First international external quality assessment scheme of nucleic acid amplification tests for the detection of Schistosoma and soil-transmitted helminths, including Strongyloides: A pilot study

Abstract

Background: Nucleic acid amplification tests (NAATs) are increasingly being used as diagnostic tools for soil-transmitted helminths (STHs; Ascaris lumbricoides, Trichuris trichiura, Necator americanus, Ancylostoma duodenale and A. ceylanicum), Strongyloides stercoralis and Schistosoma in human stool. Currently, there is a large diversity of NAATs being applied, but an external quality assessment scheme (EQAS) for these diagnostics is lacking. An EQAS involves a blinded process where test results reported by a laboratory are compared to those reported by reference or expert laboratories, allowing for an objective assessment of the diagnostic performance of a laboratory. In the current study, we piloted an international EQAS for these helminths (i) to investigate the feasibility of designing and delivering an EQAS; (ii) to assess the diagnostic performance of laboratories; and (iii) to gain insights into the different NAAT protocols used.

Methods and principal findings: A panel of twelve stool samples and eight DNA samples was validated by six expert laboratories for the presence of six helminths (Ascaris, Trichuris, N. americanus, Ancylostoma, Strongyloides and Schistosoma). Subsequently this panel was sent to 15 globally dispersed laboratories. We found a high degree of diversity among the different DNA extraction and NAAT protocols. Although most laboratories performed well, we could clearly identify the laboratories that were poorly performing.

Conclusions/significance: We showed the technical feasibility of an international EQAS for the NAAT of STHs, Strongyloides and Schistosoma. In addition, we documented that there are clear benefits for participating laboratories, as they can confirm and/or improve the diagnostic performance of their NAATs. Further research should aim to identify factors that explain poor performance of NAATs.

Fig 1. Overview of the different steps, the expert and participating laboratories of the HEMQAS pilot study.

Fig 2. The variation in the reported Cq-values across the different targets and samples.

The Cq-values reported by the different participants for the different targets is shown on the Y-axis. The boxes represent the interquartile range; the lines in the boxes represent the median values; the upper and lower whiskers represent the 95^th and the 5^th percentile, respectively; and the dots represent outliers. The X-axis represents the different samples of the HEMQAS panel (see Table 3).

Fig 3. The variation in the reported Cq-values across the different laboratories and targets.

Each dot represents the difference in Cq-value for one sample reported by one laboratory in comparison with the median Cq-values reported by the expert laboratories for that sample. These differences are shown on the Y-axis, with negative values meaning lower Cq-values compared to the median Cq-values reported across the experts, and positive values meaning higher Cq-values. The laboratories, represented by letters, are shown on the X-axis. The number of false-negative and false-positive results reported by each laboratory are shown by means of red stars and blue triangles, respectively. Laboratory M was omitted from this analysis because no positive results were reported by this laboratory.