gms | German Medical Science

GMS Zeitschrift zur Förderung der Qualitätssicherung in medizinischen Laboratorien

Gesellschaft zur Förderung der Qualitätssicherung in medizinischen Laboratorien e. V. (INSTAND e. V.)

ISSN 1869-4241

Bacterial and fungal genome detection PCR/NAT: discussion of the November 2013 distribution for external quality assessment of nucleic acid-based protocols in diagnostic medical microbiology by INSTAND e.V.

Report

  • corresponding author Udo Reischl - Institute for Clinical Microbiology and Hygiene, University Hospital Regensburg, Regensburg, Germany
  • Wulf Schneider - Institute for Clinical Microbiology and Hygiene, University Hospital Regensburg, Regensburg, Germany
  • Thomas Holzmann - Institute for Clinical Microbiology and Hygiene, University Hospital Regensburg, Regensburg, Germany
  • Martin Ehrenschwender - Institute for Clinical Microbiology and Hygiene, University Hospital Regensburg, Regensburg, Germany
  • Matthias Maaß - Labor Dr. Heidrich und Kollegen MVZ GmbH, Hamburg, Germany
  • Eberhard Straube - Institute of Medical Microbiology, University Hospital of the Friedrich Schiller University of Jena, Jena, Germany
  • Dimitrios Frangoulidis - Bundeswehr Institute of Microbiology, Munich, Germany
  • Gregor Grass - Bundeswehr Institute of Microbiology, Munich, Germany
  • Wolf Splettstösser - Bundeswehr Institute of Microbiology, Munich, Germany
  • Volker Fingerle - Bavarian State Office for Health and Food Safety, Oberschleissheim, Germany
  • Andreas Sing - Bavarian State Office for Health and Food Safety, Oberschleissheim, Germany
  • Enno Jacobs - Institute for Medical Microbiology and Hygiene, Technical University of Dresden, Dresden, Germany
  • Ingrid Reiter-Owona - Institute for Medical Microbiology, Immunology and Parasitology (IMMIP), University of Bonn, Bonn, Germany

GMS Z Forder Qualitatssich Med Lab 2014;5:Doc01

doi: 10.3205/lab000011, urn:nbn:de:0183-lab0000116

This is the English version of the article.
The German version can be found at: http://www.egms.de/de/journals/lab/2014-5/lab000011.shtml

Published: January 8, 2014

© 2014 Reischl et al.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License. You are free: to Share - to copy, distribute and transmit the work, provided the original author and source are credited. See license information at http://creativecommons.org/licenses/by-nc-nd/3.0/.


Abstract

This contribution provides an analysis report of the recent proficiency testing scheme “Bacterial and Fungal Genome Detection (PCR/NAT)”. It summarizes some benchmarks and the overall assessment of results reported by all of the participating laboratories.

A highly desired scheme for external quality assessment (EQAS) of molecular diagnostic methods in the field of medical microbiology was activated in 2002 by the German Society of Hygiene and Microbiology (DGHM) and is now organized by INSTAND e.V., Düsseldorf, Germany. This segment of the INSTAND e.V. proficiency testing program is open for diagnostic laboratories worldwide. The concept of this EQAS scheme, which is in accordance to the ”Directive of the German Medical Association for quality assurance of medical laboratory examinations“ (RiLiBÄK), part B3, is based on two validation rounds per year (spring and autumn) and a permanently expanding coverage of relevant bacterial or fungal pathogens. Briefly, next to “simply negative” samples the corresponding sets of quality control (QC) specimens may contain some strong-positive samples, samples spiked with clinical variants or species closely related to the target organisms. Further information as well as the statistically documented and discussed results of the past rounds of this proficiency testing scheme “Bacterial and Fungal Genome Detection (PCR/NAT)” can be found at the homepage of INSTAND e.V. (http://www.instandev.de). Although the preferred language of these documents is German, we are aiming to provide at least a brief discussion of the results and some key issues in English and keep the tables in a bilingual style.


Brief discussion of the current results

For the growing number of international participants we provide a brief discussion of the current results in an English version.


Examination results November 2013

RV 530: Neisseria gonorrhoeae & Chlamydia trachomatis (GO & CT)

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 # 1325304 and sample # 1325303), two samples with equal amounts of N. gonorrhoeae (sample # 1325302 and sample # 1325304; ~1x103 CFU/mL) and sample # 1325301 with ~1x104 CFU/mL of N. gonorrhoeae.

Despite relatively low amounts of C. trachomatis target cells in the positive samples #1325303 and # 1325304, only 4 false-negative results were observed among the Chlamydia trachomatis-specific results reported by the 147 participants. Among the N. gonorrhoeae-specific results, false-negative results were reported by 19 of the 144 participants for samples #1325302 and # 1325304, which contained N. gonorrhoeae target organisms in an amount of 1x103 CFU/mL. For the GO-positive sample # 1325301, which contained with 1x104 CFU/mL a ten-fold higher amount of N. gonorrhoeae target organisms, no false negative result for the detection of N. gonorrhoeae DNA was reported.

The detection of gonococcal DNA in samples which also contained C. trachomatis organisms, (or the test systems used by these participants) caused some difficulties for some participants: 12 false negative results were observed for sample # 1325304 compared to 7 false negative results for sample # 1325302. 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 # 1315302 and 1325304 (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.

Despite this infrequently observed issue regarding sensitivity, an overall very good diagnostic performance was observed. Inhibition controls were conducted by all 148 participants, and inhibition events were not reported this time.

RV 531: Chlamydia trachomatis

The current set of QC samples contained three positive samples: # 1325311 and # 1325313 with ~1x103 IFU/mL of C. trachomatis target organisms and sample # 1325314 with ~5x103 IFU/mL of C. trachomatis target organisms. Sample # 1325312 contained no target organisms but only human cells and E. coli cells.

As depicted in Tab. 2 (see Attachment 1 [Attach. 1], p. 5), the results were broadly correct for all three positive samples. For the sample #1325314 containing higher amounts of C. trachomatis DNA, 2 false-negative results were reported and for the weakly positive samples #1325311 and #1325313, 2 and 6 false-negative results were reported, respectively.

For the C. trachomatis-negative sample # 1325312, containing only non-infectious human cells and E. coli, 3 false-positive results were observed of the 102 participants. For samples # 1325312 and # 1325313 results were classified as “questionable” for one participant each. For questionable results, certificates are only issued when correct results are reported by the participant in the remaining 3 samples of RV 531.

This striking match of the current results with observations and accuracy rates in previous inter-laboratory tests can be considered as evidence of a high reliability and consistency of the used test systems and the sample processing.

Run controls were performed by all of the 102 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 specifity were observed for the C. trachomatis-specific NAT assays used by the 102 participants of the current distribution.

RV 532: Bordetella pertussis

The current set of QC samples contained one sample with a relatively high amount of Bordetella pertussis (# 1325322; 1x104 CFU/mL), one negative sample containing Bordetella parapertussis (# 1325324 with ~1x104 CFU/mL), as well as two samples containing only non-infected human cells and Escherichia coli (# 1325321 and # 1325323).

The availability of well-established commercial or in-house NAT-assays has led to a high portion of correct results. Only one of the 116 participants reported a false negative result for the sample # 1325322 (B. pertussis, 1x104 CFU/mL).

For sample # 1325324 (104 CFU/mL of B. parapertussis) 3 false-positive results were observed, which may have probably been caused by an insufficient analytical specificity of the used in-house test systems or due to a contamination during the sample preparation, amplification or detection.

Of the 116 participants, 115 included inhibition controls and inhibition events didn’t occur this time.

The amount of 104 CFU/mL of B. pertussis target organisms is significantly above the previously observed lower limit of detection for the corresponding PCR assays or test systems.

For the detection of B. pertussis, most participants used in-house test concepts with inhibition and/or positive controls. In the current distribution, 54 participating laboratories indicated the use of the IS481 insertion sequence, 10 the B. pertussis toxin coding gene and 3 the use of B. pertussis-specific ribosomal gene segments.

RV 533: Helicobacter pylori

The current set of QC samples contained three samples with a Clarithromycin-susceptible Helicobacter pylori patient strain in a kind of dilution series. Sample # 1325332 contained approximately 1x106 CFU/mL, sample # 1325333 approximately 1x105 CFU/mL and sample # 12325334 approximately 1x104 CFU/mL of the respective target organisms.

The availability of well evaluated NAT-based assays and the relatively high amount of target organisms in two of three positive samples (# 1325332: ~1x106 CFU/mL and # 1325333: ~1x105 CFU/mL) led to positive predictive values of 100%.

As noted in the test description of RV 533, clarithromycin resistance testing in the examined H. pylori isolates could be performed by participants on a voluntary basis. This molecular resistance testing is usually based on amplification and sequencing of characteristic regions within the H. pylori 23 S rDNA or the use of hybridization probes based qPCR assays. Results were communicated by 29 of the 35 participants and the results for molecular susceptibility testing were also correct, apart from one occasion.

RV 534: EHEC/STEC

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 three samples positive for EHEC: # 1325341 (E. coli, 1x105 CFU/mL, clinical isolate, stx1-, eae-, hlyA- and O157-positive), # 1325342 (E. coli, 1x104 CFU/mL, clinical isolate, stx1-, stx2-, eae-, hlyA- and O157-positive), and # 1325343 (E. coli, 1x105 CFU/mL, clinical isolate, stx2f-positive and eae-positive). The other EHEC-negative sample contained an eae- and hlyA-negative E. coli K12 strain (# 1325344).

With the exception of sample #1325343 (stx2f-positive EHEC isolate) the availability of well-established NAT-based assays and strategies for molecular differentiation resulted in consistently high accuracy rates for the three remaining samples – both for positive and negative results. Consistently correct results were reported by 98 of 104 participants.

The cause for the 3 false-negative results for stx-1 positive EHEC isolate (# 1325341) and the 6 false-negative results for stx-1- and stx-2-positive EHEC isolate (#1325342) 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, which was demonstrated impressively by the inclusion of the stx-2f-positive sample #1325343. Only 25 of the 104 participants reported positive results for the presence of genes coding for shiga toxins.

Even if the relevance of stx2f-positive EHEC isolates for human pathogenity is still controversial, this should serve once again as an example for surprising genetic diversity of bacterial isolates from the microbiological PCR routine diagnostics.

For sample # 1325344 containing an eae- and hly-negative E. coli K12 strain, fortunately only 2 of the 104 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 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 implemented by 95 of the 104 participating laboratories and the reported results were correct when performed.

RV 535: Borrelia burgdorferi

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 and B. bavariensis. B. spielmanii, a further Species with assured human-pathogenicity, seems to be rare and was so far only described from skin manifestations of Lyme borreliosis. B. bissetii, 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 distribution of QC samples contained one sample with Borrelia bissetii (# 1325351; ~1x104 organisms/mL), one sample with Borrelia spielmanii (sample # 1325353; ~1x104 organisms/mL), and one sample with Borrelia afzelii (sample # 1325354; ~1x104 organisms/mL) Sample # 1325352 contained no Borrelia organisms but only human cells and E. coli cells.

With the exception of 7 false-negative results for sample # 1325351, 4 false-negative results for sample # 1325353, 5 false-negative results for sample # 1325354, and one 7 false-positive results for sample # 1325352, correct results were reported by the 96 participating laboratories.

The participants who reported false-negative results especially for the two positive samples containing Borrelia spielmanii and Borrelia afzelii target organisms should check their NAT assay concepts including sample processing/DNA-extraction.

About 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 were observed in the current round of our external quality assessment scheme for the diagnostic detection of Borrelia burgdorferi by PCR/NAT techniques.

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 (7x), Autoimmun Diagnostika GenID Zecken Screening Kit (2x), EliGene Borrelia LC from Elisabeth Pharmacon (1x), TIB Molbiol LightMix Borrelia (1x), BactoReal B. burgdorferi from Ingenetix (1x) and Attomol Borrelia burgdorferi Realtime (1x).

RV 536: Legionella pneumophila

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, participation is promising only for 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 two positive samples with Legionella pneumophila serogroup 3 (# 1325361; ~1x106 CFU/mL and # 1325362; ~1x104 CFU/mL), as well as one sample containing Chlamydia pneumoniae (# 1325363; ~1x106 IFU/mL). Sample # 1325364 contained no target organisms but only human cells and E. coli cells.

The L. pneumophila-positive (~1x106 CFU/mL) sample # 1325361 was correctly tested positive by all of the 84 participating laboratories. Sample # 1325362, which contained a hundred-fold lower amount (~1x104 CFU/mL) of L. pneumophila reported correctly by 78 of 84 participants.

The 6 participants with false-negative results were using mainly self-developed in-house real-time PCR protocols targeting the omp-, mip-gene or 16S rDNA sequence or a nested block-cycler PCR protocol with subsequent nucleotide sequencing of the amplified 16S rDNA region. The latter concept should be sensitive enough to allow the detection of low amounts of L. pneumophila because of the nested PCR step and the large amplification volume.

Sample # 1325363 contained a significant amount of Chlamydia pneumoniae with approx. 1 x 106 IFU/mL. No cross-reactivity and false positive results of all 84 participants was observed in the respective L. pneumophila-specific PCR assay.

RV 537: Salmonella enterica

The current set of QC samples contained a kind of dilution series of Salmonella enterica serovar Typhi: sample # 1325371 contained ~1x106 CFU/mL, sample # 1325374 contained ~1x105 CFU/mL and sample # 1325373 contained ~1x104 CFU/mL. Sample # 1325372 contained no target organisms but only human cells and E. coli cells.

Only 2 false-negative results were reported for the positive samples # 1325373 and # 1325374 and no false-positive result was reported for the negative sample # 1325372.

In summary, 11 correct results for the negative sample # 1325372 as well as the positive sample # 1325371 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 (# 1325373; Salmonella enterica serovar Typhi, ~1x104 CFU/mL) was reported false negative by 1 participant. 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, as the strongly positive sample # 1325371 was preceding the negative sample and no false-positive result was reported from the 11 participants.

RV 538: Listeria spp.

The current set of QC samples contained a sample without the corresponding target organisms (# 1325384; only E. coli cells), and three samples positive for L. monocytogenes. In order to assess the analytical sensitivity of the NAT assays currently used at the participating laboratories, we decided to include also some weak positive samples in the current distribution. Relatively low numbers of L. monocytogenes cells (~104 CFU/mL and ~105 CFU/mL) were present in samples # 1325382 and # 1325381, respectively. Both were tested positive by the PCR assays applied by 23 of the 25 participants. Although the third “positive” sample contained a remarkably low amount of target organisms (# 1325383; ~103 CFU/mL of L. monocytogenes), it was nice to see that still 23 of the 25 participating laboratories were still able to detect the corresponding DNA by their Listeria-specific PCR assays.

As mentioned in the Report form, participants who are performing molecular tests covering only L. monocytogenes may indicate the corresponding results by the accessory code number 71. When the use of L. monocytogenes-specific PCR assays is indicated, we do not score (false) negative results for non-Listeria monocytogenes species (which may be present in future distributions) in the course of issuing the corresponding QC certificates. It is nice to see that correct results were reported by the majority of participating laboratories in the course of this external PCR assay validation – and, again, this indicates a remarkably high analytical sensitivity of the current L. monocytogenes-specific PCR assays.

False-positive results were observed in one case for sample # 1325384 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.

RV 539: MRSA

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 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 # 1325391 of the current set contained a mixture of S. aureus (MSSA, PVL-negative, ~1x104 CFU/mL) and a CoNS strain (S. epidermidis; mecA-positive, ~1x104 CFU/mL One sample of the current set (# 1325394) contained no target organisms but only E. coli cells. Sample # 1325393 contained a relatively high number of CA-MRSA organisms (S. aureus, mecA-positive, PVL-positive, spa:t 310; ~1x104 CFU/mL) and sample # 1325392 contained a relatively low number of typical MRSA organisms (S. aureus, mecA-positive, PVL-negative; ~1x103 CFU/mL).

The MRSA negative sample # 1325394 was tested by 240 of 241 participants with their PCR-based MRSA-specific test systems as “negative”, so only one participant observed a false positive result for sample # 1325394, which may have probably been caused by contamination with MRSA DNA during the sample preparation, amplification or detection. Fortunately, for the positive cMRSA sample # 1325393, positive results were reported by all 241 participants. Even if sample # 1325392 contained with 1x103 CFU/mL a lower amount of target organisms, with 237 positive reported results of 241 participants, positive predictive values of 99% were observed. The 4 false negative results are probably due to an insufficient analytical specificity of the used in-house test systems.

For the sample # 1325391, which contained a MSSA isolate together with a Methicillin resistant coagulase-negative Staphylococcus species, 214 of 241 participants reported their results correctly as “MRSA-negative” and 11 participants classified the results as “questionable”. 6 of these 11 participants indicated the use of test systems, which are based on a separate detection of the mecA gene and S. aureus specific target genes. With this separate detection assays, the origin of the mecA target gene cannot definitively be correlated with the S. aureus or the coagulase-negative Staphylococcus species. Regarding this aspect, “questionable” is the scientifically correct result in this case. The remainder 16 participants reported false-positive results for MRSA for sample # 1325391, containing a mixture of a MSSA isolate and a methicillin-resistant coagulase negative Staphylococcus species. These participants are encouraged to analyse the suitability of their test systems, as the described constellation is a relatively common scenario for microbiological routine diagnostic of MRSA. On the other hand, all participants, who used SCCmec based test systems, reported correct MRSA-negative results for sample #1325391.

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.

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 57 of the total 241 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 at [3] or [4]. A well evaluated protocol for the detection of PVL-positive PVL isolate can be found at [5].

In addition, commercial real-time PCR assays reliably targeting PVL-genes in MRSA and MSSA isolates are available in the meantime (for example from r-biopharm and TIB Molbiol).

RV 540: Chlamydia pneumoniae

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 # 1325404 was spiked with ~1x105 IFU/mL of C. pneumoniae whereas sample # 1325401 contained an approximately tenfold lower number of C. pneumoniae (~1x104 IFU/mL). Sample # 1325402 contained significant numbers (~105 genome copies/mL) of Mycoplasma pneumoniae organisms to assess analytical specificity. Only E. coli and non-infected human cells but no C. pneumoniae target organisms were present in sample # 1325403.

As depicted in Tab. 2 (see Attachment 1 [Attach. 1], p. 15), all participants reported correct results for the positive sample # 1325401. 93 of the 94 participants reported correctly positive results for the positive sample # 1325404, which contained a relatively high concentration of target organisms (105 IFU/mL). Affected participants are encouraged to analyse and optimize their NAT-based assays.

Only one participant reported false-positive results for negative sample # 1325402 (Mycoplasma pneumoniae) and also for the “negative” sample # 1325403, which could be due to cross-contamination events in the course of sample preparation, amplification or amplicon detection steps as cross reactivity with E. coli or Mycoplasma pneumoniae DNA is unlikely. Overall there were no noticeable problems with the current set of QC samples and a good overall correlation with the expected results was observed.

RV 541: Mycoplasma pneumoniae

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 two positive samples. A relatively high amount of M. pneumoniae (~1x105 genome copies/mL) was present in sample # 1325412 and an approximately tenfold lower amount of M. pneumoniae (~1x104 genome copies/mL) was present in sample # 1325413. Samples # 1325411 and # 1325414 were designed to monitor assay specificity: they contained a considerable amount of M. genitalium (~104 and ~105 genome copies/mL, respectively) as a related species to the target organism.

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 104 participants reported correct M. pneumoniae-positive results for sample # 1325412 containing a relatively high amount of target organisms.

The sample # 1325413 (~104 genome copies/mL) containing an approximately ten-fold lower amount of M. pneumoniae was tested correctly positive by 94 of the 104 laboratories.

Four participants reported false-positive results for the negative samples # 1325411 and #1325414, which contained considerable amounts of M. genitalium. The false-positive results could be caused by cross-contamination events in the course of sample preparation, amplification or amplicon detection steps. Cross reactivity of the used NAT-based M. pneumoniae assay with other Mycoplasma species should be analysed by the affected laboratories.

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

RV 542: Coxiella burnetii & Bacillus anthracis

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 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 # 1325422 and ~1x104 genome copies/mL in sample # 1325424), two samples with tenfold different amounts of Bacillus anthracis (sample # 1325423 with ~1x104 genome copies/mL and sample # 1325422 with ~5x104 genome copies/mL) Sample # 1325421 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 (Attachment 1 [Attach. 1], p. 17) for the Coxiella burnetii-specific results and tables 4 and 5 (Attachment 1 [Attach. 1], p. 18) for the Bacillus anthracis-specific results.

Coxiella burnetii

A relatively high amount (~104 genome copies/mL) of C. burnetii organisms were present in sample # 1325424, which consequently tested positive by all of the 20 participating laboratories. Sample # 1325422, which contained an approximately tenfold lower number of C. burnetii target organisms/mL (next to significant amounts of Bacillus anthracis DNA), was also tested positive for C. burnetii DNA by all of the 20 participants. Similar to the result observed in past distributions of our external quality assessment schemes, difficulties for the reliable detection of C. burnetii DNA may occur sporadically for samples at or below 103 genome copies/mL. As the two C. burnetii-positive samples of the current EQAS scheme carried target numbers above ~104 genome copies/mL, false-negative results are not observed this time.

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

The results for this newly introduced EQAS scheme are easily discussed. All of the 14 participants correctly reported positive results for both positive samples # 1325422 (~5x104 genome copies/mL) and # 1325423 (~1x104 genome copies/mL) and also all participants correctly reported negative results for both negative samples # 1325421 (containing E. coli and human cells) and # 1325424 (containing ~104 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).

RV 543: Francisella tularensis

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 three positive samples: a high amount of Francisella tularensis holarctica (~1x106 CFU/mL) was present in sample # 1325431, and an approximately tenfold lower amount (~1x105 CFU/mL) was present in sample # 1325433. Sample # 1325434 contained approximately 1x105 CFU/mL of a Francisella tularensis spp. novicida strain.

Similar to QC samples from past ring trials, the positive sample # 1325431 (~1x106 CFU/mL of Francisella tularensis holarctica) was correctly tested positive by all of the 15 participating laboratories. As no false-positive result was observed for the “negative” sample # 1325432 (which was positioned in direct sequence after the relatively strong positive sample # 1325431 within the set of 4), it seems that the participating laboratories have implemented functional precautions to prevent deleterious contamination events.

Also the 10-fold weaker positive sample # 1325433 (~1x105 CFU/mL of Francisella tularensis holarctica) was reliably detected by all participants with their specific PCR test systems.

As the third positive sample within the current EQAS distribution, sample # 1325434 contained about 105 CFU/mL of Francisella tularensis spp. novicida. These target organisms were reliably detected by 14 of the 15 participants, whereas a negative result for this particular F. tularensis subspecies was only observed by one participant indicating the use of an in-house PCR assay concept.

This result should not necessarily be considered as false-negative. In the current trial, we used, for the first time, DNA from the closely related “fourth” subspecies F. tularensis novicida. Strains of this subspecies have been rarely described to cause symptomatic infection in humans, but even lethal disease in immune-compromised patients has been documented. But this pathogen does not cause the typical clinical picture of tularemia in immune-competent individuals. Humans infected with F. tularensis spp. novicida do not raise anti-F. tularensis LPS (holarctica, tularensis, mediasiatica) antibodies and thus commercially available serological assays show negative results.

The taxonomy of the genus Francisella changed significantly over the last three years, the genus also comprises the pathogenic species F. philomiragia and F. hispaniensis. In the field of veterinary medicine there are two additional relevant species belonging to the genus Francisella.

At least one of the assays used by several participants will detect all of these species and subspecies. Depending on the clinical question, this property might be beneficial or adverse when interpreting the test result. We included this subspecies in the current trial in order to highlight this special difficulty in tularemia diagnostics. As of late, complete genome sequences of all relevant Francisella species and subspecies are accessible in public data bases (e.g. NCBI), allowing to check the sensitivity and specificity of all primers and probes used in in-house assays.

Due to the fact that a specific detection of F. tularensis spp. tularensis (Jellison type A) is of special concern for clinical and public health issues, we plan to include DNA from this subspecies in the near future. This would allow the participants to test their assays in terms of sensitivity or specificity detecting different F. tularensis subspecies.

Till then, any positive NAT result should be critically revisited and subtyping of the positive sample should be considered.

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.

RV 560: Pneumocystis jirovecii

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 two positive samples. A relatively high amount of Pneumocystis jirovecii (~3x105 genome copies/mL) was present in sample # 1325601 and an approximately tenfold lower amount of Pneumocystis jirovecii (~9x104 genome copies/mL) was present in sample # 1325603. The set was completed by sample # 1325602 and sample # 1325604, which contained only human cells and a considerable amount of E. coli organisms.

Fortunately, the promising results observed in the two previous rounds of our external quality assessment scheme Pneumocystis jirovecii DNA could be confirmed in the current distribution. Samples # 1325601, which contained a relatively high amount of P. jirovecii target organisms (~3x105 genome copies/mL) and # 1325603 which contained a slightly lower amount of P. jirovecii, were reported “positive” by all but one of the 59 participating laboratories.

One laboratory reported a false-negative result for sample # 1325601 but concurrently observed a correct positive result with the second positive sample # 1325603. Although this could be due to a sporadic loss of template DNA during some pre-analytical sample preparation procedures or other “simple” reasons, observation of false-negative results should give reason to check the diagnostic workflow, consider improving the sensitivity and/or checking the species coverage of the individual assay concept.

No false-positive results were observed for samples # 1325602 and # 1325604, which contained no target organisms but only human cells and E. coli cells. To sum up, the results of the present P. jirovecii distribution indicate an excellent performance of the currently employed PCR/NAT-based assay concepts as well as laboratory-specific precautions for the prevention of contamination events.

However, the limited number of results (RV 542, RV 543, RV 560), together with an insufficient reporting of the kit manufacturers applied still not allow a serious comparison of commercial tests and the very heterogeneous group of in-house PCR/NAT assay concepts with regard to analytical sensitivity, analytical specificity, susceptibility to contamination, or simply the “overall performance”.


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