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 RNAlater and were stored at 4°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.

Triplex polymerase chain reaction assay for detection of major soil-transmitted helminths, Ascaris lumbricoides, Trichuris trichiura, Necator americanus, in fecal samples.

Ascaris lumbricoides, Trichuris trichiura, and Necator americanus are medically important soil-transmitted helminths (STHs) occurring frequently worldwide including Thailand. Fecal examination using a microscope has been recommended as the gold standard for diagnosis of STH infections, but suffers from low sensitivity. Recently, highly sensitive and specific assays, such as multiplex quantitative PCR, has been established, but the high cost and need for special instruments are still barriers limiting their applications in routine diagnosis. Therefore, a conventional multiplex PCR assay, with its lower cost and greater simplicity, was developed, for the simultaneous detection of STHs in fecal samples. The multiplex PCR assay was species-specific to the three STHs, and could detect one copy of DNA target. Compared with microscopic examination of fecal samples, sensitivity and specificity of the multiplex PCR was 87% and 83%, respectively. This multiplex PCR assay provides an alternative method for routine diagnosis of STHs infection, and might be applied for epidemiological studies of STHs in endemic areas.

Acquired hookworm immunity in the golden hamster (Mesocricetus auratus) elicited by living Necator americanus third-stage infective larvae.

The aim of the study is to demonstrate and understand the acquired immunity in golden hamsters (Mesocricetus auratus) elicited by primary Necator americanus infective third-stage larvae (L3) infection. Hamsters infected with 150 L3 for 1, 2, 3, 6 and 10 weeks, were challenged with the same number of L3 and sacrificed 25 days post challenge. The primarily infected hamsters exhibited 99-100% protection against subsequent L3 challenge compared to un-infected naive hamsters. The acquired immunity was developed as early as 1 week post L3 infection and lasted up to 10 weeks. Similar protective immunity was obtained in hamsters infected with N. americanus L3 and then treated orally with a single of 100mg/kg albendazole, followed by challenge with N. americanus L3 4 and 8 weeks post-treatment. The infected hamsters exhibited a rise in IgG antibodies against L3 and juvenile adult worm antigens. Histological examination showed that challenging L3 were trapped in the skin of primarily infected hamsters and surrounded or infiltrated by different inflammatory cells. The trapped L3 were damaged and dead followed by the formation of granulomas encasing dead worms. The results demonstrate that hamsters primarily infected with N. americanus L3 develop acquired immunity against re-infection.

Suppression of inflammatory immune responses in celiac disease by experimental hookworm infection.

We present immunological data from two clinical trials where the effect of experimental human hookworm (Necator americanus) infection on the pathology of celiac disease was evaluated. We found that basal production of Interferon- (IFN-)γ and Interleukin- (IL-)17A from duodenal biopsy culture was suppressed in hookworm-infected participants compared to uninfected controls. Increased levels of CD4+CD25+Foxp3+ cells in the circulation and mucosa are associated with active celiac disease. We show that this accumulation also occurs during a short-term (1 week) oral gluten challenge, and that hookworm infection suppressed the increase of circulating CD4+CD25+Foxp3+ cells during this challenge period. When duodenal biopsies from hookworm-infected participants were restimulated with the immunodominant gliadin peptide QE65, robust production of IL-2, IFN-γ and IL-17A was detected, even prior to gluten challenge while participants were strictly adhering to a gluten-free diet. Intriguingly, IL-5 was produced only after hookworm infection in response to QE65. Thus we hypothesise that hookworm-induced TH2 and IL-10 cross-regulation of the TH1/TH17 inflammatory response may be responsible for the suppression of these responses during experimental hookworm infection.

Innate and cognate mechanisms of pulmonary eosinophilia in helminth infection.

Passage of helminth larvae through the lungs can cause pulmonary eosinophilia that may have evolved as a means of parasite attrition. If allergic responses represent a misdirected activation of this arm of the immune system, then mechanisms governing eosinophil recruitment during infection would be expected to be closely related to those seen in allergy. We studied primary Necator americanus infection and compared this to multiply-infected or vaccinated mice. The arrival of larvae in the lungs triggered rapid eosinophil recruitment, which was greatly enhanced in previously sensitized mice. Interestingly, the presence of larvae in the lung was sufficient to trigger eosinophil chemoattractant production, including the chemokines eotaxin and MIP-1alpha, and was not enhanced by prior exposure to the parasites. Infection stimulated IL-5 production in all groups; however, this and IgE production were greatly enhanced in sensitized animals. Elevated IL-5 increased bone marrow production of eosinophils, and eosinophilia was abrogated by treatment with anti-IL-5 antibody. Therefore, trapping of larvae in the pulmonary vasculature is sufficient to trigger eosinophil recruitment, by induction of chemokines and IL-5. Primed cognate Th2 immunity does not increase local chemokine production, but does increase IL-5 production, which greatly enhances the availability of eosinophils for recruitment to the lung.

Hookworm infection.

It retards growth and intellectual development in millions of children yet is largely ignored by researchers. New findings suggest excellent possibilities for a vaccine.

Necator americanus in the mouse: histopathological changes associated with the passage of larvae through the lungs of mice exposed to primary and secondary infection.

The mouse/Necator americanus model was studied to assess the histopathological changes that occur in the lungs following primary and secondary exposure to infective larvae. Groups of BALB/c mice were infected percutaneously and killed on various days post infection. Parasite numbers were counted, the bronchoalveolar leukocyte response was quantified and histological sections of lung material were examined for evidence of host protective inflammatory reactions. An increase in the inflammatory infiltration was observed between days 5 and 9 in both primary and secondary infections but was considerably more intense in re-infected animals. This involved a marked change in the character of the infiltrate, particularly in the number of eosinophils that were recovered in lavage fluid. More worms were trapped in the lungs of challenged mice, as assessed through their inability to escape from lung material incubated in vitro. Overall, the results were found to be compatible with the development of acquired resistance to N. americanus and the expression of host protective immunity during the development of challenge-infection larvae in the lungs.