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 sample # 2015304, ~1x10⁴ IFU/mL in sample # 2015303 and ~5x10³ IFU/mL in sample # 2015301), as well as three samples with different amounts of N. gonorrhoeae target organisms: ~5x10⁴ CFU/mL in sample # 2015301, ~5x10³ CFU/mL in sample # 2015304 and ~5x10² CFU/mL in sample # 2015303.

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 269 participants reported correct-positive CT results for samples # 2015301, # 2015303 and # 2015304. For the CT-negative sample # 2015302, only 2 false-positive results were noted. Among the N. gonorrhoeae-specific results, false-negative results were reported by 29 of the 267 participants for samples # 2015303 and # 2015304, which contained a relatively low number of N. gonorrhoeae target organisms (5x10² CFU/mL and 1x10³ CFU/mL, respectively) next to a high amount of C. trachomatis (5x10⁴ IU/mL). Also 6 false-positive results for the GO-negative sample # 2015302 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” is 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 # 2015301 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 269 participants.

The current set of QC samples contained three positive samples: # 2015312 with ~5x10⁴ IFU/mL of C. trachomatis target organisms, # 2015314 with ~1x10⁴ IFU/mL and # 2015311 with ~5x10³ IFU/mL. Sample # 2015313 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 # 2015311 and # 2015312. Two false-negative results were observed for sample # 2015314 of the current set. For the C. trachomatis-negative sample # 2015313, containing only non-infectious human cells and E. coli, false-positive results were observed by three laboratories among the 59 participants.

Assuming a sequential processing of the 4 individual samples of the current set, a contamination event of the “negative” sample “3” by target organism or PCR product carry-over from the positive samples “1” or “2” might have occurred within the sample prep and amplification workflow of the affected laboratories. 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 an evidence for high reliability and consistency of the applied assays and overall sample processing.

Run controls were performed by nearly all of the 59 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 59 participants.

The current set of QC samples contained one sample with a very high amount of Bordetella pertussis (# 2015323; 1x10⁶ CFU/mL), one sample with an approximately ten-fold lower number of Bordetella pertussis (# 2015321; 1x10⁵ CFU/mL), as well as two samples containing only non-infected human cells and Escherichia coli (# 2015322 and # 2015324).

The availability of well-established commercial or in-house NAT assays has led to a high portion of correct results. Only 2 of the 153 participants reported false-positive results for the B. pertussis-negative sample # 2015322, and four false-positives were observed with B. pertussis-negative sample # 2015324. The false-positivity issue is probably due to contamination events in the course of sample preparation or PCR/NAT amplification. A cross-reaction due to a possible low specificity of the used PCR/NAT test system is unlikely, because the negative samples contained only E. coli cells in the sample matrix as a kind of bacterial background. For sample # 2015323, two false-negative results were observed. With an amount of 1x10⁶ CFU/mL of B. pertussis target organisms, the false-negative results cannot be blamed on the lower limit of detection of appropriate test systems.

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, 28 participating laboratories indicated the use of the IS481 insertion sequence, 14 the pertussis toxin coding gene, and 3 participants mentioned ribosomal genes as the PCR/NAT target region. Run controls were performed by all of the 153 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-resistant Helicobacter pylori isolated from a patient in the course of an antibiotic therapy failure study in a kind of dilution series. Sample # 2015332 contained approximately 5x10⁵ CFU/mL, sample # 2015331 approximately 5x10⁴ CFU/mL, and sample # 2015334 approximately 1x10³ 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 the three Helicobacter pylori-positive samples (# 2015331 and # 2015332) as well as the reported results for the negative sample # 2015333 led to accuracy levels of 100%. Only for the very weak Helicobacter pylori-positive sample # 2015334 (~1x10³ CFU/mL), 8 false-negative results and one result classified as “questionable” were reported.

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 23S rDNA or the use of hybridization probes-based qPCR assays. Results for clarithromycin resistance were reported by 42 of the 51 participants. With two exceptions, all of the reported results were correct.

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

The current set of EQA samples contained three samples positive for EHEC: # 2015341 (E. coli, 1x10⁵ CFU/mL, clinical isolate, stx₁-, stx₂-, eae-, hlyA- and O157-positive), # 2015343 (E. coli, 5x10⁴ CFU/mL, clinical isolate, stx_2f-positive and eae-positive), and # 2015344 (E. coli, 1x10⁵ CFU/mL, clinical isolate, stx₁-positive). The other EHEC-negative sample contained an eae- and hlyA-negative E. coli K12 strain (# 2015342).

With the exception of sample # 2015343 (stx_2f-positive EHEC isolate), the availability of well-established NAT-based assays and strategies for molecular differentiation resulted in high accuracy rates for the two remaining samples. Consistently correct results were reported by 130 and 131 of the 132 participants, respectively. One of the simple reasons for false-negative results for the stx_2f-positive EHEC isolate (# 2015343) is probably the missing coverage of “rare” Shiga toxin subtypes by the common spectrum of routinely applied PCR/NAT assays or commercial EHEC PCR test kits among the participating laboratories. It is well-known that the stx_2f-encoding gene shows little if any homology to other Shiga toxin gene sequences [1]. This is impressively demonstrated by the inclusion of the stx_2f-positive sample # 2015343, where only 79 of the 132 participants reported positive results for the presence of genes coding for Shiga toxins. Even if the relevance or the pathogenic potential of stx_2f-positive EHEC isolates is still under dispute, their inclusion in the current EQA distribution could be classified as educational but not mandatory. Consequently, we have not scored those (false-)negative results for the stx_2f-positive sample # 2015343 in the course of issuing the corresponding EQAS certificates. This is characterized by the three gray-shaded boxes in Table 2 (Attachment 1 [Attach. 1], p. 8).

Except for two false-positive results with the “negative” sample # 2015342 (containing an eae- and hlyA-negative E. coli K12 strain), which are presumably caused by carry-over from the strong-positive sample 201531, the majority of the remaining results reported by the 132 participants were correct.

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 115 of the participating laboratories. Within the samples of the current distribution, all of 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 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.

The current set of QC samples contained three samples with B. burgdorferi sensu stricto organisms in our proprietary matrix: sample # 2015351 (5x10⁵ organisms/mL), sample # 2015353 (5x10⁴ organisms/mL) and sample # 2015352 (5x10³ organisms/mL). Sample # 2015354 contained no target organisms but only human cells and E. coli cells.

With the exception of 3 false-negative results for sample # 2015352 (containing a relatively low amount of 5x10³ organisms/mL of Borrelia target organisms), all participants reported correct results for the four samples of the current EQAS distribution. The false-negative results should prompt re-evaluation of the assay sensitivity.

Still 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.

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

The current set of QC samples contained two positive samples with Legionella pneumophila serogroup 14 and serogroup 1, respectively (# 2015361, ~1x10⁵ CFU/mL; and # 2015363, ~1x10⁴ CFU/mL), next to one sample containing Legionella bozemanii (# 2015364; ~5x10⁴ CFU/mL). Sample # 2015362 contained no target organisms but only human cells and E. coli cells.

The L. pneumophila-positive (~1x10⁵ and ~1x10⁴ CFU/mL) samples # 2015361 and # 2015363 were correctly tested positive by 119 of the 120 participating laboratories, respectively. Sample # 2015364, which contained ~5x10⁴ CFU/mL of Legionella bozemanii, was classified as false-positive by 11 of the participating laboratories. Observing false-positive L. pneumophila PCR results for non-pneumophila Legionella spp. should encourage the corresponding participants to review and optimize the analytical specificity of their “L. pneumophila-specific” assays and/or PCR protocols. It should be mentioned that 8 of the 11 affected laboratories mentioned the use of the commercial ampliCube Respiratory Bacterial panel (Mikrogen), which detect Legionella only on genus level. Consequently, we will consider to restrict the corresponding certificates on the detection of Legionella spp. only.

All of the 120 participants indicated the use of internal or external inhibition controls in their assay concepts, and none of the investigated samples showed inhibition.

The current set of QC samples contained two samples with Salmonella enterica serovar Typhimurium (sample # 2015371 with 1x10⁵ CFU/ml, and sample # 2015372 with 1x10⁴ CFU/ml. No target organisms but only E. coli cells were present in samples # 2015373 and # 2015374.

Only one false-negative result was reported for the positive sample # 2015372, which contained a significant number of Salmonella enterica serovar Typhimurium target organisms. For the remaining three samples, only correct-negative or -positive PCR/NAT results were reported among all of the 29 participating laboratories.

This indicates a remarkably high analytical sensitivity of the current Salmonella enterica-specific PCR assays and an improved procedure with regard to the prevention of contamination events during the individual sample preparation and PCR/NAT analytics in the participating diagnostic laboratories.

The current set of QC samples contained one sample without the corresponding target organisms (# 2015383, only E. coli cells), one sample positive for L. monocytogenes (# 2015381 with ~1x10⁵ CFU/mL) and one sample with Listeria ivanovii (# 2015384 with ~1x10⁴ CFU/mL) as a Listeria species other than L. monocytogenes. The fourth sample (# 2015382) this time contained a Streptococcus pneumoniae strain (~5x10⁴ CFU/mL). The Listeria monocytogenes-containing sample (#2015381) was correctly reported positive by all but one of the 42 participating diagnostic laboratories. In addition, the “negative” E. coli-containing sample # 2015383 was correctly identified as negative by all participants. Thirty-seven of the 42 participants indicated the use of Listeria monocytogenes-specific PCR/NAT assays, which is reflected by the high number of “false-negative” results for sample # 2015384, containing 1x10⁴ CFU/mL of Listeria ivanovii organisms. However, as noted in the protocol booklet, participants using L. monocytogenes-specific PCR/NAT assays may indicate this fact in the electronic report form. 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. Despite of one sample containing an MSSA isolate together with a methicillin-resistant coagulase-negative Staphylococcus species and one sample containing a mecC-positive MRSA isolate, no “difficult” or “interesting” sample was included into the current panel. All 298 participants consistently reported correct results for 3 of 4 samples this time.

Sample # 2015391 of the current set contained a mixture of S. aureus (MSSA, PVL-negative, ~1x10⁵ CFU/mL) and a CoNS strain (S. epidermidis, mecA-positive, ~1x10⁵ CFU/mL). One sample of the current set (# 2015394) contained no target organisms but only E. coli cells. Sample # 2015392 contained a relatively high number of a mecC-positive and methicillin-resistant S. aureus isolate (MRSA, PVL-negative, spa:t10009; ~5x10⁵ CFU/mL), and sample # 2015393 contained a typical MRSA isolate (MRSA, PVL-negative, ~5x10⁴ CFU/mL).

The MRSA-negative sample # 2015394 was correctly reported negative by 278 of 281 participants with their MRSA-specific PCR/NAT assays. Only three participants observed a false-positive result, which may probably have been caused by contamination with MRSA DNA during the sample preparation, amplification or detection. Fortunately, for the positive MRSA sample # 2015393, positive results were reported by nearly all of the 281 participants. Three false-negative results were reported, and one participant classified their results as “questionable”. Affected participants are encouraged to analyze and optimize their PCR/NAT-based assays, because the amount of MRSA target organisms (5x10⁴ CFU/mL) was not abnormally low.

For sample # 2015391, which contained an MSSA isolate together with a Methicillin-resistant coagulase-negative Staphylococcus species, 252 of all 281 participants reported their results correctly as “MRSA-negative” and 8 participants classified the results as “questionable”. Five of these 8 participants indicated the use of test systems which are based on a separated detection of the mecA gene and S. aureus-specific target genes. In this case, the origin of the mecA target gene cannot definitively be correlated with the S. aureus or the coagulase-negative Staphylococcus species. Regarding this aspect, “questionable” is the scientifically correct result for these assays. The remaining 21 participants reported false-positive results for MRSA for sample # 2015391, containing a mixture of an MSSA isolate and a methicillin-resistant coagulase-negative Staphylococcus species. These participants are encouraged to analyze the suitability of their test systems, as the described constellation is a relatively common scenario for the molecular detection of MRSA in clinical swabs or other sample types. As depicted in Table 3 (Attachment 1 [Attach. 1], p. 14), most of the participants indicating the use of SCCmec-based PCR/NAT assays for the detection of MRSA reported correct (MRSA-negative) results for sample # 2015391.

The overall poor assay performance for MRSA-positive sample # 2015392 is quickly explained after taking a closer look at the genetic constellation of this particular clinical isolate: an S. aureus patient isolate, where the phenotypic resistance for Oxacillin is not encoded by the “usual” mecA gene but the genetically distinct mecC gene. These sporadically observed Oxacillin-resistant S. aureus strains harbor all commonly used genetic markers on the genome level, including the SCCmec-orfX region. But on the nucleotide sequence level, these MRSA strains clearly differ from isolates harboring the “popular” mecA gene in their genome and are carrying a resistance-mediating mecC gene instead with a strongly divergent gene sequence. The different nucleotide sequence inevitably lead to false-negative results for MRSA in all commercial or in-house SCCmec-based PCR test systems, which were not adapted to the detection of the mecC gene. As expected, the MRSA isolate in sample # 2015392 was only tested MRSA-positive by participants using PCR/NAT assays that have been “udated” for the inclusion of the mecC gene in the meantime. Even if such mecC-positive MRSA isolates are not frequently encountered in routine practice, this EQAS distribution should draw attention to the possible occurrence of such mecC-positive MRSA isolates.

Since the need to cover such MRSA isolates is still under dispute, their inclusion in the current panel is still classified as educational but not mandatory. Consequently, we have not scored those (false-)negative MRSA results in sample # 2015392 in the course of issuing the corresponding EQAS certificates. This is characterized by the three gray-shaded boxes in Table 2 (Attachment 1 [Attach. 1], p. 13).

Overall, it should be noted that, again, a pleasingly large proportion of participants reported a correct result, predominantly correctly positive results for one positive sample and correctly negative findings for the 2 MRSA-negative samples. This indicates excellent sample workup that manages to avoid the risk of contamination and carry-over events through laboratory-specific prevention measures.

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 87 of the total 281 participating laboratories, and within the current distribution, the results for the molecular PVL testing were correct in all but one case. Additional information can be found 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, a number of commercial real-time PCR assays reliably targeting PVL genes in MRSA and MSSA isolates are now available.

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. 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 # 2015402 was spiked with ~5x10⁵ IFU/mL of C. pneumoniae, whereas sample # 2015403 contained an approximately hundred-fold lower amount of C. pneumoniae (~1x10⁴ IFU/mL). Sample # 2015404 contained significant numbers of Haemophilus influenzae (~1x10⁵ CFU/mL). Only E. coli and human cells were present in sample # 2015401.

As depicted in Table 2 (Attachment 1 [Attach. 1], p. 15), all of the 138 current participants reported correct positive results for the very strong positive C. pneumoniae sample # 2015402 (5x10⁵ IFU/mL) and also for sample # 2015403, which contained an approximately fifty-fold lower number of target organisms. For both of the C. pneumoniae-negative samples in the current distribution, # 2015401 and # 2015404 (where the latter sample contained significant amounts of Haemophilus influenzae, which in a sense represents an assay specificity challenge), all but one laboratories reported correct-negative results. The single sporadically observed false-positive result could be due to simple (cross-)contamination events in the course of sample processing and extraction. From the methodical point of view, a severe cross-reactivity of C. pneumoniae-specific NAT/PCR assays with H. influenzae DNA seems to be unlikely. This false-positive result, 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 samples like BAL or other respiratory materials. 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. As a consequence, diagnostic assays designed for M. pneumoniae antigen detection in clinical specimens or other serological assays will fail due to the low number of M. pneumoniae-infected cells in individual samples of the RV 541 distributions.

The current set of QC samples contained two M. pneumoniae-positive samples.

A relatively high amount of M. pneumoniae (~5x10⁵ genome copies/mL) was present in sample # 2015411, and an approximately hundred-fold lower amount of M. pneumoniae (~5x10³ genome copies/mL) was present in sample # 2015414. Sample # 2015413 was designed to monitor assay specificity: it contained a considerable amount of Chlamydia pneumoniae organisms (~5x10⁵ IFU/mL). The set was completed by sample # 2015412, which contained only human cells and a considerable amount of E. coli organisms.

As observed during the past distributions of our EQAS scheme for Mycoplasma pneumoniae PCR/NAT detection, the availability of well-established commercial or in-house PCR/NAT assays has led to a high percentage of correct results. Among the M. pneumoniae-specific results reported by the 160 participants, all but 2 laboratories reported correct-positive results for the relatively high positive sample # 2015411, and all but 6 participants reported correct-positive PCR/NAT results for the approximately hundred-fold weaker M. pneumoniae-positive sample # 2015414.

Sample # 2015413, which contained 5x10⁵ IFU/mL of Chlamydia pneumoniae, was tested correctly negative by 153 of the 160 participants. Six participants reported false-positive results for the C. pneumoniae sample, which could be due to shortcomings in analytical specificity or just cross-contamination events in the course of sample preparation, amplification or amplicon detection steps. The affected laboratories are encouraged to improve their diagnostic workflow or to check the analytical specificity of their PCR/NAT assays. Likewise, this hint holds true for the 6 participants who reported false-positive results for sample # 2015412 of the current set (no target organisms but only non-infected human cells and E. coli cells).

With the exception of one participant, who indicated inhibition events with all 4 samples, no other noticeable problems were experienced 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 external quality assessment scheme (EQAS) 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. 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. 17) contained two samples with different amounts of C. burnetii organisms (~1x10⁴ genome copies/mL in sample # 2015423 and ~1x10⁵ genome copies/mL in sample # 2015421), one sample with ~1x10⁴ genome copies/mL of B. anthracis strain UR-1 (sample # 2015421) and one sample with ~1x10⁵ genome copies/mL of a B. anthracis STI vaccine strain (sample # 2015424). Sample # 2015422 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. 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: The relatively high amount (1x10⁵ genome copies/mL) of C. burnetii organisms in sample # 2015421 was correctly reported by all of the current participants. The ten-fold lower concentration of target organisms in sample # 2015423 was correctly identified as “positive” by 47 of the 48 participating laboratories. The two “negative” samples (#2015422 contained only E. coli, and # 2015424 contained only B. anthracis) were correctly reported as negative by all but two participants, and two participants classified their results as “questionable”. Laboratories who reported false-positive results for the latter two samples, which could be due to shortcomings in analytical specificity or just cross-contamination events in the course of sample preparation, amplification or amplicon detection steps, are encouraged to improve their diagnostic workflow or to check the analytical specificity of their PCR/NAT assays.

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

Bacillus anthracis: All participants correctly reported negative results for the samples # 2015422 and # 2015423 which did not contain B. anthracis target organisms. The positive sample # 2015424 containing ~1x10⁵ genome copies/mL of B. anthracis strain Sti was correctly reported by all of the 27 participating laboratories. This particular strain is positive for the B. anthracis-specific markers rpoB (or dhp61) and pagA and also contains the “protective antigen, lethal and edema factor” encoding plasmid pXO1, but not the virulence plasmid pXO2.

The second positive sample # 2015421 (~1x10⁴ genome copies/mL of B. anthracis patient strain UR-1) was correctly reported by all but one of the 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 program 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 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 # 2015433 and ~1x10⁴ CFU/mL in sample # 2015434), two samples with different amounts of B. melitensis DNA (~1x10⁵ CFU/mL in sample # 2015432 and ~1x10⁴ CFU/mL in sample # 2015434). Sample # 2015431 contained only human cells and a considerable amount of E. coli organisms.

Francisella tularensis: Similar to many of the past distributions, the positive samples # 2015433 (~1x10⁵ CFU/mL of F. tularensis subsp. tularensis) and # 2015434 (~1x10⁴ CFU/mL of F. tularensis subsp. tularensis) were correctly tested positive by all of the 33 participating laboratories, respectively. As a notable improvement to some of our previous EQAS distributions, also the F. tularensis-negative samples # 2015431 and # 2015432 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 concepts, and none of the investigated samples showed inhibition.

Brucella spp.: In accordance to the previously discussed result constellation of F. tularensis-specific NAT/PCR assays, the B. melitensis-positive samples # 2015432 and # 2015434 as well as the B. melitensis-negative samples # 2015431 and # 2015433 were correctly reported by the 30 participating laboratories within the current distribution of RV 542. None of the participants observed an inhibition of the nucleic acid amplification. 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 # 2015441 contained Klebsiella pneumoniae with an OXA-232 gene (~1x10⁷ genome copies/mL), sample # 2015442 contained cultured organisms from the Enterobacter cloacae complex with an IMI-2 gene (~1x10⁷ genome copies/mL), sample # 2015444 contained an NDM-1-positive Serratia marcescens isolate (~1x10⁷ genome copies/mL). The fourth sample # 2015443 was designed as negative control and contained only E. coli without carbapenemase genes.

All but two participating laboratories reported sample # 2015441 and sample #201544 as “carbapenemase-positive”. The third “positive” sample # 2015442 (Enterobacter cloacae complex with an IMI-2 gene) was correctly reported by only 14 of the 89 participants. Apparently, most of the assays currently used for molecular detection of carbapenemase genes lack this IMI-2 target. Consequently, we have not scored those (false-)negative results for the IMI-2 gene in sample # 2015442 in the course of issuing the corresponding EQAS certificates. This is characterized by the three gray-shaded boxes in Table 2 (Attachment 1 [Attach. 1], p. 21). This limitation should be kept in mind, however, when discrepancies between phenotypic and molecular testing of carbapenem susceptibility arise. IMI-2 belongs to the group of serine-carbapenemases and is predominantly found in Enterobacter spp. Its number has increased over the past few years in Germany and it is often associated with a colistin resistance.

For sample # 2015443, which contained carbapenemase-negative E. coli K12, two false-positive and one questionable result were 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 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 three Clostridium difficile-positive samples: sample # 2015452 with ~5x10⁵ CFU/mL, sample # 2015453 with ~5x10⁴ CFU/mL, and sample # 2015451 with ~5x10³ CFU/mL. Sample # 2015454 contained only human cells and a considerable amount of E. coli organisms. The samples # 2015452 and # 2015453 containing relatively high amounts of C. difficile (5x10⁵ CFU/mL and ~5x10⁴ CFU/mL) were correctly reported as “positive” by 179 and 177 of the 181 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 participant reporting a false-positive result for sample # 2015454, containing only E. coli, but no target organism. A 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. Sample # 2015451 with the lowest amount of target organisms (~5x10³ CFU/mL) was correctly identified as positive by 177 participants. Again a false-negative result should prompt re-assessment of the sensitivity of the used test system. All but 2 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 # 2015464 of the current set contained a relatively high amount of Enterococcus faecium vanA (~1x10⁴ CFU/mL), and sample # 2015461 contained an approximately five-fold higher amount of Enterococcus faecium vanB (~5x10⁴ CFU/mL). Sample # 2015463 contained Enterococcus faecalis (~1x10⁵ CFU/mL), and sample # 2015462 contained no target organisms but only human cells and E. coli cells.

All of the 68 participating laboratories correctly reported positive results for sample # 2015464. The second ‘positive’ sample (# 2015461) was correctly classified by 65 participants. Of note, the reported dedicated vanA/vanB identifications for these two samples were with a single exception correct. Compared to previous EQAS distributions, the number of false-positive results for the ‘negative’ samples (# 2015462 and # 2015463) increased. 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 88 participants are depicted in Tables 2 to 9 (Attachment 1 [Attach. 1], p. 24–28), and a good overall correlation between the expected results (Table 1, Attachment 1 [Attach. 1], p. 24) and the reported results was observed. Briefly, only sporadic false-negative or false-positive results were observed. For example, false-positive Trichomonas vaginalis DNA and Gardnerella vaginalis DNA results were reported by one participant for sample # 2015474 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 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. 29). A relatively high amount of Pneumocystis jirovecii (~1x10⁵ organisms/mL) was present in sample # 2015601, and an approximately ten-fold lower amount of Pneumocystis jirovecii (~1x10⁴ organisms/mL) was present in sample # 2051604. The set was completed by P. jirovecii-negative samples # 2015602 and # 2015603, which contained only human cells and a considerable amount of E. coli organisms next to the lyophilization matrix. Sample # 2015601 of the current distribution, which contained the highest amount of P. jirovecii target organisms (~1x10⁵ organisms/mL), and sample # 2015604 with a ten-fold lower concentration of P. jirovecii, were classified as “positive” by 117 and 107 of the 118 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 sample # 2015602, and one laboratory for sample # 2015603, both containing no target organisms but only E. coli cells and our sample matrix. All but one of the participants 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.