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 (~5x10⁵ IFU/mL in sample # 1725302, ~1x10⁵ IFU/mL in sample # 1725303 and 5x10⁴ IFU/mL in sample # 1725304) and three samples with different amounts of N. gonorrhoeae target organisms: ~5x10⁶ CFU/mL in sample # 1725303, ~5x10⁴ CFU/mL in sample # 1725301 and ~5x10³ CFU/mL in sample # 1725304.

Despite the relatively low amounts of C. trachomatis target organisms in the positive sample # 1725304, all but one of the 191 participants reported correct positive CT results. For the two samples with a five- or tenfold higher amount of C. trachomatis (# 1725303 and # 1725302), only 1 false-negative result for each sample was observed in the current distribution. Among the N. gonorrhoeae-specific results, false-negative results were reported by only 7 of the 190 participants for sample # 1725304, which contained a relatively low number of N. gonorrhoeae target organisms (5x10³ CFU/mL) next to a high amount of C. trachomatis (5x10⁴ IU/mL). Also 3 false-positive results for the GO-negative sample # 1725302 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 organism or PCR products of the positive samples “1” and/or the very high positive sample “3” 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 samples # 1725301 and # 1725304 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 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 191 participants.

Tables 4 to 7 (Attachment 1 [Attach. 1], p. 2-3) were included this time to enable a detailed evaluation of the C. trachomatis- and GO-specific NAT components of combined GO/CT test systems. In 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: # 1725314 with ~1x10⁵ IFU/mL of C. trachomatis target organisms, # 1725311 with ~5x10⁴ IFU/mL of C. trachomatis target organisms, and sample # 1725312 with ~1x10⁴ IFU/mL of C. trachomatis target organisms. Sample # 1725313 of the current set contained no target organisms but only human cells and E. coli cells. As depicted in Tab. 2 (Attachment 1 [Attach. 1], p. 4), the majority of the results reported for the negative sample # 1725313 and the three CT-positive samples # 1725311, # 1725312 and # 1725314 were correct.

Also for the C. trachomatis-positive sample # 1725312, containing the lowest amount of target organisms within the current distribution, no false-negative result was observed among the 74 participants. This striking match of the current results with observations and accuracy rates over the last couple of years can be considered as an evidence for a high reliability and consistency of the applied assays and overall sample processing.

Run controls were implemented and performed by all of the 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 various C. trachomatis-specific NAT assays.

The current set of EQA samples contained one sample with a relatively high amount of Bordetella pertussis (# 1725321; 5x10⁴ CFU/mL), one sample with an approximately tenfold lower number of Bordetella pertussis (# 1725324; 5x10³ CFU/mL), one negative sample containing Bordetella parapertussis (# 1725322 with ~5x10⁵ CFU/mL), as well as one sample containing only human cells and Escherichia coli (# 1725323).

The availability of well-established commercial or in-house NAT assays has led to a high portion of correct results. Only 1 of the 126 participants reported a false-positive result for the B. parapertussis sample # 1725322 and one participant classified this sample as “questionable”. The false-positivity issue is probably due to contamination events in the course of sample preparation or low specifity of the used PCR/NAT test system. For sample # 1725324 (5x10³ CFU/mL of B. pertussis) 17 false-negative results were observed. With an amount of slightly above 10³ CFU/mL of B. pertussis target organisms, the lower limit of detection of appropriate PCR/NAT test systems is obviously reached or at least touched to a certain extent. Therefore we have not scored those (false) negative results for the weak-positive B. pertussis sample in the course of issuing the corresponding EQAS certificates. This is also characterized by the three gray-shaded boxes for sample # 1725324 in Tab. 2 (Attachment 1 [Attach. 1], p. 5).

However, for participants who have reported false-negative B. pertussis results with sample # 1725321 or questionable B. pertussis results with the negative sample # 1725323, it is strongly recommended to initiate appropriate measures to improve the analytical specificity of their assay concepts. Except the sporadic false-positive or false-negative results briefly discussed above, all of the remaining results reported by the 126 participants were correct.

For the specific and sensitive detection of B. pertussis, most participants used self-developed (in-house) PCR/NAT test systems with inhibition and/or positive controls. In the submitted report forms 85 participating laboratories indicated the IS481 insertion sequence as target, 11 the pertussis toxin coding gene and 2 participants are targeting ribosomal genes. Run controls were performed by 123 participants and inhibition events were not observed among the samples of the current distribution.

The current set of EQA samples contained two samples with a relatively high amount of target organisms. Sample # 1725333 contained approximately 5x10⁶ CFU/ mL of a Clarithromycin-susceptible Helicobacter pylori patient strain, and sample # 1725331 contained approximately 5x10⁵ CFU/ mL of a Clarithromycin-resistant Helicobacter pylori isolated from a patient in the course of an antibiotic therapy failure study. Only one of the 38 participants reported a false-negative result for sample # 1725333, while no false-negative results were reported for sample # 1725331. Only two false-positive PCR/NAT results were reported for sample # 1725332 of the current distribution, which contained a culture suspension of the related species Helicobacter mustelae (~5x10⁵ CFU/ mL). Sample # 1725334, which contained only E.coli culture suspension, was correctly reported as “negative” by all but two of the participants.

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 36 of the 38 participants and all but one of the reported results of molecular susceptibility testing 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: # 1725341 (E. coli, 5x10⁴CFU/mL, clinical isolate, stx₁-, stx₂-, eae-, hlyA- and O157-positive), # 1725343 (E. coli, 5x10⁴ CFU/mL, clinical isolate, stx_2f-positive and eae-positive), and # 1725344 (E. coli, 5x10³ CFU/mL, clinical isolate, stx_2d-positive). The other EHEC-negative sample contained an eae- and hlyA-negative E. coli K12 strain (# 1725342).

With the exception of samples # 1725344 (stx_2f-positive EHEC isolate) and # 1725344 (containing a relatively low amount of an stx_2d-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 111 of 113 participants.

With an amount of slightly above 10³ CFU/mL of stx-positive EHEC target organisms, the lower limit of detection of appropriate PCR/NAT test systems is obviously reached or at least touched to a certain extent for the weak-positive sample # 1725344.

The reason for the 49 false-negative results for the stx_2f-positive EHEC isolate (# 1725343) 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. This is impressively demonstrated by the inclusion of the stx_2f-positive sample # 1725343 where only 88 of the 113 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. With an amount of slightly above 10³ CFU/mL of stx-positive EHEC target organisms, the lower limit of detection of appropriate PCR/NAT test systems is obviously reached or at least touched to a certain extent for sample # 1725344. Therefore we have not scored those (false) negative results for the stx_2f-positive sample and for the weak-positive EHEC sample # 1725344 in the course of issuing the corresponding EQAS certificates. This is characterized by the three gray-shaded boxes for both samples in Tab. 2 (Attachment 1 [Attach. 1], p. 7).

Except two false-positive results with the “negative” sample # 1725342 (containing an eae- and hly-negative E.coli K12 strain), which are presumably due to carry-over from the strong positive sample # 1725341, all of the remaining results reported by the 113 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 106 of the 113 participating laboratories. With three exceptions, 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 scheme (EQAS) also other B. burgdorferi genotypes or genospecies will be present in individual samples.

Short recapitulation: So far 21 different species belonging to the B. burgdorferi sensu lato complex were described, that naturally present genetic differences in commonly used target genes. For specificity testing, B. miyamotoi was included. This species was first described in 1994 from Japan, belongs to the relapsing fever group spirochetes but is transmitted by the same hard ticks as B. burgdorferi s.l. B. miyamotoi meanwhile was detected from Ixodes ticks from the USA, Asia and Europe. In 2011 first human cases were described from Russia, later on from the USA, Japan and Europe. Symptoms comprise fever, chills, headache, muscleache, arthralgias and nausea. Diagnosis relies on stained blood smears and PCR. For therapy doxycyclin is recommended, in more severe cases ceftriaxone or penicillin G.

The current distribution of QC samples contained one sample with Borrelia spielmanii (# 1725352; ~5x10⁴ organisms/mL), one sample with Borrelia miyamotoi (sample # 1725353; ~5x10⁴ organisms/mL) and one sample with Borrelia afzelii (sample # 1725354; ~1x10⁵ organisms/mL). Sample # 1725351 contained a low number of Borrelia afzelii (~5x10² organisms/mL).

With the exception of 3 false-negative results for sample # 1725352 (~5x10⁴ B. spielmanii organisms/mL) and 34 false-negative results as well as 5 results classified as “questionable” for sample # 1725351 (containing a very low amount of Borrelia target organisms), all participants reported correct results for the 3 positive samples. The three false-negative results for sample # 1725352 should prompt serious re-evaluation of the assay sensitivity.

The B. burgdorferi sensu lato complex “negative” sample # 1725353 containing ~5x10⁴ organisms/mL of Borrelia miyamotoi was classified false-positive by 68 and “questionable” by one of the 93 participating laboratories. Potentially, this is either due to cross-reactivity with this genetically very closely related spirochete or due to contamination events during sample preparation or analysis.

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 EQA scheme is designed exclusively for the testing of NAT-based methods and protocols for direct detection of low amounts of Legionella pneumophila from appropriate clinical specimens. Individual samples may contain relatively small amounts of the corresponding target organism. For this reason, participation is promising only for diagnostic laboratories, which have established a highly sensitive and specific PCR-/NAT-based method for the detection of L. pneumophila DNA or who want to evaluate their method with the help of an external quality control scheme.

The current set of QC samples contained two positive samples with Legionella pneumophila serogroup 2 (# 1725362; ~5x10⁵ CFU/mL and # 1725361; ~5x10³ CFU/mL) next to one sample containing Legionella londiniensis (# 1725363; ~5x10⁴ CFU/mL). Sample # 1725364 contained no target organisms but only human cells and E. coli cells.

The L. pneumophila-positive sample # 1725362 was correctly tested positive by 97 of the 98 participating laboratories. Sample # 1725362, which contained a relative low amount of L. pneumophila target organisms in the current distribution, was reported positive by only 54 of the 98 participating laboratories. Although sample # 1725362 was not considered in the course of issuing the corresponding EQA certificates, laboratories with negative results should consider to investigate and to improve the analytical sensitivity of their individual PCR/NAT assay concepts.

Sample # 1425362, which contained ~5x10⁴ CFU/mL of Legionella londiniensis, was classified false-positive by one of the participating laboratories. Observing false-positive L. pneumophila PCR results for non-pneumophila Legionella spp. should encourage the correspoding participants to review and optimize the analytical specificity of their “L. pneumophila-specific” assays and/or PCR protocols. Briefly, L. londiniensis has been frequently isolated from aqueous environments but not from clinical specimens. A single case of Legionnaires' disease in a hematopoietic stem cell transplant recipient caused by this organism is described, which confirms that L. londiniensis can be an opportunistic pathogen [3].

All but one of the 98 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 positive samples with Salmonella enterica. A relatively high amount of the corresponding target organism was present in sample # 1725371 (Salmonella enterica serovar Enteritidis, ~1x10⁴ CFU/ mL) and a similar amount was present in sample # 1725373 (Salmonella enterica serovar Typhimurium, ~1x10⁴ CFU/ mL). No target organisms but only E. coli cells were present in samples # 1725372 and # 1725374.

Only one false-negative result was reported for the positive sample # 1725373, 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 23 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 a sample without the corresponding target organisms (# 1725383; only E. coli cells), two samples positive for L. monocytogenes (# 1725384 with ~1x10⁵ CFU/mL and # 1725381 with ~1x10⁴ CFU/mL) and one sample with Neisseria meningitidis (# 1725382) as a Listeria species other than L. monocytogenes.

The Listeria monocytogenes-containing samples (# 1725381 and # 1725384) were correctly reported positive by all but two participants. In addition, the “negative” E. coli-containing sample # 1725383 as well as the “negative” N. meningitidis sample # 1725382 were correctly identified as negative by all of our participating laboratories. Thirty-one of the 32 participants indicated the use of Listeria monocytogenes-specific PCR/NAT assays. Although this particular EQA scheme is formally focussed on Listeria spp., and may therefore contain non-Listeria monocytogenes species from time to time, it is noted in the report form 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 and/or community acquired (CA)-MRSA DNA in typical 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 natural background of human cells and other components. Here it is important to note that NAT assays designed for MRSA culture confirmation purposes may fail due to the low number of MRSA organisms in individual samples of the EQA panels. Despite of one sample containing an MSSA isolate together with a methicillin-resistant coagulase-negative Staphylococcus species, no “difficult” or “interesting” sample was included into the current panel. Sample # 1725392 contained a cMRSA or CA-MRSA isolate (MRSA, PVL-positive, spa:t 310; ~5x10⁵ CFU/mL) which tested positive with the MRSA-specific assays in 268 of the 272 participating laboratories. The 4 false-negative results for this sample could be due to an insufficient analytical sensitivity of the used in-house or commercial PCR/NAT test systems or certain problems in the preanalytical or analytical sample workup.

The MRSA-negative sample # 1725393, which contained no target organisms but only E. coli cells, was correctly tested negative by 267 of 272 participants with their PCR-based MRSA-specific test systems.

The other one of the remaining MRSA-negative samples of the current set (# 1725394), contained a mixture of S. aureus (MSSA, PVL-negative, ~5x10⁴ CFU/mL) and a CoNS strain (S. epidermidis; mecA-positive, ~5x10⁴ CFU/mL), and was correctly tested negative by only 264 of the 272 participants. It should be noted that the S. aureus strain in the latter sample does not contain any residual segments of SCCmec cassettes or one of these well-known mecA deletions.

So the 5 participants who observed a false positive result for sample # 1725393 and the 8 participants who observed a false positive result for sample # 1725391 should carefully investigate their PCR/NAT assay concepts and workflows for potential contaminatione events with MRSA DNA or residual amplicon carry-over from previous rounds of PCR analysis during sample preparation, amplification or detection. Think that we are all aware about clinical consequences of reporting false-positive MRSA results….

For the sample # 1725394 of the current distribution, which contained a mixture of S. aureus (MSSA, PVL-negative, ~1x10⁵ CFU/mL) and a Methicillin-resistant coagulase-negative Staphylococcus species (S. epidermidis; mecA-positive, ~1x10⁵ CFU/mL), 245 of 272 participants reported correct MRSA-negative results and 9 participants classified the results as “questionable”. Six of these 9 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 the separate detection of mecA and S.aureus target sequences, the origin of the mecA target gene can not 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 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 18 participants, who reported false-positive MRSA results for sample # 1725394, are strongly encouraged to analyse the appropriateness of their PCR/NAT 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, skin or wound swabs. On the other hand, all participants, who used SCCmec-based PCR test systems, reported correct MRSA-negative results for sample # 1725394.

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.

Also, an optional molecular detection of putative pathogenicity factor PVL (Panton-Valentine Leukocidin) or its coding gene lukF/S-PV was possible in the concept of our EQA scheme for MRSA/cMRSA. Corresponding results were reported by 57 of the 272 participating laboratories and within the current distribution the results for the molecular PVL testing were correct in all but two cases. Additional information can be found in [4] or [5]. A well evaluated protocol for the detection of PVL-positive PVL isolate can be found in [6].

In addition, commercial real-time PCR assays reliably targeting PVL genes in MRSA and MSSA isolates are available from a number of 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 (clinical) sample material. With the development and composition of the corresponding sample materials we intended to mimic the situation of processing typical clinical samples like BAL or other respiratory specimens. So the lyophilized samples usually contain low amounts of target organisms in a natural background of human cells and other components. As a consequence, diagnostic assays designed for C. pneumoniae antigen detection in clinical specimens or other serological assays will fail due to the low number of C. pneumoniae-infected cells in individual samples of the QC set. The current set of QC samples contained two samples positive for C. pneumoniae. Sample # 1725403 was spiked with ~1x10⁶ IFU/mL of C. pneumoniae, whereas sample # 1725402 contained an approximately hundred fold lower amount of C. pneumoniae (~1x10⁴ IFU/mL). Sample # 1725401 contained significant numbers of Haemophilus influenzae (~1x10⁴ CFU/mL). Only E. coli and human cells were present in sample # 1725404.

As depicted in Tab. 2 (Attachment 1 [Attach. 1], p. 13), with one exception all of the 109 current participants reported correct results for the very strong C. pneumoniae-positive sample # 1725403 (1x10⁶ IFU/mL !). For sample # 1725402, which contained an approximately hundred-fold lower number of target organisms, two false-negative results were reported among the 109 participants. For both of the C. pneumoniae-negative samples in the current distribution, # 1725404 and # 1725401 (which contained Haemophilus influenzaeas a kind of assay specificity challenge), all but one laboratories reports correct negative results. The two sporadically observed false-positive results could be due to simple (cross-)contamination events in the course of sample processing and extraction, as a molecular or technical cross-reactivity of C. pneumoniae-specific NAT/PCR assays with E. coli or H. influenzae DNA is unlikely. Certainly, a false-positive result should 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 two positive samples. A relatively high amount of M. pneumoniae (~1x10⁵ genome copies/mL) was present in sample # 1725413. An approximately tenfold lower amount of M. pneumoniae (~1x10⁴ genome copies/mL) was present in sample # 1725411. The set was completed by samples # 1725412 and # 1725414, which contained only human cells and a considerable amount of E. coli. With the exception of 3 laboratories, all participants correctly reported the sample # 1725413 as positive. The second “positive” sample with a ten-fold lower amount of the target organism (# 1725411) was correctly identified by 116 participants. The E. coli-containing “negative” samples were correctly reported by 119 and 122 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. 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 (Tab. 1: Attachment 1 [Attach. 1], p. 15) contained two samples with different amounts of C. burnetii DNA (~1x10⁴ genome copies/mL in sample # 1725424 and ~1x10⁵ genome copies/mL in sample # 1725422) and two samples with different amounts of B. anthracis strain UR-1 DNA (~1x10⁴ genome copies/mL in sample # 1725421 and ~1x10⁵ genome copies/mL in sample # 1725422). Sample # 1725423 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. 15) for the C. burnetii-specific results and Tables 4 and 5 (Attachment 1 [Attach. 1], p. 16) for the B. anthracis-specific results.

Coxiella burnetii: The relatively high amount (1x10⁵ genome copies/mL) of C. burnetii organisms in sample # 1725422 was correctly reported by 30 of the 31 participants. The participant reporting a false-negative result should reassess the performance of the test system used and evaluate processes of sample work-up and analysis. The ten-fold lower concentration of the pathogen in sample # 1725424 was correctly identified by all participating laboratories. The two “negative” samples (# 1715422 contained only E. coli and # 1715421 contained only B. anthracis) were correctly reported as negative by all participants. Overall, there were no noticeable problems with the current set of QC samples and a good correlation with the expected results was observed.

Bacillus anthracis: With the exception of one “questionable” result, all participants correctly reported negative results for the samples # 1725423 and # 1725424 which did not contain the target organism. The “positive” sample # 1725421 containing ~1x10⁴ genome copies/mL B. anthracis strain “UR-1” was correctly reported by all 15 participants. The second positive sample # 1725422 (~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 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 (Tab. 1: Attachment 1 [Attach. 1], p. 17) contained three positive samples: a high amount of Francisella tularensis spp tularensis (~5x10⁵ CFU/mL) was present in sample # 1725434, an approximately ten lower amount (~5x10⁴ CFU/mL) was present in sample # 1725433, and ~1x10⁴ CFU/mL of Francisella tularensis spp. novicida was present in sample # 1725432.

Both samples with the highest amount of target organism (# 1725433 and # 1725434) were correctly identified as “positive” by all participants (Tables 2 and 3: Attachment 1 [Attach. 1], p. 17). Even with pathogen amounts of ~1x10⁴ CFU/mL (sample # 1725432) 15 out of 18 labs were able to detect Francisella DNA. No false-positive results were observed for the “negative“ sample # 1725431. Overall, these results corroborate the lower limits of detection observed in our previous EQAS distributions. Although the number of participating laboratories is still not very high, the results of the present distribution indicate that the lower limit of detection is about or slightly below 10⁴ organisms/mL when using currently employed and well evaluated PCR/NAT-based assay concepts for the detection of F. tularensis DNA in a sample harbouring even non-target DNA as it is given in clinical specimens.

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 Tab. 1 (Attachment 1 [Attach. 1], p. 18), the current set contained three samples with different carbapenem-resistant Enterobacteriaceae: sample # 1725441 contained an Citrobacter freundii complex isolate with two carbapenemase genes: KPC-3 and VIM-4 (~1x10⁷ genome copies/mL), sample # 1725442 contained an Serratia marcescens isolate with a OXA-48 gene (~1x10⁶ genome copies/mL) and sample # 1725443 contained an Enterobacter cloacae complex isolate with a NDM-7 gene (~1x10⁶ genome copies/mL). The fourth sample # 1725444 was designed as negative control and contained only E. coli without carbapenemase genes. All 62 participating laboratories reported sample # 1725441 (Citrobacter freundii complex carrying a KPC-3 and VIM-4 carbapenemase) as “carbapenemase-positive”. Notably, all participants were also able to detect carbapenemase genes in sample # 1725442 (S. marcescens carrying OXA-48). The third “positive” sample # 1725443 (containing Enterobacter cloacae complex with a NDM-7 gene) was correctly reported by 58 of the 62 participants, 4 laboratories missed the NDM-7 gene. A false-negative result for carbapenemase gene-positive samples should prompt investigations regarding the coverage of carbapenemase genes by the test system used.

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 three Clostridium difficile-positive samples: sample # 1725453 with ~1x10⁵ CFU/mL, sample # 1725451 with ~1x10⁴ CFU/mL, and sample # 1725452 with ~5x10³ CFU/mL. Sample # 1725454 contained only human cells and a considerable amount of E. coli organisms.

The samples # 1725451 and # 1725453 containing relatively high amounts of C. difficile (1x10⁴ CFU/mL and ~1x10⁵ CFU/mL) were with one exception correctly reported as “positive”. 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 # 1725454, containing only E. coli, but no target organism. As cross-reaction of the applied test system with E. coli DNA is unlikely, probably cross-contamination during the process of sample preparation and analysis is causative. Sample # 1725452 with the lowest amount of target organism (~5x10³ CFU/mL) was correctly identified as positive by 93 participants. Again a false-negative result should prompt re-assessment of the sensitivity of the used test system. 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 # 1725463 of the current set contained a relatively high amount of Enterococcus faecium vanB (~5x10⁵ CFU/mL) and sample # 1725462 contained an approximately hundred lower amount Enterococcus faecium vanA (~5x10³ CFU/mL). Samples # 1725461 and # 1725464 contained no target organisms but only human cells and E. coli. Of the 38 participating laboratories, 37 correctly reported positive results for the samples # 1725462 and 1725463. Of note, the reported dedicated vanA/vanB identifications for these two samples were all correct. All laboratories correctly reported sample # 1725464 as “negative”. The false-positive result for sample # 1725461 should prompt investigations of the laboratory processes regarding sample processing. 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, 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 suitable clinical sample material. With the development of diagnostic material similar to clinical samples 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 Tab. 1: Attachment 1 [Attach. 1], p. 21)). A relatively high concentration of Pneumocystis jirovecii (~1x10⁵ organisms/mL) was present in sample # 1725602, whereas in sample # 1725604 Pneumocystis jirovecii (~5x10³ organisms/mL ) was present at an approximately ten-fold lower concentration. The set was completed by samples # 1725601 and # 1725603 which contained only human cells and a considerable amount of E. coli organisms.

Sample # 1725602, which contained P. jirovecii target organisms (~1x10⁵ organisms/mL) at highest concentration and sample # 1725604 with a twenty-fold lower concentration of P. jirovecii, were both reported “positive” by 86 and 85 of the 87 participating laboratories, respectively. Although this could be due to a loss of template DNA during pre-analytical sample preparation procedures or other reasons like an insufficient lysis procedure of the almost intact target organisms in the lyophilized sample material, observation of false-negative results should certainly trigger reassessment of the diagnostic workflow, sensitivity and/or specificity of the individual assay concept. The Pneumocystis-negative samples (# 1725601 and # 1725603, containing only E. coli and a suspension of human cells) were correctly classified “negative” by all but one participant, respectively. In general, observing false-positive or questionable results for negative samples within our EQAS panels should definitely prompt investigations regarding all processes involved in sample preparation and analysis in order to optimize the applied PCR/NAT workflows, test kits or assay protocols.