcal strains were included into the current panel.

Sample # 1825391 of the current distribution contained a mixture of S. aureus (MSSA, PVL-negative, ~5x10⁴ CFU/mL) and a CoNS strain (S. epidermidis; mecA-positive, ~5x10⁴ CFU/mL).

Sample # 1825392 contained a MRSA isolate (MRSA, PVL-negative; ~5x10⁵ CFU/mL) which tested positive with the MRSA-specific assays in 292 participating laboratories.

Sample # 1825394 contained a MRSA isolate (MRSA, PVL-negative; ~1x10⁴ CFU/mL) which tested positive with the MRSA-specific assays in 283 participating laboratories. One sample of the current set (# 1825393) contained no MRSA target organisms but only a clinical isolate of an oxacillin-sensible CoNS strain (S. epidermidis; mecA-negative, ~1x10³ CFU/mL).

The MRSA negative sample # 1825393 was tested correctly negative by 280 of 294 participants with their PCR-based MRSA-specific test systems, so only 11 participants reported a false positive result for sample # 1825393, which may have probably been caused by contamination with MRSA DNA during the sample preparation, amplification or detection. Fortunately, for the relatively strong positive MRSA sample # 1825392, positive results were reported by almost all participants. Even when sample # 1825394 contained a slightly lower amount of MRSA target organisms (1x10⁴ CFU/mL), correct positive results were still reported by 283 participants, leading to positive predictive values of above 96%. The 11 false negative results are probably due to an insufficient analytical specificity of the used in-house (or LDT-) PCR/NAT test systems or problems during 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. 14), no serious problems can be seen for individual commercial assay concepts. So any “problems” are obviously due to the technical or manual workflow of individual operators.

For the sample # 1825391, which contained a MSSA isolate together with a Methicillin resistant coagulase-negative Staphylococcus species, 272 of 294 participants reported their results correctly as “MRSA-negative” and 7 participants classified the results as “questionable”. Five of these 7 participants indicated the use of test systems, which are based on a separate detection of the mecA gene and S.aureus specific target genes. With this separate detection assays, the origin of the mecA target gene cannot definitively be correlated with the S. aureus or the coagulase-negative Staphylococcus species. Regarding this aspect, “questionable” is the scientifically correct result in the case when using duplex or multiplex PCR/NAT assays, which are correlating quantitative results and using a kind of software algorithm for confirming or ruling out the presence of mecA-positive S. aureus organisms in the investigated sample. The remaining 15 participants reported false-positive results for MRSA for sample # 1825391, containing a mixture of a MSSA isolate and a methicillin-resistant coagulase negative Staphylococcus species. These participants are encouraged to analyse the appropriateness of PCR/NAT their test systems or assay concepts since the simultaneous presence of MSSA ond mecA-positive CoNS organisms is a relatively common scenario for microbiological routine diagnostics of nasal swabs. On the other hand, almost all participants, who used SCCmec based test concepts, reported correct MRSA-negative results for sample # 1825391.

Overall, it should be noted that a pleasingly large proportion of participants reported correct results for all 4 samples of the current EQAS distribution. This indicates excellent sample workup functioning of laboratory-specific prevention measures to avoid the risk of contamination and carry-over events. Moreover, an optional molecular detection of putative pathogenicity factor PVL (Panton-Valentine Leukocidin) or its coding gene lukF/S-PV is possible in this EQAS scheme. Corresponding results were reported by 55 of the total 294 participating laboratories and within the current distribution the (negative) results for molecular PVL testing were correct in all but one case. Additional information can be found at Linde et al. [4] or Witte et al. [5]. A well evaluated protocol for the detection of PVL-positive PVL isolate can be found at Reischl et al. [6]. In addition, commercial real-time PCR assays reliably targeting PVL-genes in MRSA and MSSA isolates are available from a number if diagnostic companies in the meantime (e.g., r-biopharm or TIB Molbiol).

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. So the lyophilized samples usually contain low amounts of target organisms in a natural background of human cells and other components. As a consequence, diagnostic assays designed for C. pneumoniae antigen detection in clinical specimens or other serological assays will fail due to the low number of C. pneumoniae infected cells in individual samples of the QC set.

The current set of QC samples contained two samples positive for C. pneumoniae. Sample # 1825401 was spiked with ~5x10⁵ IFU/mL of C. pneumoniae whereas sample # 1825403 contained an approximately tenfold lower number of C. pneumoniae (~5x10⁴ IFU/mL). Sample # 1825402 contained significant numbers (~5x10⁴ 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 # 1825404.

As depicted in table 2 (Attachment 1 [Attach. 1], p. 15), all but one of the 120 participants reported correct results for the strong positive sample # 1825401, 117 of the 120 participants reported correctly positive results for the slightly weaker positive sample # 1825403, which still contained a relatively high concentration of C. pneumoniae target organisms (5x10⁴ IFU/mL). Participants who missed to detect C. pneumoniae DNA in these two samples are encouraged to analyse and optimize their NAT-based assays. Three participants reported false-positive results for the negative sample # 1825404 (only E. coli and non-infected human cells), which could probably be due to cross-contamination events in the course of sample preparation, amplification or amplicon detection steps.

Moreover, it is good to see that no false-positive results were reported for the C. pneumoniae negative sample # 1825402 (Mycoplasma pneumoniae). Overall there were no noticeable problems with the current set of QC samples and a good overall correlation with the expected results.

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 only one positive sample with a relatively high amount of M. pneumoniae (# 1825412; ~1x10⁵ genome copies/mL). Sample # 1825413 was designed to monitor assay specificity: it contained a considerable amount of M. hominis (~1x10⁵ genome copies/mL) as a related species to the target organism. The set was completed by samples # 1825411 and # 1825414, 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 M. 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. The M. pneumoniae containing sample (#1825412) was correctly reported by 134 of the 138 participants, respectively. The E. coli-containing samples (# 1825411 and # 1825414) were correctly reported by 136 and 135 participants, respectively. Sample # 1825413 contained M. hominis (~10⁵ genome copies/mL), and was erroneously reported positive by four laboratories. This may indicate lacking species-specificity of the test systems and trigger further investigations.

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

The current set of QC samples (table 1, Attachment 1 [Attach. 1], p. 17) contained two samples with different amounts of C. burnetii DNA (~1x10³ genome copies/mL in sample # 1825421 and ~1x10⁵ genome copies/mL in sample # 1825423) and two samples with different amounts of B. anthracis strain UR-1 DNA (~1x10⁴ genome copies/mL in sample # 1825421 and ~1x10⁵ genome copies/mL in sample # 1825422). Sample # 1825424 contained only human cells and a considerable amount of E. coli organisms. For convenient data presentation and analysis, we decided to depict the PCR/NAT-results for each target organisms within this combined EQAS scheme in two separate tables: please see tables 2 and 3 (Attachment 1 [Attach. 1], p. 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 three laboratories, all participants correctly reported ‘positive’ results for the C. burnetii-containing samples # 1825421 (1x10³ genome copies/mL) and # 1825423 (1x10⁵ genome copies/mL). The two “negative” samples (# 1825422 contained only B. anthracis and # 1825424 contained only E. coli) were correctly reported as negative by 33 and 34 of the 36 participants, respectively. These false negative and positive results should be noticed by the affected participants to control preparation and handling protocols and the applied detection configuration.

Bacillus anthracis: All participants correctly reported negative results for the samples # 1825423 and # 1825424 which did not contain the target organism. The “positive” sample # 1825421 containing ~1x10⁴ genome copies/mL B. anthracis strain “UR-1” was correctly reported by all 19 participants. The second positive sample # 1825422 (~1x10⁵ genome copies/mL of B. anthracis strain “UR-1”) was also correctly reported by all 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).

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 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 (table 1, Attachment 1 [Attach. 1], p. 19) contained two positive samples: ~5x10⁴ CFU/mL of Francisella tularensis spp. tularensis was present in sample # 1825431 and ~1x10⁴ CFU/mL of Francisella tularensis spp. novicida was present in sample # 1825432, two samples with different amounts of Brucella melitensis DNA (~1x10⁵ CFU/mL in sample # 1825434 and ~1x10⁴ CFU/mL in sample # 1825432). Sample # 1825433 contained only human cells and a considerable amount of E. coli organisms.

Francisella tularensis: Similar to QC samples from past distributions, the positive samples # 1825431 (~5x10⁴ CFU/mL of Francisella tularensis spp. tularensis) and # 1825432 (~1x10⁴ CFU/mL of Francisella tularensis spp. novicida) were correctly tested positive by 21 and 16 of the 21 participating laboratories, respectively. All laboratories reported correct results for the ‘negative’ samples # 1825433 and # 1825434.

Brucella spp.: The positive samples # 1825432 and # 1825434 were correctly reported by 16 and 19 laboratories, respectively. In both samples without target organism (# 1825432 and #1825433), a total of 2 ‘false-positive’ results were reported. None of the participants observed an inhibition of the nucleic acid amplification.

Since the two samples with low amounts of F. tularensis as well as Brucella spp. DNA were missed by the same two laboratories, a targeted inspection and improvement of the individual DNA preparation procedures is strongly recommended.

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. 21), the current set contained four samples with different carbapenem-resistant Enterobacteriaceae: sample # 1825441 contained a Escherichia coli with a OXA-244 gene (~1x10⁷ genome copies/mL), sample # 1825442 contained an Klebsiella pneumoniae isolate with two carbapenemase genes: NDM-1 and OXA-48 (~1x10⁷ genome copies/mL) and sample # 1825444 contained a Citrobacter freundii-complex isolate with a GES-5 gene (~1x10⁷ genome copies/mL). The fourth sample # 1825443 was designed as negative control and contained only E. coli without carbapenemase genes.

86 of the 88 participating laboratories reported sample # 1825441 (E. coli carrying a OXA-244 carbapenemase) as “carbapenemase positive”. Notably, all but 3 participants were able to detect carbapenemase genes in sample # 1825442 (K. pneumoniae carrying NDM-1 and OXA-48). The third “positive” sample # 1815444 (containing C. freundii with a GES-5 gene) was not correctly reported by 76 of the 88 participants. Apparently, most test systems used were not able to detect GES-5. The E. coli-containing sample # 1825443 was correctly identified as ‘carbapenemase-negative’ by 86 laboratories.

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

The current set of QC samples contained two Clostridium difficile positive samples: sample # 1825451 with ~1x10⁵ CFU/mL, and sample # 1825454 with ~1x10⁴ CFU/mL. Samples # 1825452 and # 1825453 contained only human cells and a considerable amount of E. coli organisms. The “positive” samples # 1825451 and # 1815454 were correctly reported as “positive” by all but one participating laboratory. For the two ‘negative’ samples # 1825452 and # 1825453, 1 ‘questionable’ and 1 false-positive result were reported, respectively. All participants included controls to detect inhibitions of the PCR reaction. Significant inhibitory events were not reported.

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

Sample # 1825461 of the current set contained a relatively high amount of Enterococcus faecalis vanA (~1x10⁵ CFU/mL) and sample # 1825463 contained a similar amount of Enterococcus faecium vanB (~1x10⁵ CFU/mL). Sample # 1825464 contained Enterococcus faecalis (~1x10⁴ CFU/mL) and sample # 1825462 contained no target organisms but only human cells and E. coli cells. Of the 55 participating laboratories, 55 and 54 correctly reported positive results for the samples # 1825461 and 1825463, respectively. Of note, 44 participants reported dedicated vanA/vanB identification for these two samples, of which 43 were correct. We were pleased to see that also for the “negative” samples #1825462 and # 1825464, all but one participant reported correct results. A false-positive result in the analysis of these samples should once again prompt evaluation of the test system and/or the workflow in the laboratory. This is especially important when considering the impact of molecular VRE detection on the clinical management of patient. All participants included controls to detect inhibitions of the PCR reaction. Significant inhibitoric 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. As the current and the forthcoming distribution (November 2018) are classified as “pilot studies”, 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 34 registered participants are depicted in table 2 (Attachment 1 [Attach. 1], p. 25) and a good overall correlation between the expected results (table 1, Attachment 1 [Attach. 1], p. 25) and the reported results was observed.

The current report form (and also the forthcoming ones) of RV 547 distributions contain an extra field for a simple 7-digit code, where participants can specify the theoretical pathogen spectrum of their individual assay concepts. This will help to fairly consider the broad spectrum of different commercial and in-house PCR/NAT assays regarding species coverage, differentiation and multiplex capabilities. Hence the resulting certificates will be individually adapted and tailored to the covered species portfolio and/or detection capabilities of the participant’s individual assay concept.

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

The latest set of QC samples contained two positive specimens (see table 1, Attachment 1 [Attach. 1], p. 26). A relatively high amount of Pneumocystis jirovecii (~1x10⁵ organisms/mL) was present in sample # 1825602 and an approximately tenfold lower amount of Pneumocystis jirovecii (~1x10⁴ organisms/mL) was present in sample # 1825604. The set was completed by P. jirovecii-negative samples # 1825601 and # 1825603, which contained only human cells and a considerable amount of E. coli organisms next to the lyophilization matrix. Sample # 1825602, which contained the highest amount of P. jirovecii target organisms (~1x10⁵ organisms/mL) and sample # 1825604 with a twenty-fold lower concentration of P. jirovecii, were classified as “positive” by 103 and 99 of the 104 participating laboratories, respectively. Although this could be due to a loss of template DNA during pre-analytical sample preparation procedures or other reasons, observation of false-negative results should encourage the affected laboratories to check the diagnostic workflow, consider improving the sensitivity and/or analysing the species coverage of the individual assay concept. Only one laboratory reported a false-positive result for sample # 1825603, containing only E. coli. The second P. jirovecii-negative sample # 1825601 was correctly reported “negative” by all of the 104 participants.

The yet limited number of participants in the trials RV 542 to RV 546 and 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 supply some details of their P. jirovecii-specific PCR/NAT assays or names of commercial kits.