Date Published: December 18, 2014
Publisher: Public Library of Science
Author(s): Tawin Inpankaew, Fabian Schär, Virak Khieu, Sinuon Muth, Anders Dalsgaard, Hanspeter Marti, Rebecca J. Traub, Peter Odermatt, Ricardo J. Soares Magalhaes. http://doi.org/10.1371/journal.pntd.0003313
Abstract: BackgroundMicroscopy-based identification of eggs in stool offers simple, reliable and economical options for assessing the prevalence and intensity of hookworm infections, and for monitoring the success of helminth control programs. This study was conducted to evaluate and compare the diagnostic parameters of the Kato-Katz (KK) and simple sodium nitrate flotation technique (SNF) in terms of detection and quantification of hookworm eggs, with PCR as an additional reference test in stool, collected as part of a baseline cross-sectional study in Cambodia.Methods/Principle FindingsFecal samples collected from 205 people in Dong village, Rovieng district, Preah Vihear province, Cambodia were subjected to KK, SNF and PCR for the detection (and in case of microscopy-based methods, quantification) of hookworm eggs in stool. The prevalence of hookworm detected using a combination of three techniques (gold standard) was 61.0%. PCR displayed a highest sensitivity for hookworm detection (92.0%) followed by SNF (44.0%) and quadruple KK smears (36.0%) compared to the gold standard. The overall eggs per gram feces from SNF tended to be higher than for quadruple KK and the SNF proved superior for detecting low egg burdens.Conclusion/SignificanceAs a reference, PCR demonstrated the higher sensitivity compared to SNF and the quadruple KK method for detection of hookworm in human stool. For microscopic-based quantification, a single SNF proved superior to the quadruple KK for the detection of hookworm eggs in stool, in particular for low egg burdens. In addition, the SNF is cost-effective and easily accessible in resource poor countries.
Partial Text: Human hookworms are estimated to infect between 576–740 million people globally and are responsible for a global burden of 3.2 million disability-adjusted life years , . Hookworms are a leading cause of iron deficiency anemia and protein malnutrition, especially among pre- and school-aged children and untreated infections are known to result in adverse maternal-fetal outcomes in pregnant women . The principal intervention strategy for hookworm infection is periodic mass drug administration of humans with the benzimidazole drugs, albendazole or mebendazole.
In the present study, three diagnostic techniques (KK, SNF and PCR) were assessed for the qualitative and two techniques (KK and SNF) for the quantitative detection of hookworm eggs in fecal samples from humans in Cambodia. Direct comparison of the three diagnostic techniques showed that the PCR assay had a superior sensitivity compared to the SNF, the single, duplicate and quadruplicate KK techniques. The KK when performed in duplicate with stool samples collected over two consecutive days provided a higher sensitivity (36.0%) for diagnosing hookworm infection when compared to one day KK (day 1 or day 2) alone (27.2% and 20.0%). The ten individuals that were positive by KK and negative by SNF were also found negative on PCR. Therefore it is likely that these 10 positives were false positives on the quadruplicate KK, which is yet another disadvantage of this diagnostic approach. The field of view is poor compared to the SNF and fecal artifacts can be mistaken as helminth eggs. The PCR results are likely explained by two factors: (i) false negatives – the inability to amplify these samples could be associated with failure to remove PCR inhibitors in human stool following DNA extraction , (ii) false-positive coproscopy results, i.e. that Trichostrongylus eggs detected in stool were misidentified as hookworm eggs. Trichostrongylus columbricformis which is present in humans in neighboring countries such as Lao People’s Democratic Republic  and Thailand  produce eggs very similar to hookworms. Although there are no published reports of human infection with this species in Cambodia, T. columbricformis infection in humans cannot be disregarded because molecular identification of other strongylid nematodes was not attempted in this study.