Formalin-ethyl Acetate Concentration Technique Research Articles

Diagnosis of early infection and post chemotherapeutic treatment by copro-DNA detection in experimental opisthorchiasis

Opisthorchis viverrini is considered as a carcinogenic parasite which is responsible for cholangiocarcinoma in Southeast Asia. Effective treatment and control of the parasite to reduce the risk of cancer requires efficient diagnostic methods. Because of the limitations involved in human studies, the present work is aimed at comparing diagnostic performance of copro-DNA detection by PCR and fecal examination by formalin-ethyl acetate concentration technique (FECT) during the course of O. viverrini infection and postcurative chemotherapy in experimentally infected hamsters. A manual method of DNA preparation from fecal specimens previously established in human studies was used in PCR analysis. Following infection with varying doses of metacercariae (5, 10, 25, and 50 cysts/animal), PCR analyses were positive as early as 3weeks post-infection while FECT were negative. PCR tests were comparable to FECT regardless of intensity of infection beginning from 4 to 12weeks post infection. In chemotherapeutic experiments, with reference to the presence of worm in liver, treatment failures were detected by PCR but not FECT in a group of hamsters infected with 10 cysts/animal. PCR and FECT both detected residual infection at 1 and 2weeks post-treatment in the group of animals infected with five cysts per animal. High concordant results between diagnoses by PCR, FECT, and worm burden indicated that PCR is suitable for an early diagnosis, evaluation of drug efficacy, and also re-infection post-treatment.

Differentiating Entamoeba histolytica, Entamoeba dispar and Entamoeba moshkovskii using nested polymerase chain reaction (PCR) in rural communities in Malaysia

BackgroundIn this study, a total of 426 human faecal samples were examined for the presence of Entamoeba histolytica, Entamoeba dispar, Entamoeba moshkovskii infection via a combination of microscopic examination and nested polymerase chain reaction (PCR) targeting 16S ribosomal RNA of Entamoeba species.MethodsFaecal sample were collected from 426 participants in five rural villages in Peninsular Malaysia. The faecal samples were processed by direct wet smear and formalin ethyl acetate concentration technique followed by iodine staining and examined via microscopy for the presence of Entamoeba species and other intestinal parasites. Microscopically positive samples for Entamoeba species cysts were further characterized using a Nested Polymerase Chain Reaction (Nested-PCR) targeting 16S-like ribosomal RNA gene. The data entry and analysis was carried out using the SPSS software (Statistical Package for the Social Sciences) program for Windows version 17 (SPSS, Chicago, IL, USA).ResultsBased on single faecal examination, overall prevalence of Entamoeba infection was 17.6% (75/426). Females (19.1%) were more commonly infected compared to males (15.9%). Comparison by age groups showed that adults (23.9%) had higher infection rates than children (15.3%). The PCR results showed that 52 out of 75 microscopy positive samples successfully generated species-specific amplicons. The infection with E. histolytica (75.0%; 39/52) was the most common, followed by E. dispar (30.8%; 18/52) and E. moshkovskii (5.8%; 3/52). Of these, 33 (63.5%) were shown to contain only E. histolytica, 10 (19.2%) contained E. dispar and 3 (5.8%) contained only E. moshkovskii. Mixed infection with E. histolytica and E. dispar was found in 6 (11.5%) samples.ConclusionsThe present study essentially emphasized the benefit of molecular techniques in discriminating the pathogenic Entamoeba species from the non-pathogenic for accurate diagnosis and better management of amoebiasis. The presence of E. moshkovskii is of great public health concern as it was the first time it has been reported in Malaysia.

Helminth and intestinal protozoa infections, multiparasitism and risk factors in Champasack province, Lao People's Democratic Republic.

BackgroundDetailed investigations of multiparasitism are scarce in the Mekong River basin. We assessed helminth (trematode, nematode, and cestode), and intestinal protozoa infections, and multiparasitism in random population samples from three different eco-epidemiological settings in Champasack province, southern Lao People's Democratic Republic (Lao PDR), and determined underlying risk factors.MethodologyTwo stool samples were collected from 669 individuals aged ≥6 months over consecutive days and examined for helminth infections using the Kato-Katz method. Additionally, one stool sample per person was subjected to a formalin-ethyl acetate concentration technique for diagnosis of helminth and intestinal protozoa infections. Questionnaires were administered to obtain individual and household-level data pertaining to behavior, demography and socioeconomic status. Risk factors for hepato-biliary and intestinal parasitic infections and multiparasitism were determined using multiple logistic regressions analyses.Principal FindingsMultiple species intestinal parasite infections were common: 86.6% of the study participants harbored at least two and up to seven different parasites concurrently. Regarding nematode infections, hookworm was the most prevalent species (76.8%), followed by Ascaris lumbricoides (31.7%) and Trichuris trichiura (25.0%). Regarding trematodes, Opisthorchis viverrini and Schistosoma mekongi infections were found in 64.3% and 24.2% of the participants, respectively. Infections with intestinal protozoa were rare.Conclusions/SignificanceThere is a pressing need to intensify and sustain helminth control interventions in the southern part of Lao PDR. Given the high prevalence with nematode and trematode infections and the extent of multiparasitism, preventive chemotherapy is warranted. This intervention should be coupled with health education and improved access to clean water and adequate sanitation to consolidate morbidity control and enhance sustainability.

Prevalence of Giardia lamblia in diarrheic children in Almadinah Almunawarh, KSA

Giardia lamblia is one of the most common intestinal parasites of humans. It is a flagellated, teardrop-shaped parasite which has two life forms, the trophozoite and the cyst. This study was carried out in Almadinah Almunawarh in 2011 to determine the prevalence of giardiasis in diarrheic children. Three methods were used in this study: direct microscopic examination of stool samples, formol-ethyl acetate concentration technique and rapid dipstick immunodiagnostic technique (detection of Giardia copro-antigen). Out of 889 stool specimens collected from children, giardiasis was detected in 1.9% of them. The age group 6 – 8 years had the highest rate (41.2%) and 4–6 years the lowest (29.4%). Also, males had a higher rate than females (52.9%) and (47.1%), respectively. Diagnosis of giardiasis should rely on microscopic examination of stool samples. Concentration techniques are to be done if available, together with sensitive and specific immunichromatographic tests.

Investigation on Opisthorchis felineus occurrence and life cycle in Italy

Opisthorchiasis is a fish borne parasitic infection caused by helminths of the genus Opisthorchis (Digenea, Opisthorchiidae), affecting humans and other fish-eating mammals. Despite Opisthorchis felineus was first described in Italy in 1884, no cases of human opisthorchiasis were reported in this country until 2004; from then on, 4 outbreaks due to this species have been recorded in Central Italy. Following the more relevant of these outbreaks, involving 34 people in August 2007, snails, fishes and fecal samples collected from the Bolsena and Bracciano lakes (Central Italy) were analyzed in order to define the cycle of O. felineus in the area and investigate its prevalence in the different hosts. Pools of 20–40 snails each (4983 specimens altogether) of the genus Bithynia were analyzed by PCR for parasite DNA detection. Eight hundred and ninety-four fish belonging to 12 species were collected from the two lakes and tested for metacercariae both by muscle compression and digestion techniques. Eighty-seven fecal samples of 5 putative definitive host species were collected very close to the two lakes and tested for parasite eggs detection by formalin–ethyl acetate concentration technique. Identification at the species level of metacercariae and eggs, respectively, from fish and stool was confirmed by PCR analysis and sequencing. O. felineus DNA was detected in 0.08% (overall minimum infection rate) of snails of the genus Bithynia from the two lakes. The tench, Tinca tinca, was the only fish found infested in both lakes (prevalence 88.5%). O. felineus eggs were found only in cat feces (prevalence 46.4%). The tench represents the only threat for the human consumption in the study area while Coregonus sp., the most economically important species for the local fishery and frequently consumed raw marinated, resulted to be not infected. The high prevalence recorded both in fish and in definitive host suggests a widespread and massive presence of the parasite in the area. Further studies are needed to better investigate the possible role of some cyprinids species as intermediate hosts, in order to check their safety for human consumption.

Rapid Molecular Detection of Opisthorchis viverrini in Human Fecal Samples by Real-Time Polymerase Chain Reaction

Real-time fluorescence resonance energy transfer (FRET) polymerase chain reaction (PCR) supplemented with melting curve analysis is a highly sensitive and fast method offering a high throughput. We report the development of a real-time FRET PCR for molecular detection of Opisthorchis viverrini in human fecal samples. The diagnostic sensitivity, specificity, accuracy, and positive and negative predictive values of this method were 97.5%, 100%, 98.9%, 100%, and 98.2%, respectively. The sensitivity was not significantly different from that of the quantified formalin-ethyl acetate concentration technique, the gold standard (P > 0.05). The procedure has potential for diagnosis of human opisthorchiasis in disease-endemic areas, for large epidemiologic investigations involving at risk populations, and monitoring eradication programs of the liver fluke, which causes hepatobiliary diseases and induces cholangiocarcinoma.

Feconomics®: A Simple, Novel and Fast Technique for Stool Concentration in Parasitology Laboratory

Summary Feconomics® is a new ready-to-use kit for fecal concentration that eliminates the need for centrifugation and floatation by using absorbent beads. To assess its efficacy in the diagnosis of intestinal parasites, a comparative, double-blind study was conducted in the Parasitology Laboratory of Celal Bayar University Medical School. Stools (Group I, n=251) submitted for routine ova and parasite examination were concentrated with both routine formalin ethyl acetate concentration (FEAC) technique and Feconomics®. Since the number of helminthes identified in the stool samples of patients were very low, helminthes obtained from the animal models in the laboratory were included (Group II, n=11). The iodine-stained samples of all stools and some of the positive samples stained with Gomori’s trichrome and Kinyoun’s acid fast stain were read by specialists. In Group I, 103 of 251 (41.04%) samples were found to be positive for one or more intestinal parasites; among them, 76 (30.28%) and 96 (38.25%) stools were found to be positive with FEAC and Feconomics®, respectively, and the difference was significant (P=0.000). Same parasites were identified with both methods among all 11 samples in Group II. There was no difference between the methods for the morphological integrity and visual appearances of the parasites having cyst or egg forms; yet, it was noticed that the vegetative forms of the parasites were only identified with Feconomics®. Review of our data indicated that Feconomics® may be suggested as a fast and effective fecal concentration method for Parasitology laboratories owing to the identification of higher number of parasites compared to FEAC, and parasites with only vegetative forms such as Dientamoeba fragilis.

Diversity of human intestinal helminthiasis in Lao PDR

Food-borne trematodiasis is an emerging public health problem, including in Lao PDR. We investigated the diversity of intestinal helminthes and polyparasitism in patients with hepatobiliary or intestinal symptoms in hospital and community-based surveys. Stool samples from 232 individuals aged >or=15 years were examined by the Kato-Katz method (three samples) and a formalin ethyl-acetate concentration technique (one sample). Opisthorchis viverrini and minute intestinal flukes (MIF) were common, with prevalences of 86.2% and 62.9%, respectively. Hookworm was the predominant soil-transmitted helminth (65.9%). The prevalences of Taenia spp., Strongyloides stercoralis and Trichuris trichiura were 22.8%, 10.3% and 8.6%, respectively. Additionally, 97 individuals were purged; O. viverrini and Haplorchis taichui were found in 95 and 76 participants, respectively. Other trematodes included Phaneropsolus bonnei (22.7%), Prosthodendrium molenkampi (14.4%), Haplorchis pumilio (5.2%), Haplorchis yokogawai (3.1%) and Echinochasmus japonicus (3.1%). Co-infection with O. viverrini and MIFs was rampant (81.4%). Polytrematode infection is highly prevalent in Lao PDR and hence requires urgent attention.

Detecting Blastocystis using parasitologic and DNA-based methods: a comparative study

Few studies have targeted the relative performance of diagnostic methods used for the detection of Blastocystis, a unicellular organism often present in fecal specimens from individuals with and without gastrointestinal symptoms. Aims of this study included a comparison of the formol ethyl acetate concentration technique (FECT), permanent trichrome staining of feces fixed in sodium acetate–acetic acid–formalin (SAF-PST), xenic in vitro culture (XIVC), and polymerase chain reaction (PCR) regarding Blastocystis screening of 107 samples from 93 patients with suspected enteroparasitic disease. Compared with PCR, the sensitivity/specificity of XIVC, SAF-PST, and FECT was 89%/100%, 82%/100%, and 50%/100%, respectively. False-negative results generated by the FECT and SAF-PST appeared to be associated with Blastocystis sp. subtype 3. A comparison of results obtained by dideoxy sequencing of positive PCR products amplified from DNA extracted directly from feces and DNA extracted from 5- and 28-day-old XIVC of 10 randomly chosen Blastocystis isolates showed no disparities, indicating that XIVC has very little or no impact on subtype distribution or variation within a given specimen. It is recommended that short-term XIVC be used for cost-effective screening of fresh fecal specimens for Blastocystis infection to generate valid prevalence estimates and to identify isolates for molecular characterization in studies aiming to illuminate the molecular epidemiology of Blastocystis.

The prevalence of Dientamoeba fragilis in patients with suspected enteroparasitic disease in a metropolitan area in Denmark

The prevalence of Dientamoeba fragilis in patients from a metropolitan area in Denmark was determined by examination of paired stool samples using two techniques: a formol ethyl-acetate concentration technique with unpreserved faeces and a permanent staining technique on faeces preserved with sodium acetate-acetic acid-formalin (SAF). Using the SAF permanent staining technique and the formol ethyl-acetate concentration technique, 25% and 15% of the specimens, respectively, were parasite-positive. D. fragilis was detected in 12 of the 103 patients, only two of whom harboured other recognised pathogenic parasites. Overall, D. fragilis had a remarkably high prevalence in the metropolitan area of Denmark investigated.

DETECTION OF BLASTOCYSTIS HOMINIS IN UNPRESERVED STOOL SPECIMENS BY USING POLYMERASE CHAIN REACTION

Blastocystis hominis is a common enteric parasite of worldwide distribution. Its pathogenetic potential has not yet been established, although numerous case reports suggest that B. hominis may cause the development of various gastrointestinal symptoms and disorders. The detection of the parasite in stool specimens is conventionally done by microscopy of direct smears, fecal concentrates, or permanently stained smears; however, morphology-based diagnosis is problematic. The aim of this study was to develop and evaluate a polymerase chain reaction (PCR) technique for the direct detection of B. hominis in human stool samples. Primers were based on small subunit ribosomal DNA and able to detect > or =32 parasites/200 mg stool artificially spiked with cultured B. hominis. In the evaluation of 43 clinical specimens, the PCR was tested against the formol ethyl acetate concentration technique (FECT) and a culture technique, proving 100% test specificity and a significantly higher sensitivity than the FECT. The PCR method is recommended for screening clinical specimens for B. hominis infection and for use in prevalence studies.

Heart Failure Caused by Hookworm Infection Possibly Associated with Organic Food Consumption

An 87-year-old man with heart failure caused by severe anemia was referred to our hospital. Gastroenteroscopy revealed the existence of several parasites in the duodenum. Examination of the stool by a formalin-ethyl acetate concentration technique detected hookworm eggs. After a single dose of pyrantel pamoate, his symptoms including dyspnea on exertion and edema diminished. And also hemoglobin and B-type natriuretic peptide (BNP) had improved dramatically. Hookworm infection is an extremely rare cause of heart failure, and furthermore this parasitic infection is not common in advanced countries. We suggest the possible relationship between parasite infection and organic foods.

Evaluation of PCR based coprodiagnosis of human opisthorchiasis

In this study, a recently developed PCR test for the detection of Opisthorchis viverrini in human faecal samples was evaluated using two parasitological methods as references. During a survey of foodborne trematodes (FBT) in the Vientiane Province, Lao PDR, 85 samples were collected and evaluated for FBT eggs by the Kato Katz (KK) technique, the formalin ethyl acetate concentration technique (FECT) and a PCR analysis for the distinction between O. viverrini and other FBT. The two parasitological methods did not differ in the ability of detecting FBT eggs, and a single KK reading was characterized by a sensitivity of 85% when compared to two FECT readings. The PCR tested positive only in cases where eggs had been demonstrated by parasitological examination. However, the PCR tested negative in some samples with very high egg counts. Demonstrating a PCR sensitivity of approximately 50% in samples with faecal egg counts > 1000, the previously reported PCR sensitivity based on in vitro studies was not supported. It is believed that technical problems rather than diagnostic reference related issues were responsible for the relatively low PCR performance. Further studies should aim at optimizing DNA extraction and amplification, and future PCR evaluation should include specificity control such as the scanning electron microscopy of eggs in test samples or the expulsion of adult trematodes from PCR tested individuals.

Comparison of the Quantitative Formalin Ethyl Acetate Concentration Technique and Agar Plate Culture for Diagnosis of Human Strongyloidiasis

The quantitative formalin ethyl acetate concentration technique (QFEC) was compared to agar plate culture (APC) for the detection of Strongyloides stercoralis larvae. QFEC could substitute for APC only when the parasite load was higher than 50 larvae per g of stool. This study serves as a good reminder to those conducting stool exams about the sensitivity and specificity limitations of both techniques.

Epidemiology of Strongyloides stercoralis in north-east Thailand: application of the agar plate culture technique compared with the enzyme-linked immunosorbent assay

Cross-sectional surveys of parasitic infection were performed using the agar plate culture technique (APCT) and modified formalin-ethyl acetate concentration technique (MFECT) to assess the true prevalence of Strongyloides stercoralis relative to other parasites in north-east Thailand. The enzyme-linked immunosorbent assay (ELISA) for diagnosis of S. stercoralis infection was used to estimate the seroprevalence for comparison with coproprevalence. Faecal and serum samples were collected from study participants during October—November 2000. Within the sample population of 332 rural northeast Thais from 3 communities, S. stercoralis was the most common parasitic infection (average 28.9%, range 27.7–30.3%) as determined by APCT; by MFECT the average was 5.4% (range 1.8–8.6%). Other intestinal parasites by order of prevalence were Opisthorchis viverrini (average 14.2%, range 8.6–9.4%), hookworm (average 12.3%, range 4–20.2%), Echinostoma sp. (7.5%), Giardia intestinalis (0.9%), Trichuris trichiura (0.6%), and Taenia sp., Hymenolepis nana and Entamoeba coli (all 0.3%). In an analysis of a subset of the sample population for which serum samples were available ( n = 120), coproprevalence by APCT was 33.3% (range 27–53.8%) and seroprevalence was 47.5% (range 29.7–57.9%) by modified unit-based ELISA and 34.2% (range 21.6–42.1%) by conventional optical density (OD)-based ELISA. Taking APCT as the reference method for diagnosis of strongyloidiasis, the sensitivity and specificity of the OD-based ELISA were 65% and 81.3%, respectively, and of the unit-based ELISA were 77.5% and 71.3%, respectively. Our results indicate that S. stercoralis is the predominant parasite in rural north-east Thailand, and that APCT and ELISA should be used as complementary diagnostic methods for community-based parasite surveys, at least among those in high-risk groups.

Coprotest® quantitativo: quantificação de ovos de helmintos em amostras fecais utilizando-se sistema de diagnóstico comercial

A técnica coproparasitológica de concentração em formol acetato de etila foi empregada para a quantificação de ovos de helmintos. O método quantitativo proposto foi padronizado utilizando-se o sistema comercial Coprotest® e amostras fecais contendo diferentes cargas de ovos de Ascaris lumbricoides. Para a comparação do Coprotest® quantitativo com outros métodos de quantificação de ovos, foi preparada em laboratório uma série de amostras fecais, com carga decrescente de ovos de A. lumbricoides, Trichuris trichiura e Schistosoma mansoni. Discutem-se as vantagens de se empregar um método capaz de detectar maior número de espécies de helmintos, além de protozoários, e que permita, concomitantemente, estimar a intensidade das infecções por geo-helmintos e S. mansoni nas populações. O Coprotest® quantitativo mostrou ser de aplicação viável, fornecendo resultados comparáveis a outros métodos quantitativos já descritos na literatura.

Evaluation of the optimal number of faecal specimens in the diagnosis of cryptosporidiosis in AIDS and immunocompetent patients

This study determines the optimal number of faecal samples that should be examined in order to minimize the occurrence of false-negative results in the diagnosis of cryptosporidiosis using routine techniques. A total of 23,023 faecal samples from 10,870 patients submitted for parasitological examination were processed by the formalin-ethyl acetate concentration technique and stained using a modified Ziehl-Neelsen method. Cryptosporidiosis was diagnosed in 232 patients (2.13%), 44 of whom were infected by HIV, the prevalence rate in this population group being 15.54%. The increase in the number of diagnoses obtained by the examination of n and (n-1) specimens was evaluated statistically. This study found that three is the optimal number of faecal samples that should be examined when cryptosporidiosis is suspected in immunocompetent patients; whereas, only two samples are required for diagnosing this protozoosis in AIDS patients.

Hemo-De as substitute for ethyl acetate in formalin-ethyl acetate concentration technique

In comparative studies, Hemo-De (PMP Medical Industries, Inc., Irving, Tex.) was found to be a suitable replacement for ethyl acetate in the Formalin-ethyl acetate concentration technique. With essentially equivalent recovery rates for both procedures, the Formalin-Hemo-De concentration technique is considered to be the preferred technique because Hemo-De is less toxic and less flammable and does not present disposal problems, and its cost is approximately one-fourth that of ethyl acetate.