Diagnostic performance of a single and duplicate Kato-Katz, Mini-FLOTAC, FECPAKG2 and qPCR for the detection and quantification of soil-transmitted helminths in three endemic countries

Abstract

Background: Because the success of deworming programs targeting soil-transmitted helminths (STHs) is evaluated through the periodically assessment of prevalence and infection intensities, the use of the correct diagnostic method is of utmost importance. The STH community has recently published for each phase of a deworming program the minimal criteria that a potential diagnostic method needs to meet, the so-called target product profiles (TPPs).

Methodology: We compared the diagnostic performance of a single Kato-Katz (reference method) with that of other microscopy-based methods (duplicate Kato-Katz, Mini-FLOTAC and FECPAKG2) and one DNA-based method (qPCR) for the detection and quantification of STH infections in three drug efficacy trials in Ethiopia, Lao PDR, and Tanzania. Furthermore, we evaluated a selection of minimal diagnostic criteria of the TPPs.

Principal findings: All diagnostic methods showed a clinical sensitivity of ≥90% for all STH infections of moderate-to-heavy intensities. For infections of very low intensity, only qPCR resulted in a sensitivity that was superior to a single Kato-Katz for all STHs. Compared to the reference method, both Mini-FLOTAC and FECPAKG2 resulted in significantly lower fecal egg counts for some STHs, leading to a substantial underestimation of the infection intensity. For qPCR, there was a positive significant correlation between the egg counts of a single Kato-Katz and the DNA concentration.

Conclusions/significance: Our results indicate that the diagnostic performance of a single Kato-Katz is underestimated by the community and that diagnostic specific thresholds to classify intensity of infection are warranted for Mini-FLOTAC, FECPAKG2 and qPCR. When we strictly apply the TPPs, Kato-Katz is the only microscopy-based method that meets the minimal diagnostic criteria for application in the planning, monitoring and evaluation phase of an STH program. qPCR is the only method that could be considered in the phase that aims to seek confirmation for cessation of program.

Trial registration: ClinicalTrials.gov NCT03465488.

Conflict of interest statement

The FECPAKG2 technology was produced and distributed by Techion Group Ltd, of which ET is an employee and GM is managing director. Both hold stocks in Techion Group Ltd. The Mini-FLOTAC device is a commercial product distributed by GC, LR and MPM through the University of Napoli Federico II. However, the affiliations of ET, GM, GC, LR and MPM did not play any role in the preparation and submission of this manuscript. All other authors declared that they have no competing interests.

Figures

Fig 1. Number of subjects withheld at recruitment, baseline and follow-up, and for the statistical data analysis.

STH: soil-transmitted helminth; n: number of subjects; FECs: fecal egg counts expressed in eggs per gram of stool (EPG), 2x KK: duplicate Kato-Katz.

Fig 2. Clinical sensitivity of five diagnostic methods as a function of egg excretion.

The bar plots represent the clinical sensitivity of single Kato-Katz (blue), qPCR (white), duplicate Kato-Katz (grey), Mini-FLOTAC (black) and FECPAKG2 (red) across seven infection intensity categories. The range in fecal egg counts (FECs; expressed as eggs per gram of stool (EPG)) is shown on the X-axis. These FECs correspond with the highest FECs across the microscopic methods (Kato-Katz, Mini-FLOTAC and FECPAKG2). The Y-axis represent the clinical sensitivity in percentage. The number samples for each infection category (n) is shown above the bars. The WHO defined classes of infections intensities (low (L), moderate (M) and high (H)) are shown on top. The dotted horizontal line represents a clinical sensitivity of 95%.

Fig 3. Agreement in fecal egg counts by the four microscopic methods.

The scatterplots illustrate the agreement in fecal egg counts (FECs, expressed in eggs per gram of stool (EPG) based on a single Kato-Katz (1x KK) with those obtained with a duplicate Kato-Katz (grey dots), Mini-FLOTAC (black dots) and FECPAKG2 (red dots) for A. lumbricoides (n = 540), T. trichiura (n = 889) and hookworm (n = 675). In each panel, the Spearman’s correlation coefficient (Rs) is given. The striped diagonal line represents the line of equivalence.

Fig 4. Agreement in infection intensity classification by four microscopic method.

Each bar represents all cases classified by a single Kato-Katz (1x KK; reference method) into low, moderate or heavy infection intensity using the WHO threshold criteria (Table 2). The Y-axis of each bar represents the proportion of these cases that are categorized as low, moderate and heavy intensity infections by means of a duplicate Kato-Katz (2x KK; top row plots), Mini-FLOTAC (middle row plots) and FECPAKG2 (bottom row plots). The white color indicates cases classified as low infection intensity, grey indicates cases classified as moderate infection intensity and black indicates cases categorized as heavy infection intensity, by either duplicate Kato-Katz, Mini-FLOTAC or FECPAKG2. There is a full agreement in classifying the intensity of infection with the reference method when the bars representing low, moderate and high infection intensities by the reference method are completely white, grey and black, respectively. As an example, of all heavy intensity hookworm infections, only approximately 20% are classified correctly as heavy when using the FECPAKG2, and approximately 60% and 20% are misclassified as low and moderate, respectively.

Fig 5. Agreement in fecal egg counts by Kato-Katz and genomic concentration by qPCR.

The scatterplots illustrate the agreement in fecal egg counts (FECs; expressed in eggs per gram of stool (EPG)) based on a single Kato-Katz and the DNA concentration (expressed in number of genome equivalents per ml (GE/ml)) based on qPCR for Ascaris (n = 540), Trichuris (n = 889) and hookworm (n = 675). In each panel, the Spearman’s correlation coefficient (RS) is shown.