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 two samples with similar amounts of C. trachomatis (~1x10³ IFU/mL and ~5x10³ IFU/mL; sample # 1315303 and sample # 1315301), two samples with equal amounts of N. gonorrhoeae (sample # 1315302 and sample # 1315304; ~1x10⁴ CFU/mL) and sample # 1315301 with ~1x10³ CFU/mL of N. gonorrhoeae.

Despite relatively low amounts of C. trachomatis target cells in the positive samples #1315301 and # 1315303, among the Chlamydia trachomatis-specific results reported by the 170 participants, only 7 false-negative results were observed. Among the N. gonorrhoeae-specific results, false-negative results were reported by 25 of the 170 participants for sample # 1315301, which contained N. gonorrhoeae target organisms in an amount of 1x10³ CFU/mL, next to 5x10³ CFU/mL of C. trachomatis organisms. For GO-positive samples # 1315302 and #1315304, which contained with 5x10⁴ CFU/mL similar amounts of N. gonorrhoeae target organisms, interestingly only 4 participants failed to detect N. gonorrhoeae DNA and only 8 false-negative results were reported from 170 participants. The reason for this finding, which was similarly observed in the November distribution, remains unclear. Competition of certain of primer sequences present in the complex reaction mixture could be a possible explanation. But one could assume that for chlamydial and gonococcal (duplex) NAT systems, specific amplification of specific genomic regions of these two different target organisms is realized via two different primer sets and the use of consensus primers is avoided. Since the amount of target organisms in sample # 1315301 (1x10³ CFU/mL) could not be considered as “extremely low“, false negative results should encourage the participants to review and optimize their respective gonococcal specific NAT-based test system.

The current set of QC samples contained two samples with similar amount (~1x10³ IFU/mL and ~5x10³ IFU/mL) of C. trachomatis target organisms (# 1315314 and # 1315312) and two samples without target organisms (# 1315311 and # 1315313) containing only non-infected human cells and Escherichia coli.

As depicted in Tab. 2 of statistical analysis (see Attachment 1 [Attach. 1], p. 4) for both positive samples the results were correct while for sample # 1315314 false-negative results were reported by only 2 of the 115 participants. For both C. trachomatis-negative samples # 1315311 and # 1315313, only 3 false-positive and 3 results classified as “questionable” were observed of the 115 participants. This striking match of the current results with observations and accuracy rates in previous interlaboratory tests can, at least indirectly, be considered as evidence of a high reliability and consistency of the used test systems and the processing of the samples.

Run controls were performed by 113 of the 115 participants and inhibition events were observed at 2 of the 460 test results. 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 specifity were observed for the C. trachomatis-specific NAT assays used by the 115 participants of the current distribution.

The current set of QC samples contained one sample with a relatively high amount of Bordetella pertussis (# 1315322; 1x10⁵ CFU/mL), one sample with an approximately hundredfold lower number of Bordetella pertussis (# 1315323; 1x10³ CFU/mL), as well as two samples containing only non-infected human cells and Escherichia coli (# 1315321 and # 1315324).

The availability of well-established commercial or in-house NAT-assays has led to a high portion of correct results. Only 1 of the 145 participants reported false-positive results for the B. pertussis-negative sample # 1315321. The false-positivity issue is probably due to contamination events in the course of sample preparation. A cross reaction due to a possible low specificity of the used PCR/NAT test system is unlikely, because the sample contained only E. coli cells. For sample # 1315323 (10³ CFU/mL of B. pertussis) 6 false-negative results were observed. With an amount of 10³ CFU/mL of B. pertussis target organisms the lower limit of detection of corresponding PCR assays or test systems is obviously reached.

For the detection of B. pertussis, most participants used in house test concepts with inhibition and/or positive controls. In the current distribution, 71 participating laboratories indicated the use of the IS481 insertion sequence, 12 the B. pertussis toxin coding gene and 3 the use of B. pertussis-specific ribosomal gene segments.

The current set of QC samples contained two samples with a relatively high amount of target organisms. Sample # 1315331 contained approximately 1x10⁶ CFU/ml of a Clarithromycin-susceptible Helicobacter pylori patient strain, and sample # 1315333 contained approximately 1x10⁴ CFU/ml of a Clarithromycin-resistant Helicobacter pylori. Only one of the 48 participants reported false-negative results for sample # 1315332, which contained only a suspension of E. coli. Therefore, cross reaction caused by low specifity of the used test is unlikely. Sample # 1315334 contained a culture suspension of Campylobacter jejuni (~1x10⁵ CFU/ml), which therefore was correctly reported “negative“ by all of the 48 participants – indicating an overall good analytical specificity of the used PCR test systems.

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 the amplification and sequencing of characteristic areas within the H. pylori 23 S rDNA or the sequence analysis of this genomic region by means of hybridization probes. Results were communicated by 32 of the 48 participants, and 27 of the results for molecular susceptibility testing were also 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 QC samples contained two samples positive for EHEC: # 1315344 (E. coli, 1x10⁵ CFU/mL, clinical isolate, stx₁-, stx₂-, eae-, hlyA- and O157-positive), and # 1315343 (E. coli, 1x10⁴ CFU/mL, clinical isolate, stx_2c-positive, EAEC positive, stx₂-negative, eae-negative, and hlyA-negative). Sample # 1315341 contained ~1x104 CFU/mL of Salmonella enterica serovar typhi. The other EHEC-negative sample contained an eae- and hlyA-negative E. coli K12 strain (# 1315342).

Sample # 1315343 contained a very interesting stx_2c-positive EHEC isolate that contains genetic markers for enteroaggregative E. coli (EAEC) in addition to the shiga toxin gene. Even if this pathogenetic constellation is rather unusual, it is a good example for the surprising variety of clinical strains which may be encountered in microbiological routine PCR diagnostics of EHEC.

By the way, a number of PCR protocols have been developed for the genotypic detection of EAEC strains. Large plasmids, which are present in most EAEC strains and carrying genes coding for bundle-forming fimbriae and the production of EAEC heat-stable enterotoxin 1 (EAST1), are representing potential targets for a specific and sensitive detection. PCR assays targeting a cryptic open reading frame, which is adjacent to the plasmid replicon, turned out to be practicable as screening tests by detecting EAEC strains with a specificity and sensitivity of about 90%.

But now back to the current EHEC external quality assessment scheme: the availability of well-established NAT assays lead to high accuracy rates for correct results and 103 of the 110 participants reported correct results. The cause for the 5 false-negative results for stx-1c positive EHEC (# 1315343) and the 3 false-negative results for stx-1- and stx-2-positive EHEC isolate (#1315344) remains unclear. Maybe the common spectrum of stx-1 and stx-2 genes is not fully covered by the applied test systems. A false negative result in some PCR test systems could have been expected for stx-2f, because of the known little homology to other shiga toxin gene sequences, but a stx-2f EHEC positive isolate was not included this time. Interestingly, mainly commercial assays were affected producing false-negative results of sample # 1315343 and # 1315344. For sample # 1315341 containing significant amounts of Salmonella enterica Serovar Typhi, fortunately none of the 110 participants reported false positive results.

Since 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 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 were performed by 99 of the 110 participating laboratories and the reported results were correct when performed.

Due to numerous requests, here is 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 18 different species belonging to the B. burgdorferi sensu lato complex were described, that naturally present genetic differences regarding popular target genes. Of special interest – since of assured human pathogenicity and widely distributed in Europe – are B. burgdorferi sensu stricto, B. afzelii, B. garinii, B. spielmanii and B. bavariensis. B. bissettii, B. lusitaniae und B. valaisiana are regarded as possibly human pathogen. Regarding OspA especially B. garinii showed a striking heterogeneity with 5 genetic distinguishable “genotypes”.

The current set of QC samples contained a kind of dilution series from Borrelia garinii organisms. A relatively high amount of Borrelia garinii (~1x10⁶ organisms/mL) was present in sample # 1315353, which consequently was tested positive by 117 of the 119 participating laboratories. Sample # 1315352 contained ~1x10⁵ Borrelia garinii organisms per mL (detected by 112 participants) and sample # 1315351 contained ~1x10⁴ organisms/mL still detected by 108 participants. For sample # 1315354, which contained a very low amount of target organisms (~1x10³ organisms per mL), reported positive results dropped to 83 of the 119 participants. The last sample can be regarded as an educational one, the aim for the (near) future for the ambitious PCR.

The participants who reported false-negative results especially for the two high positive samples should check their NAT assay concepts including sample processing/DNA-extraction.

Only just under half of the participating laboratories used self-developed (in-house) tests with inhibition and/or positive controls. No inhibition and also no obviously significant differences to commercially available kits regarding sensitivity could be observed.

Next to the three commercial assays provided with a designated code number (Qiagen artus Borrelia LC Kit (Code [20]), Demeditec GenFlow (Code [21]) and LightMix Borrelia from TIB Molbiol (Code [22]), participants indicated the use of the following commercial assays or kits on their report form: GeneProof Borrelia burgdorferi PCR Kit (9x), EliGene Borrelia LC from Elisabeth Pharmacon (3x), Autoimmun Diagnostika GenID Zecken Screening Kit (2x), TIB Molbiol LightMix Borrelia (1x), EliGene Borrelia RT from Elisabeth Pharmacon (1x), Artus Borrelia LC Kit (1x), BactoReal B. burgdorferi from Ingenetix (1x), Attomol Borrelia burgdorferi Realtime (1x), Attomol Borrelia burgdorferi-DNA-LINA (1x), RapidSTRIPE Borrelia Assay from Analytik Jena (1x), und Borrelia real time PCR kit from Gerbion (1x).

Due to numerous requests: this ring 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 a participation is promising for only for such diagnostic laboratories, which have established a highly sensitive and specific PCR-based method for the direct detection of L. pneumophila DNA or who want to evaluate this method in the course of an external quality control.

The current set of QC samples contained only two positive samples with Legionella pneumophila serogroup 3 (# 1315363; ~1x10⁵ CFU/mL and # 1315364; ~1x10⁴ CFU/mL), as well as one sample containing Legionella micdadei (# 1315362; ~5x10⁴ CFU/mL). Sample # 1315361 contained no target organisms but only human cells and E. coli cells.

The L. pneumophila-positive (~1x10⁵ CFU/mL) sample # 1315363 was correctly tested positive by all of the 98 participating laboratories. Sample # 1315364, which contained (~1x10⁴ CFU/mL) L. pneumophila reported correctly by 92 of 98 participants.

Sample # 1315362 contained considerable amounts (5x10⁴ CFU/mL) of Legionella micdadei, whose DNA showed no cross reactivity for 91 participants. The 6 participants with false-positive results were using mainly self-developed in-house real-time PCR protocols with omp-, mip-gene or 16S rDNA as target sequence.

However, colleagues who observed a cross-reaction with their in-house assays and are aiming at a high analytical performance should consider improving the sensitivity and/or checking the species coverage of their individual assay concepts.

Participants who reported false-positive results used mainly in house real-time PCR protocols with 16S rDNA-, omp- and mip-coding genes or a nested block-cycler PCR protocol with subsequent nucleotide sequencing of the amplified 16S rDNA region, which should allow however the distinction between L. pneumophila and L. micdadei in any case, since these species clearly differ at the level of the ribosomal marker genes. The presence of contamination events however is unlikely due to sample order.

The current set of QC samples contained three positive samples with Salmonella enterica. A relatively high amount of the corresponding target organism was present in sample # 1315374 (Salmonella enterica serovar Enteritidis, ~5x10⁵ CFU/ml), a similar amount was present in sample

# 1315371 (Salmonella enterica serovar Typhi, ~1x10⁵ CFU/ml), and an approximately tenfold lower amount was present in sample # 1315372 (Salmonella enterica serovar Typhi, ~1x10⁴ CFU/ml). No target organisms but only E. coli cells were present in sample # 1315373.

Only 3 false-negative result was reported for the positive samples # 1315372 and # 1315374, one false-positive result was reported for the negative sample # 1315373.

In summary, 10 correct results for the negative sample # 1315373 as well as the positive sample # 1315371 with relatively high amount of target organisms were reported correctly for the NAT-based detection of Salmonella by the 11 participants. Only the slightly weaker positive sample (# 1315372; Salmonella enterica serovar Typhi, ~ 1x10⁴ CFU/mL) was reported false negative by 2 participants and for # 1315374 (Salmonella enterica serovar Enteritidis ~ 5x10⁵ CFU/mL) one false negative result was reported.

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 two samples without the corresponding target organisms (# 1315382 and # 1315383; only E. coli cells), one sample positive for L. monocytogenes (# 1315384 with 1x10⁴ CFU/ml) and one sample with Listeria ivanovii (# 1315381) as a Listeria species other than L. monocytogenes.

In order to test the specificity of individual test systems, the sample # 1315381 contained relatively high amounts of non-monocytogenes species: L. ivanovii. In contrast to some of the previous rounds of our external quality assessment schemes, where the almost exclusive use of L. monocytogenes-specific NAT test systems had led to no positive results, the sample with L. ivanovii was reported positive for Listeria spp. by three of a total of 31 participants. Once again, this constellation is caused by the almost exclusive use of L. monocytogenes-specific NAT-testsystems. On the other hand, these results indicate a pleasingly high specificity of the used L. monocytogenes-specific test systems in the laboratories of 30 participants.

In order to assess the analytical sensitivity and check the lower detection limit of the currently used NAT assays, we decided to include also weak positive samples in the current distribution. Relatively low numbers of L. monocytogenes cells (~1x10⁴ CFU/mL) were present in sample # 1315384 and the corresponding DNA preparations tested positive by the PCR assays applied by 30 of the 31 participants.

No false-positive results were observed for samples # 1315382 and # 1315383 containing only human and E. coli cells, so it seems that the majority of participating laboratories have implemented functional precautions to prevent deleterious contamination events.

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 QC set.

Despite of the “interesting“ sample # 1315393, no “difficult” sample was included into the current panel. Almost consistently correct PCR results were observed for 3 of the 4 samples of the current panel by a total of 259 participants with their NAT-based test systems.

Sample # 1315393 of the current set contained a relative low amount of La-MRSA organisms (PVL-negative, ~1x10³ CFU/ml).

Sample # 1315391 contained a methicillin-resistent S. aureus isolate (MRSA, PVL-negative, ~1x10⁴ CFU/ml). Sample # 1315394 contained a clinical isolate of a methicillin-resistent S. haemolyticus strain (mecA-positive, PVL-negative (~1x10⁴ CFU/mL). Negative sample # 1315392 consisted of human cells with considerable amounts of E. coli cells.

The two MRSA negative samples, # 1315394 and # 1315392 were tested by 257 or 252 respectively of 259 participants with their PCR-based MRSA-specific test systems as “negative“ and only one participant observed a false positive result for sample # 1315392. One participant reported a result classified as “questionable”. Sample # 1315394 (mecA-positive S. haemolyticus) was reported with a false positive result by 7 participants. These 7 false positive results may have been caused probably most likely occurring in isolated events of contamination with MRSA DNA during the sample preparation, amplification or detection or by insufficient analytical specificity of the used test system.

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

The apparent “lack of recognition“ for the MRSA positive sample # 1315393 is quickly explained on closer inspection. This sample contained a rather exotic S. aureus patient isolate, which belongs to the group of so-called lifestock associated (LA)-MRSA. These S. aureus isolates with Oxacillin resistance, are typically of MLST type 398 and contain a functional mecA gene on the genomic level, but differ slightly in the nucleotide sequence of SCCmec-orfX region from typical MRSA isolates. These differences may under certain circumstances lead to false negative results for LA-MRSA with some commercial or in-house SCCmec-based PCR test systems and can so not be clearly identified as MRSA.

As obvious from the naming, LA-MRSAs can be found in the agricultural or veterinary field and are gaining importance from a diagnostic perspective (for example in the definition of “Risk groups“ for PCR-based MRSA screenings). For further information: [1], [2], [3].

For the current set of QC samples correctly positive results for the LA-MRSA-positive sample # 1315393 were observed by 246 participants, a false negative result was reported by 10 participants and 3 participants have classified the result as ”questionable”. These latter 3 participants reported the use of the Roche LC MRSA Advanced test. Since a different (~ 4°C lower) melting point of the hybridization probes is observed in this real time PCR assay when LA-MRSA organisms are present (see the above mentioned Eurosurveillance paper), the “false-negative“ classification of the results with sample # 1315393 are a simple consequence of the underlying assay result interpretation software. However, due to the relatively low amount of target organisms in the sample material, a negative result was not assessed as “false negative” irrespectively of the PCR/NAT test system used.

Altogether the observed results indicate, apart from sample # 1315393, a high reliability of the NAT-based direct detection of MRSA in clinical specimens. In the context of our test series the 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 184 of the total 259 participating laboratories and within the current distribution the results for the molecular PVL testing were correct in all but one case.

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 # 1315402 was spiked with ~1x10⁶ IFU/ml of C. pneumoniae whereas sample # 1315404 contained an approximately thousand fold lower number of C. pneumoniae (~1x10³ IFU/ml). Sample # 1315401 contained significant numbers of Legionella pneumophila organisms to assess analytical specificity. Only E. coli and non-infected human cells but no C. pneumoniae target organisms were present in sample # 1315403.

As depicted in Tab. 2 (see Attachment 1 [Attach. 1], p. 14), all participants reported correct results for the positive sample # 1315402. For positive sample # 1315404 with a lower concentration of target organisms (10³ IFU/ml), still 104 from the 109 participants observed correctly positive results. Due to the relatively low amount of target organisms, results were not assessed in the course of issuing the certificates, but the 5 affected laboratories are encouraged to improve their diagnostic workflow or to check the analytical specificity of their PCR/NAT assays. Comparable results from former rounds of our external quality assessment schemes (November 2008 and April 2009) let assume, that the “true“ lower limit of detection is more in the scale of 10² IFU/mL of C. pneumoniae in appropriate sample material.

No participants reported false-positive results for sample # 1315401 (Legionella pneumoniae) and only 2 false-positive results were observed for the “negative“ sample # 1315403, which could be due to cross-contamination events in the course of sample preparation, amplification or amplicon detection steps. Overall there were no noticeable problems with the current set of QC samples and a good overall correlation with the expected results was observed.

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 want to mimic the situation of processing typical clinical samples like BAL or other respiratory materials. 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. As a consequence, diagnostic assays designed for M. pneumoniae antigen detection in clinical specimens or other serological assays will fail due to the low number of M. pneumoniae infected cells in individual samples of the RV 541 distributions.

The current set of QC samples contained three positive samples. A relatively high amount of M. pneumoniae (~1x10⁶ genome copies/mL) was present in sample # 1315414, an approximately tenfold lower amount of M. pneumoniae (~1x10⁵ genome copies/mL) was present in sample # 1315412 and an approximately hundred fold lower amount of M. pneumoniae (~1x10⁴ genome copies/mL) was present in sample # 1315413. The set was completed by sample # 1315411, 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 Mycoplasma pneumoniae PCR/NAT detection, the availability of well-established commercial or in-house PCR/NAT-assays has led to a surprisingly high percentage of correct results, at least for 3 of the 4 samples of the current set.

With the exception of one laboratory, all 119 participants reported correct M. pneumoniae-positive results for the relatively high positive sample # 1315414 and no participant reported false-negative results for sample # 1315412, which contained 10⁵ M. pneumoniae genome copies/mL.

The weak positive sample # 1315413 (10⁴ genome copies/mL) was tested correctly positive by 109 of the 119 participants and 2 participants classified their results as “questionable”.

One participant reported false-positive results for the negative sample # 1315411, which could be due to cross-contamination events in the course of sample preparation, amplification or amplicon detection steps.

Overall, it seems that the participating laboratories have implemented functional precautions to prevent deleterious contamination events.

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 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 contained two samples with different amounts of Coxiella burnetii organisms (~1x10³ genome copies/mL in sample # 1315421 and ~1x10⁵ genome copies/mL in sample # 1315422), two samples with ten-fold different amounts of Bacillus anthracis (sample # 1315421 with ~5x10³ genome copies/mL and sample # 1315424 with ~5x10⁴ genome copies/mL) Sample # 1315423 contained only human cells and a considerable amount of E. coli organisms.

For the ease of data presentation and analysis, we decided to depict the PCR/NAT results for the two different target organisms within this combined EQAS scheme in two separate tables: please see Tab. 2 and 3 for the Coxiella burnetii-specific results and Tab 4 and 5 for the Bacillus anthracis-specific results (Attachment 1 [Attach. 1], p. 16 and 17).

A relatively high amount (10⁵ genome copies/mL) of C. burnetii organisms were present in sample # 1315422, which consequently tested positive by all of the 21 participating laboratories. Sample # 1315421, which contained an approximately hundred-fold lower number of C. burnetii target organisms per mL, was also tested positive for C. burnetii DNA by nearly all of the 21 participants. Similar to the result observed in past distributions of our external quality assessment schemes, difficulties for the reliable detection of C. burnetii DNA occurred sporadically for samples at or below 10³ genome copies per mL – this target load should probably be considered as the lower limit of detection for this pathogen using current PCR/NAT assay concepts. Overall, there were no noticeable problems with the current set of QC samples and a good correlation with the expected results was observed.

The results for this newly introduced ring test is quickly presented and discussed. All of the 10 participants correctly reported positive results for both positive samples # 1315421 (5x10³ genome copies /mL) and # 1315424 (5x10⁴ genome copies /mL) and also all participants correctly reported negative results for both negative samples #1315423 (containing E. coli and human cells) and # 1315422 (103 genome copies of Coxiella burnetii in a suspension of human cells).

After this very successful round of external quality assessment, “standardized samples“ are now 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 (Prof. Reischl).

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 contained only two positive samples: a high amount of Francisella tularensis holarctica (~1x10⁶ CFU/mL) was present in sample # 1315432, and an approximately hundred-fold lower amount (~1x10⁴ CFU/mL) was present in sample # 1315433.

Similar to QC samples from past ring trials, positive sample #1315432 was correctly tested positive by all of the 11 participating laboratories. As no false-positive results were observed for the “negative“ samples # 1315431 and # 1315434, it seems that the participating laboratories have implemented functional precautions to prevent deleterious contamination events.

Also the weaker positive sample # 1315433 (~1x10⁴ CFU/mL of F. tularensis) of the current set was reliably detected by almost all participants with their specific PCR test systems and only two of the 11 participants (one has indicated the use of a commercial test system and one indicated the use of a proprietary in house PCR assay) reported false negative results. Overall, these results corroborate the 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.

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 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 contained a kind of dilution series of Pneumocystis jirovecii organisms in the sample matrix: sample # 1315601 contained about 3x10⁶ genome copies/mL, sample # 1315602 about 4x10⁵ genome copies/mL and sample # 1315603 about 3x10⁴ genome copies/mL of Pneumocystis jirovecii. Sample # 1315604 of the current set contained no target organisms but only non-infected human cells and E. coli cells.

Fortunately, the promising results of the November 2012 external quality assessment scheme could also be confirmed in the current distribution. Samples # 1315601, which contained a relatively large amount of P. jirovecii target organisms (~ 3x10⁵ genome copies per mL) and # 1315602 which contained an approximately ten-fold lower amount of P. jirovecii, were reported “positive“ by all of the 66 participating laboratories. Even for the sample # 1315603, containing relatively low amounts of target organisms, 65 correctly positive results were reported by the 66 participants. Only one participant reported a false-negative result, which occurred probably due to the use of an in house NAT-based test system showing insufficient analytical sensitivity.

No false-positive results were observed for sample # 1315604, which contained no target organisms but only human cells and E. coli cells. To sum up, the result constellation of the present P. jirovecii distribution indicates an excellent performance of the currently employed PCR/NAT-based assay concepts as well as laboratory-specific precautions for the prevention of contamination events.

The relatively small numbers of participants with RV 542, RV 543 and RV 560 still not allows a serious comparison between certain commercial tests, prefabricated kits and the very heterogeneous group of in house PCR/NAT assay concepts regarding analytical sensitivity, analytical specificity, susceptibility to contamination, or simply the “overall performance”.