Comparison of the egg recovery rates and limit of detection for soil-transmitted helminths using the Kato-Katz thick smear, faecal flotation and quantitative real-time PCR in human stool

Abstract

Background

Monitoring the success of soil-transmitted helminth (STH) control programs relies on accurate diagnosis and quantitative assessment of infection prevalence and intensity. As preventative chemotherapeutic program coverage for STH expands, the necessity of gaining insights into the relative or comparative sensitivities, in terms of limits of detection (LOD) and egg-recovery-rates (ERR) for microscopy and quantitative polymerase chain reaction qPCR-based diagnostic techniques becomes imperative to inform suitability for their intended use for large scale STH monitoring and treatment efficacy studies.

Methodology/Principal findings

The diagnostic performance in terms of ERR and LOD of the Kato-Katz (KK) thick smear technique, sodium nitrate (NaNO ₃) faecal floatation (FF) and qPCR for the accurate detection and enumeration of STH eggs were calculated and expressed in eggs per gram (EPG), by experimentally seeding parasite-free human faeces with Ascaris spp., Trichuris spp. and Necator americanus eggs representing low, medium and high intensity infections. The efficiency of NaNO ₃ flotation was also calculated over a range of specific gravities (SpGr) for the optimum recovery of STH eggs. FF of SpGr 1.30 recovered 62.7%, 11% and 8.7% more Trichuris spp., Necator americanus and Ascaris spp. eggs respectively, than the recommended SpGr of 1.20. All diagnostic methods demonstrated strong direct correlation to the intensity of seeded EPG. KK and FF (SpGr 1.30) resulted in significant lower ERRs compared to qPCR ( p <0.05). qPCR demonstrated significantly (p <0.05) greater sensitivity with an ability to detect as little as 5 EPG for all three STH, compared to 50 EPG by KK and FF (SpGr 1.30).

Conclusions/Significance

This study compares the diagnostic parameters in terms of LOD and ERRs of STHs for the KK, FF and qPCR. These results indicate that the diagnostic performance of qPCR assays should be considered by control programs in the phase that aims to seek confirmation of transmission break and cessation of preventive chemotherapy in low-transmission settings, in line with the control targets of the WHO neglected tropical diseases 2030 Roadmap.

Author summary

STH infections predominately affect resource-poor communities and negatively impact on child and maternal health. Diagnostics play a critical role in guiding and informing existing STH control programs and the implementation and evaluation of intervention strategies. The KK technique is most commonly used as per WHO guidelines and is the basis for determining quantitative thresholds for low, moderate and heavy infections. More recently, FF and laboratory-based quantitative PCR techniques have provided alternative options of diagnosing STH infections. A number of studies correlating the relative sensitivity, diagnostic agreement, and egg enumeration of these techniques by comparing field-generated diagnostic data have attempted to correlate quantitative outputs to those defined by the WHO, with little resolution. Moreover, as large-scale deworming programs levels scale up, the necessity to apply techniques that can precisely detect light infections is of outmost importance to establish preventive chemotherapy end-points using more accurate means. In this study we compared the LOD and ERR of seeded non-infected human faecal samples with Ascaris, Trichuris and Necator spp. eggs using KK, FF and qPCR. When compared to copro-microscopy methods, qPCR showed the highest sensitivity for the detection of light-intensity infections and was more accurate in enumerating the original number of eggs per gram of seeded faeces. In addition to this, the results indicated that the diagnostic performance of both KK and FF (using solution at a SpGr of 1.30) were equally efficient and should be considered with their diagnostic limitations in mind, when aiming to monitor STH infections in low-transmission settings.