Comparison of four DNA extraction and three preservation protocols for the molecular detection and quantification of soil-transmitted helminths in stool

Background : A DNA extraction and preservation protocol that yields sufficient and qualitative DNA is pivotal for the success of any nucleic acid amplification test (NAAT), but it still poses a challenge for soil-transmitted helminths (STHs), including Ascaris lumbricoides, Trichuris trichiura and the two hookworms (Necator americanus and Ancylostoma duodenale). In the present study, we assessed the impact of different DNA extraction and preservativation protocols on STH-specific DNA amplification from stool. Methodology and principal findings : In a first experiment, DNA was extracted from 37 stool samples with variable egg counts for T. trichiura and N. americanus applying two commercial kits, both with and without a prior bead beating step. The DNA concentration of T. trichiura and N. americanus was estimated by means of qPCR. The results showed clear differences in DNA concentration across both DNA extraction kits, which varied across both STHs. They also indicated that adding a bead beating step substantially improved DNA recovery, particularly when the FECs were high. In a second experiment, 20 stool samples with variable egg counts for A. lumbricoides, T. trichiura and N. americanus were preserved in either 96% ethanol, 5% potassium dichromate or RNA later and were stored at 4 degrees C for 65, 245 and 425 days. DNA was extracted using the DNeasy Blood & Tissue kit with a bead beating step. Stool samples preserved in ethanol proved to yield higher DNA concentrations as FEC increased, although stool samples appeared to be stable over time in all preservatives. Conclusions : The choice of DNA extraction kit significantly affects the outcome of NAATs. Given the clear benefit of bead beating and our validation of ethanol for (long-term) preservation, we recommend that these aspects of the protocol should be adopted by any stool sampling and DNA extraction protocol for downstream NAAT-based detection and quantification of STHs

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

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 duodenaleandA.ceylanicum),Strongyloides stercoralisandSchistosomain 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,StrongyloidesandSchistosoma). 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,StrongyloidesandSchistosoma. 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. Author summary Tests that detect parasite DNA in human stool are increasingly being used for the diagnosis of infections with intestinal worms, including schistosomiasis. To ensure the quality in diagnostic testing of these parasitic worms, it is important that laboratories evaluate the diagnostic performance of their DNA-based tests. This can best be achieved by participating in an external quality assessment scheme (EQAS). 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. Currently, such an EQAS for parasitic worms is lacking. We therefore piloted an international EQAS for the diagnosis of parasitic worms involving 15 laboratories in Africa, Asia, Australia, Europe, and North America. Although most laboratories performed well, we could clearly identify those laboratories that may need to improve their test protocol. We found that laboratories were using many different test protocols, and further research should aim to verify whether this has an impact on the performance of the diagnostic outcomes

Monitoring the therapeutic efficacy and the emergence of anthelmintic drug resistance in the Ethiopian national deworming program targeting human soil-transmitted helminthiasis

Soil-transmitted helminths (STHs), including Ascaris lumbricoides (roundworm), Trichuris trichiura (whipworm), Ancylostoma duodenale and Necator americanus (hookworms) are the most prevalent of all neglected tropical diseases. They affect the most impoverished people living in tropical and subtropical countries. To control the morbidity of these worms, administration of anthelmintic drugs to high-risk populations, the so-called preventive chemotherapy (PC) programs, are rolled out at level that is unprecedented in history in many endemic countries, including Ethiopia. To ensure the success of these large-scale deworming programs, it is of utmost importance that the therapeutic efficacy is periodically assessed to detect any treatment failure that may arise due to the emergence of anthelmintic resistance (AR)

Longitudinal assessment of the exposure to Ascaris lumbricoides through copromicroscopy and serology in school children from Jimma Town, Ethiopia

We previously demonstrated that serology holds promise as an alternative diagnostic tool to copromicroscopy to monitor and evaluate deworming programs targeting soil-transmitted helminths (STHs)

Characterization of the β-tubulin gene family in Ascaris lumbricoides and Ascaris suum and its implication for the molecular detection of benzimidazole resistance

Background The treatment coverage of control programs providing benzimidazole (BZ) drugs to eliminate the morbidity caused by soil-transmitted helminths (STHs) is unprecedently high. This high drug pressure may result in the development of BZ resistance in STHs and so there is an urgent need for surveillance systems detecting molecular markers associated with BZ resistance. A critical prerequisite to develop such systems is an understanding of the gene family encoding beta-tubulin proteins, the principal targets of BZ drugs. Methodology and principal findings First, the beta-tubulin gene families of Ascaris lumbricoides and Ascaris suum were characterized through the analysis of published genomes. Second, RNA-seq and RT-PCR analyses on cDNA were applied to determine the transcription profiles of the different gene family members. The results revealed that Ascaris species have at least seven different beta-tubulin genes of which two are highly expressed during the entire lifecycle. Third, deep amplicon sequencing was performed on these two genes in more than 200 adult A. lumbricoides (Ethiopia and Tanzania) and A. suum (Belgium) worms, to investigate the intra- and inter-species genetic diversity and the presence of single nucleotide polymorphisms (SNPs) that are associated with BZ resistance in other helminth species; F167Y (TTC>TAC or TTT>TAT), E198A (GAA>GCA or GAG>GCG), E198L (GAA>TTA) and F200Y (TTC>TAC or TTT>TAT). These particular SNPs were absent in the two investigated genes in all three Ascaris populations. Significance This study demonstrated the presence of at least seven beta-tubulin genes in Ascaris worms. A new nomenclature was proposed and prioritization of genes for future BZ resistance research was discussed. This is the first comprehensive description of the beta-tubulin gene family in Ascaris and provides a framework to investigate the prevalence and potential role of beta-tubulin sequence polymorphisms in BZ resistance in a more systematic manner than previously possible