Elsevier

Acta Tropica

Volume 126, Issue 2, May 2013, Pages 110-114
Acta Tropica

An improved DNA isolation technique for PCR detection of Strongyloides stercoralis in stool samples

https://doi.org/10.1016/j.actatropica.2013.02.003Get rights and content

Abstract

Strongyloides stercoralis is a nematode that causes severe infections in immunocompromised patients. The low parasitic burden of chronically infected patients makes diagnosis difficult to achieve by conventional methods. Here, an in-house (IH) method for the isolation of parasite DNA from stools and a PCR assay for the molecular diagnosis of S. stercoralis were optimized. DNA yield and purity improved with the IH method which included a step of incubation of stool samples with a glycine–SDS buffer and mechanical disruption prior to DNA extraction. For the PCR assay, the addition of bovine serum albumin was required to neutralize inhibitors present in stool. The analytical sensitivity of the PCR using DNA as template, isolated with the IH method, was superior to the commercial one. This study demonstrates that a combined method that adds the step of glycine–SDS buffer incubation plus mechanical disruption prior to DNA isolation with the commercial kit increased PCR sensitivity to levels of the IH method. Finally, our assay was tested on 17 clinical samples. With the IH method for DNA isolation, a S. stercoralis specific band was detected by PCR in the first stool sample in all patients (17/17), while with the commercial kit, our S. stercoralis-specific band was only observed in 7 samples. The superior efficiency of the IH and combined methods over the commercial kit was demonstrated when applied to clinical samples with low parasitic burden. These results show that the DNA extraction procedure is a key to increase sensitivity of the S. stercoralis PCR assay in stool samples. The method developed here could help to improve the molecular diagnosis of S. stercoralis.

Highlights

► DNA yield and purity improved with the IH method. ► IH method includes incubation of stool samples in glycine–SDS buffer plus mechanical disruption. ► S. stercoralis was detected in all patients by the IH method but only in 9/17 by APC in the first sample. ► The higher efficiency of the IH method is demonstrated in clinical samples with low parasitic burden.

Introduction

The diagnosis of Strongyloides stercoralis, endemic in tropical and subtropical regions, is difficult to achieve owing to its life cycle. This nematode has alternate parasitic and free-living life styles. Soil or auto-infective filariform larvae (L3) penetrate the skin, enter the bloodstream and finally get into the small intestine to dwell as parthenogenetic females. Autoinfection is responsible for chronic infections in people living out of endemic areas for decades. In these persons, larvae excretion is fluctuating and low. Most chronic infections are asymptomatic, sometimes with eosinophilia as the only laboratory finding. Immunocompromised people may experience accelerated autoinfection causing hyperinfection or dissemination syndromes where mortality reaches roughly 80% (Siddiqui and Berk, 2001, Keiser and Nutman, 2004, Concha et al., 2005). These patients can suffer bacteraemia, bacterial meningitis, bacterial abscesses, diarrhea and pneumonia due to the erratic migration of enterobacteria-carrying autoinfectant stage L3. In this setting, larvae are easily detected in samples. These clinical presentations can be observed following immunosuppressive therapy (e.g. corticosteroids) as in transplant recipients or autoimmune disease patients. Accordingly, current practice guidelines recommend the screening for Strongyloides infection before transplantation or immunosuppressive therapy (Tomblyn et al., 2009; Ramanathan and Nutman 2008; Roxby et al., 2009). However, physicians out of endemic areas do not routinely check for S. stercoralis in asymptomatic patients missing opportunity of diagnosis (Boulware et al., 2007). To date, patients with suspected strongyloidosis are controlled by direct observation of larvae in stool. Those with negative results, but having eosinophilia and history of residence in endemic areas, receive ivermectin as empiric treatment (Tomblyn et al., 2009). Nevertheless, recent studies have raised concerns on the efficacy of the current therapy dosage (Bisoffi et al., 2011, Ramanathan and Nutman, 2008). Direct observation of larvae in stools has low sensitivity, and serology may provide false negative or indeterminate results thus limiting the use of these methods as screening tests (Keiser and Nutman, 2004, Segarra-Newnham, 2007, Vadlamudi et al., 2006). Agar plate culture (APC) is currently the most sensitive method without reaching 100% sensitivity. It is laborious and requires at least 7-day culture follow-up for best results (Segarra-Newnham, 2007, Repetto et al., 2010).

Molecular methods can be useful for the early diagnosis and etiological treatment of strongyloidosis in those asymptomatic patients that will undergo immunosuppressive therapy (Verweij et al., 2009, Gordon et al., 2011). However, optimization of DNA extraction and PCR assays for the detection of intestinal nematodes in stools are challenging due to the complexity of nematode cuticle and inhibitors that can be present in stools (Wilson, 1997, Al-Soud Waleed and Rådstrom, 2000). Thus, the aim of this study was to develop an in-house (IH) method for nematode DNA isolation to improve the accuracy of S. stercoralis diagnosis by PCR.

Section snippets

Materials and methods

This study was approved by the Ethics Committees of both, the School of Medicine and the Medical School Hospital (Hospital de Clínicas José de San Martin) at University of Buenos Aires. Informed consents were signed by all participants before sample collection. All stool samples were obtained from patients above 18 years old.

Optimization of an IH method for DNA isolation of stools

Different protocols of DNA extraction of purified S. stercoralis L3 larvae were assayed. The DNA yield was significantly higher (P < 0.01) with the IH method, where samples were incubated with GTES followed by mechanical disruption (2.13 ± 1.34 μg/ml; n = 7), compared to those where this step was omitted (0.25 ± 0.20 μg/ml; n = 7). Moreover, by adding this step, the A260/280 ratio rose from values below 1.2 to 1.8 ± 0.02 reflecting an enhanced DNA purity. When the IH method was applied to stools from healthy

Discussion

The results presented here show that S. stercoralis DNA obtained from stool samples by an IH method is suitable for a PCR technique, and could improve the sensitivity of diagnosis. A combination of GTES plus a strong mechanical disruption was required for successful DNA recovery. Stools may contain bacterial proteases, nucleases, cell debris and bile acids which can inhibit DNA amplification by PCR. The ability of BSA to bind and neutralize PCR inhibitors has been demonstrated previously (

Funding

This work was supported by grants from the Agencia Nacional de Promoción Científica y Tecnológica (ANPCyT) from Argentina and the Universidad de Buenos Aires.

Conflict of interest

All authors declare no conflict of interest.

Acknowledgments

The authors thank D. Bomparola and A. Gómez Raccio for the submission of bacterial, fungi and rotavirus samples and Dr. L. Belaunzarán for valuable help with the artwork. CDAS, SIC, VST and SMGC are members of the Research Career of the Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Argentina.

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