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Evaluation of Two DNA Extraction Methods for Detection of Strongyloides stercoralis Infection.

ABSTRACT: Strongyloides stercoralis is present worldwide, but its prevalence is still uncertain, mainly due to the lack of sensitivity of diagnostic methods. Molecular techniques are under development, but a standardized protocol is still unavailable. We compared the sensitivity of real-time PCR, using two extraction protocols, with that of the Baermann technique. Samples were collected in the framework of the baseline screening of a randomized clinical trial evaluating moxidectin against S. stercoralis in Lao People's Democratic Republic. Two stool samples from each participant were processed by the Baermann method, and one subsample was processed by PCR. DNA was extracted using the QIAamp DNA stool minikit based on the standard protocol for the QIAamp DNA minikit (QIA) and using a modification of the QIA procedure (POL). Subsequently, all extracted samples were analyzed by real-time PCR. Overall, 95 samples were analyzed by the three diagnostic methods. Sixty-nine (72.6%) samples were positive according to the Baermann method, 25 (26.3%) by the QIA method, and 62 (65.3%) by the POL method. The sensitivities were 86% (95% confidence interval [CI], 76.7 to 92.9), 31.0% (95% CI, 21.3 to 42.6), and 78.0% (95% CI, 66.8 to 86.1) for the Baermann, QIA, and POL methods, respectively. The sensitivities calculated for each day of the Baermann method separately were 60% (48.4 to 70.8%) and 64% (52.2 to 74.2%) for days 1 and 2, respectively. In conclusion, the POL method revealed a good performance and was comparable to the Baermann test performed on two stool samples and superior to the Baermann method performed on one stool sample. Additional studies are needed to standardize a PCR protocol for S. stercoralis diagnosis.

SUBMITTER: Barda B

PROVIDER: S-EPMC5869848 | biostudies-other | 2018 Apr

REPOSITORIES: biostudies-other

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Evaluation of Two DNA Extraction Methods for Detection of Strongyloides stercoralis Infection.

Barda Beatrice B Wampfler Rahel R Sayasone Somphou S Phongluxa Khampheng K Xayavong Syda S Keoduangsy Khonsavanh K Schindler Christian C Keiser Jennifer J

Journal of clinical microbiology 20180326 4

<i>Strongyloides stercoralis</i> is present worldwide, but its prevalence is still uncertain, mainly due to the lack of sensitivity of diagnostic methods. Molecular techniques are under development, but a standardized protocol is still unavailable. We compared the sensitivity of real-time PCR, using two extraction protocols, with that of the Baermann technique. Samples were collected in the framework of the baseline screening of a randomized clinical trial evaluating moxidectin against <i>S. ste ...[more]

PMID: 29367294

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Project description:BackgroundStrongyloides stercoralis is endemic in tropical and subtropical regions, but reports of infections in central and northern Europe have been recently increasing. Infections occur mainly in humans and dogs. In dogs, both dog-adapted and zoonotic S. stercoralis genotypes seem to occur. Clinical manifestations mainly include gastrointestinal and respiratory signs. The severity of the disease can vary greatly and depends on the immune status of the host. The infection is potentially fatal in immunosuppressed individuals, either medically induced or due to an underlying disease, in which hyperinfections and disseminated infections with extraintestinal parasite dissemination may occur.MethodsDiagnosis was based on coproscopy, including flotation and the Baermann funnel technique, histology of small intestinal biopsies and molecular analysis of mitochondrial cytochrome oxidase subunit I (COI) and hypervariable regions I and IV (HVR I and HVR IV) of the nuclear 18S rDNA loci.ResultsTwo independent cases of severe canine S. stercoralis infection in Austria are presented. In both cases, S. stercoralis was detected in histological sections of the small intestine and with the Baermann funnel technique. Molecular analysis revealed strains with zoonotic potential. Case 1 was a 1-year-old female French bulldog with a long history of respiratory and gastrointestinal signs, severe emaciation and apathy before S. stercoralis infection was diagnosed. Treatment with moxidectin (2.5 mg/kg body weight [BW], oral route) did not eliminate the infection, but treatment with ivermectin (0.2 mg/kg BW, subcutaneously) was successful. Case 2 consisted of two 2-month-old Pomeranian puppies, one female and one male, from a litter of four, which died soon after presenting dyspnoea and haemorrhagic diarrhoea (female) or torticollis (male); S. stercoralis infection was first diagnosed post-mortem.ConclusionMore attention should be paid to this nematode because although it appears to be rare in Austria, it is easily overlooked on standard coproscopy unless a Baermann funnel technique is used, and even then, it can be missed. Moxidectin is not always successful in eliminating the infection, and treatment with ivermectin should be considered in cases of infection.

Project description:Detecting infections of Strongyloides stercoralis is arduous and has low sensitivity. Clinically this is a major problem because chronic infections may disseminate in the host and lead to a life threatening condition. Epidemiologically, S. stercoralis is often missed in surveys as it is difficult to identify by standard stool examination procedures. We present, for the first time, evidence that the infection can be detected in filtered urine samples collected and processed in the field and subsequently assayed for the presence of parasite DNA. Urine specimens (∼40mL) were collected from 125 test and control individuals living in rural and peri-urban regions of Northern Argentina. From the same individuals, fresh stool specimens were processed using three different copropological methods. Urine specimens were filtered in the field through a 12.5cm Whatman No. 3 filter. The filters were dried and packed individually in sealable plastic bags with desiccant and shipped to a laboratory where DNA was recovered from the filter and PCR-amplified with primers specific to a dispersed repetitive sequence. Prevalence of S. stercoralis infection by stool culture and direct examination was 35/125 (28%), In contrast, PCR-based detection of parasite-specific trans-renal DNA in urine indicated that 56/125 (44.8%) carried the parasite. Of the patients that tested positive for urine-based parasite DNA, approximately half also tested positive in their stool specimens. There were 6.4% of cases where parasite larvae were seen in the stool but no DNA was amplified from the urine. As proof of principle, DNA amplification from urine residue reveals significantly more cases of S. stercoralis infection than the current standard stool examination techniques. Additional work is required to establish the relative utility, sensitivity and specificity of urine-based analysis compared to parasitological and nucleic acid detection from stool for clinical and epidemiological detection for S. stercoralis infection.

Project description:BACKGROUND:Strongyloidiasis is a gut infection with Strongyloides stercoralis which is common world wide. Chronic infection usually causes a skin rash, vomiting, diarrhoea or constipation, and respiratory problems, and it can be fatal in people with immune deficiency. It may be treated with ivermectin or albendazole or thiabendazole. OBJECTIVES:To assess the effects of ivermectin versus benzimidazoles (albendazole and thiabendazole) for treating chronic strongyloides infection. SEARCH METHODS:We searched the Cochrane Infectious Diseases Group Specialized Register (24 August 2015); the Cochrane Central Register of Controlled Trials (CENTRAL), published in the Cochrane Library; MEDLINE (January 1966 to August 2015); EMBASE (January 1980 to August 2015); LILACS (August 2015); and reference lists of articles. We also searched the metaRegister of Controlled Trials (mRCT) using 'strongyloid*' as a search term, reference lists, and conference abstracts. SELECTION CRITERIA:Randomized controlled trials of ivermectin versus albendazole or thiabendazole for treating chronic strongyloides infection. DATA COLLECTION AND ANALYSIS:Two review authors independently extracted data and assessed risk of bias in the included trials. We used risk ratios (RRs) with 95% confidence intervals (CIs) and fixed- or random-effects models. We pooled adverse event data if the trials were sufficiently similar in their adverse event definitions. MAIN RESULTS:We included seven trials, enrolling 1147 participants, conducted between 1994 and 2011 in different locations (Africa, Southeast Asia, America and Europe).In trials comparing ivermectin with albendazole, parasitological cure was higher with ivermectin (RR 1.79, 95% CI 1.55 to 2.08; 478 participants, four trials, moderate quality evidence). There were no statistically significant differences in adverse events (RR 0.80, 95% CI 0.59 to 1.09; 518 participants, four trials, low quality evidence).In trials comparing ivermectin with thiabendazole, there was little or no difference in parasitological cure (RR 1.07, 95% CI 0.96 to 1.20; 467 participants, three trials, low quality evidence). However, adverse events were less common with ivermectin (RR 0.31, 95% CI 0.20 to 0.50; 507 participants; three trials, moderate quality evidence).In trials comparing different dosages of ivermectin, taking a second dose of 200 μg/kg of ivermectin was not associated with higher cure in a small subgroup of participants (RR 1.02, 95% CI 0.94 to 1.11; 94 participants, two trials).Dizziness, nausea, and disorientation were commonly reported in all drug groups. There were no reports of serious adverse events or death. AUTHORS' CONCLUSIONS:Ivermectin results in more people cured than albendazole, and is at least as well tolerated. In trials of ivermectin with thiabendazole, parasitological cure is similar but there are more adverse events with thiabendazole.

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Evaluation of Two DNA Extraction Methods for Detection of Strongyloides stercoralis Infection.

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Evaluation of Two DNA Extraction Methods for Detection of Strongyloides stercoralis Infection.

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