Multi-parallel qPCR provides increased sensitivity and diagnostic breadth for gastrointestinal parasites of humans: field-based inferences on the impact of mass deworming

Abstract

BackgroundAlthough chronic morbidity in humans from soil transmitted helminth (STH) infections can be reduced by anthelmintic treatment, inconsistent diagnostic tools make it difficult to reliably measure the impact of deworming programs and often miss light helminth infections.MethodsCryopreserved stool samples from 796 people (aged 2–81 years) in four villages in Bungoma County, western Kenya, were assessed using multi-parallel qPCR for 8 parasites and compared to point-of-contact assessments of the same stools by the 2-stool 2-slide Kato-Katz (KK) method. All subjects were treated with albendazole and all Ascaris lumbricoides expelled post-treatment were collected. Three months later, samples from 633 of these people were re-assessed by both qPCR and KK, re-treated with albendazole and the expelled worms collected.ResultsBaseline prevalence by qPCR (n = 796) was 17 % for A. lumbricoides, 18 % for Necator americanus, 41 % for Giardia lamblia and 15 % for Entamoeba histolytica. The prevalence was <1 % for Trichuris trichiura, Ancylostoma duodenale, Strongyloides stercoralis and Cryptosporidium parvum. The sensitivity of qPCR was 98 % for A. lumbricoides and N. americanus, whereas KK sensitivity was 70 % and 32 %, respectively. Furthermore, qPCR detected infections with T. trichiura and S. stercoralis that were missed by KK, and infections with G. lamblia and E. histolytica that cannot be detected by KK. Infection intensities measured by qPCR and by KK were correlated for A. lumbricoides (r = 0.83, p < 0.0001) and N. americanus (r = 0.55, p < 0.0001). The number of A. lumbricoides worms expelled was correlated (p < 0.0001) with both the KK (r = 0.63) and qPCR intensity measurements (r = 0.60).ConclusionsKK may be an inadequate tool for stool-based surveillance in areas where hookworm or Strongyloides are common or where intensity of helminth infection is low after repeated rounds of chemotherapy. Because deworming programs need to distinguish between populations where parasitic infection is controlled and those where further treatment is required, multi-parallel qPCR (or similar high throughput molecular diagnostics) may provide new and important diagnostic information.

An extensive burden of giardiasis associated with intestinal schistosomiasis and anaemia in school children on the shoreline of Lake Albert, Uganda

“…Use of the Quik-Chek RDT revealed that, whilst no cryptosporidiosis was encountered, a considerable number of children at Runga and Bugoigo were patently infected with clinically relevant Giardia. This was later confirmed by real-time PCR which, by having greater diagnostic sensitivity 29,[40][41][42] , found a larger proportion of children with some evidence of infection.…”

Towards interruption of schistosomiasis transmission in sub-Saharan Africa: developing an appropriate environmental surveillance framework to guide and to support ‘end game’ interventions

“…Others include schistosome-snail investigations around Lake Victoria. More broadly, the application of DNA-based assays in medical malacology [72] also coincided with the rise in DNA diagnostic assays [19], such as real-time PCR approaches with TaqMan® probes, for medical diagnostics [73, 74]. It is precisely that these medical diagnostic assays have become accepted, standardised and largely routine, that they can be used to spur on interests in environmental surveillance of schistosomes by inspection of water filtrates, environmental samples as well as in field-caught or sentinel snails [65, 66], see Table 1.…”

Molecular Diagnostics for Soil-Transmitted Helminths

“…Early on in the use of molecular diagnostics for fecal pathogens, there were significant sensitivity problems when DNA extraction techniques that were not specifically designed to remove these inhibitors48 were used. PCR inhibitors are largely removed without problem using 1) more recent in-house developed protocols,27,49 2) tissue kits with additional inhibitor removal steps,24,50 and 3) stool and soil-engineered kits for DNA extraction 19,21,43,50–53…”

“…Several large studies have shown a clear relationship between stool DNA concentration as determined by qPCR and egg counts 19,21,43. Interestingly, there is also a good correlation between DNA quantification in the stool (presumably reflecting egg DNA) and the number of expelled adult worms after albendazole treatment 21. The evaluation of the existing data on A. lumbricoides across multiple studies (Table 1) has been aided enormously by the fact that all of the studies have targeted the internal transcribed spacer 1 region (see Table 2).…”