Rapid detection of Opisthorchis viverrini and Strongyloides stercoralis in human fecal samples using a duplex real-time PCR and melting curve analysis

Human opisthorchiasis caused by the liver fluke Opisthorchis viverrini is an endemic disease in Southeast Asian countries including the Lao People’s Democratic Republic, Cambodia, Vietnam, and Thailand. Infection with the soil-transmitted roundworm Strongyloides stercoralis is an important problem worldwide. In some areas, both parasitic infections are reported as co-infections. A duplex real-time fluorescence resonance energy transfer (FRET) PCR merged with melting curve analysis was developed for the rapid detection of O. viverrini and S. stercoralis in human fecal samples. Duplex real-time FRET PCR is based on fluorescence melting curve analysis of a hybrid of amplicons generated from two genera of DNA elements: the 162 bp pOV-A6 DNA sequence specific to O. viverrini and the 244 bp 18S rRNA sequence specific to S. stercoralis, and two pairs of specific fluorophore-labeled probes. Both O. viverrini and S. stercoralis can be differentially detected in infected human fecal samples by this process through their different fluorescence channels and melting temperatures. Detection limit of the method was as little as two O. viverrini eggs and four S. stercoralis larvae in 100 mg of fecal sample. The assay could distinguish the DNA of both parasites from the DNA of negative fecal samples and fecal samples with other parasite materials, as well as from the DNA of human leukocytes and other control parasites. The technique showed 100% sensitivity and specificity. The introduced duplex real-time FRET PCR can reduce labor time and reagent costs and is not prone to carry over contamination. The method is important for simultaneous detection especially in areas where both parasites overlap incidence and is useful as the screening tool in the returning travelers and immigrants to industrialized countries where number of samples in the diagnostic units will become increasing.     

Factors Affecting Recovery of Strongyloides stercoralis Larvae: an Approach to a Newly Modified Formalin-Ether Concentration Technique for Diagnosis of Strongyloidiasis

To improve the diagnosis efficiency of human strongyloidiasis by using formalin-ether concentration technique (FECT), the effects of various factors on the recovery rates of Strongyloides stercoralis larvae were comparatively evaluated. Fresh stool and a short time exposure of larvae to formalin yielded significantly higher numbers of larvae than preserved stool and 10-min exposure. Likewise, straining through wire mesh yielded a significantly higher number of larvae recovered than straining through gauze did. In addition, centrifugation for 5 min for separation of larvae from debris yielded a significantly greater number of larvae recovered than centrifugation for 2 min did. The efficacies of the five versions of FECT with different factors-FECT 1, FECT 2, FECT 3, FECT 4, and FECT 5-were then compared. It was found that FECT 5 was 1.8, 2.0, 1.9, and 1.4 times more effective than FECT 1, FECT 2, FECT 3, and FECT 4, respectively. FECT 5 is a modified FECT method, whose modifications included using fresh stool without a preservative substance; a short-time rather than 10-min formalin exposure; and the use of wire mesh instead of gauze.Strongyloidiasis caused by Strongyloides stercoralis is a harmful infectious disease for immunosuppressed patients (5). The most accurate method for diagnosis of strongyloidiasis, which is widely used in areas where this disease is endemic, is to detect S. stercoralis larvae in stool, whereas detection by indirect methods, especially immunological techniques, are not as effective (11). Agar plate culture (APC) has consistently been found to be 1.6 to 6.0 times more effective than formalin-ether concentration technique (FECT) (1, 8). However, APC has many disadvantages: it is time-consuming and costly, has a tendency of risk of larval infection (7, 8), and demonstrates a particular inability to detect 10% (6) to 20% (12) of positive samples detected by FECT. Thus, if the efficacy of FECT can be improved, a more precise diagnosis of strongyloidiasis will be obtained. Many laboratory personnel believe that variations in the skill and experience of examiners caused the effectiveness of APC shown to be 1.6 to 6.0 times greater than FECT (1). However, there could possibly be some concealed factors causing the lower sensitivity of FECT.A pilot study was thus conducted using a large number of S. stercoralis larvae in diluted stool samples from patients with strongylodiasis (kept for 2 months in 10% formalin) for the capability of larval counting. FECT was performed, after which four layers were formed: ether, plug of debris, formalin and sediment. The results showed that 62.5% of larvae were in the debris plug, 32.5% floated in the formalin layer, and only 5% settled in the sediment. The diluted stool samples were also comparatively filtered either through two layers of wet gauze or two layers of wire mesh (0.7- by 0.7-mm and 1.2- by 1.2-mm pore sizes). The results revealed that a significantly lower number of larvae were recovered in the experiment using gauze than with the two wire meshes. The fine wire mesh also yielded a significantly lower number of larvae than the coarse one did. This work therefore aims to compare various factors affecting the number of larvae dwelling in fresh stool that may affect the recovery rate of S. stercoralis larvae and to determine suitable modification conditions of the FECT method to obtain a more precise diagnosis of strongyloidiasis.     

Albendazole Stimulates the Excretion of Strongyloides stercoralis Larvae in Stool Specimens and Enhances Sensitivity for Diagnosis of Strongyloidiasis

We succeeded in stimulation of excretion of Strongyloides stercoralis larvae in stool by oral administration of a single dose of 400 mg albendazole to strongyloidiasis patients. This result overcame the false-negative results of stool examination due to low larval numbers. Stool samples were collected from 152 asymptomatic strongyloidiasis patients in the morning, prior to eating. After breakfast, they were given a dose of 400 mg albendazole, and stool samples were collected the following morning. Agar plate culture (APC), modified formalin-ether concentration technique (MFECT), and direct-smear (DS) methods were used to examine stool specimens within 3 h after defecation. The results before and after albendazole was taken were compared. All APCs that were positive became negative after albendazole administration, while MFECT showed a 1.4- to 18.0-fold increase in larval numbers in 97.4% (148/152) of the samples. The DSs were positive in 3 out of 3 smears at a larval number of ≥45 larvae per g (lpg) of stool, and in 1or 2 out of 3 smears at a larval number between 35 and 44 lpg. At a larval number of <35 lpg, the DS became negative. Interestingly 90.5% (19/21) of the samples that were negative by all methods before albendazole administration became positive by MFECT after the treatment. Thus, MFECT can be effectively used for diagnosis of strongyloidiasis with prior administration of albendazole to the subject.Strongyloidiasis is a helminthic infection caused by Strongyloides stercoralis, a worm that is particularly dangerous for immunosuppressed patients. Although many methods are presently being used to diagnose this disease, a gold standard for the detection of larvae in the stool is still needed (14). Parasitological methods include agar plate culture (APC) (10), the Baermann method (4), the formalin-ether concentration technique (FECT) (13), the quantitative formalin ethyl acetate concentration technique (QFECT) (9), and the newly modified FECT (MFECT) (1). However, each method has its own disadvantages, resulting in false negativity. In addition, female S. stercoralis parasites, unlike other intestinal roundworms, embed in the mucosa of the small intestine, where they lay embryonated eggs that immediately hatch (8). Thus, the larvae are often scanty and irregularly excreted (11, 15). Repeated stool examination is therefore recommended for the diagnosis of strongyloidiasis (6). During an intestinal-helminth survey by direct fecal smear (DS) examination, some participants tested negative for Strongyloides larvae. After albendazole treatment for Ascaris lumbricoides, Trichuris trichiura, and hookworms, the stools were reexamined. Surprisingly, stools that were previously negative for S. stercoralis larvae became positive (data not shown). It was then considered possible that albendazole treatment could increase the sensitivity of stool examination methods for diagnosis of strongyloidiasis. Thus, this research aimed to enhance the sensitivity for the diagnosis of strongyloidiasis by using a single 400-mg oral dose of albendazole to stimulate the excretion of larvae into the stool for easier detection by MFECT and/or DS, particularly in cases where strongyloidiasis was strongly suspected, such as in patients with unexplained chronic diarrhea and patients returning from areas where strongyloidiasis is endemic.     

Molecular identification of Ascaris lumbricoides and Ascaris suum recovered from humans and pigs in Thailand,Lao PDR,and Myanmar

Parasitology Research - Ascaris lumbricoides is the largest roundworm known from the human intestine while Ascaris suum is an internal parasite of pigs. Ascariasis, caused by Ascaris lumbricoides,...