The effect of formalin fixation on the polymerase chain reaction characterization of Entamoeba histolytica

Ramos, Fernando; Zurabián, Rimma; Morán, Patricia; Ramiro, Manuel; Gómez, Alejandro; Clark, C. Graham; Melendro, Emma I.; Garcı́a, Gabriela; Ximénez, Cecilia

doi:10.1016/s0035-9203(99)90045-7

Cited by 25 publications

(12 citation statements)

References 9 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…PVA and SAF preserve trophozoites and cysts, and formalin preserves cysts for examination in wet mounts. However, methods of fixation of feces with fixatives or preservatives may result in a decreased sensitivity of PCR with time (143,192). A few groups have, however, shown good results using formalin-fixed samples for PCR (147,157).…”

Section: Complexity Of Fecal Samplesmentioning

confidence: 99%

“…Ethanol is a simple transport medium that preserves amebic DNA. The most widely used reagent for the preservation of fecal samples is 10% buffered formalin solution (120); however, this solution is reported to hamper product amplification by PCR because of the interfering nature of the fixative, which perfuses the organisms and reacts with DNA (143). Consequently, freezing a fresh fecal specimen at Ϫ20°C before extraction of DNA is a better strategy, as it does not affect the sensitivity of the molecular assays (52,105,123).…”

Section: Complexity Of Fecal Samplesmentioning

confidence: 99%

See 1 more Smart Citation

Laboratory Diagnostic Techniques for Entamoeba Species

et al. 2007

View full text Add to dashboard Cite

SUMMARY The genus Entamoeba contains many species, six of which (Entamoeba histolytica, Entamoeba dispar, Entamoeba moshkovskii, Entamoeba polecki, Entamoeba coli, and Entamoeba hartmanni) reside in the human intestinal lumen. Entamoeba histolytica is the causative agent of amebiasis and is considered a leading parasitic cause of death worldwide in humans. Although recent studies highlight the recovery of E. dispar and E. moshkovskii from patients with gastrointestinal symptoms, there is still no convincing evidence of a causal link between the presence of these two species and the symptoms of the host. New approaches to the identification of E. histolytica are based on detection of E. histolytica-specific antigen and DNA in stool and other clinical samples. Several molecular diagnostic tests, including conventional and real-time PCR, have been developed for the detection and differentiation of E. histolytica, E. dispar, and E. moshkovskii in clinical samples. The purpose of this review is to discuss different methods that exist for the identification of E. histolytica, E. dispar, and E. moshkovskii which are available to the clinical diagnostic laboratory. To address the need for a specific diagnostic test for amebiasis, a substantial amount of work has been carried out over the last decade in different parts of the world. The molecular diagnostic tests are increasingly being used for both clinical and research purposes. In order to minimize undue treatment of individuals infected with other species of Entamoeba such as E. dispar and E. moshkovskii, efforts have been made for specific diagnosis of E. histolytica infection and not to treat based simply on the microscopic examination of Entamoeba species in the stool. The incorporation of many new technologies into the diagnostic laboratory will lead to a better understanding of the public health problem and measures to control the disease.

show abstract

Section: Complexity Of Fecal Samplesmentioning

confidence: 99%

Section: Complexity Of Fecal Samplesmentioning

confidence: 99%

Laboratory Diagnostic Techniques for Entamoeba Species

et al. 2007

View full text Add to dashboard Cite

show abstract

“…Microscopic examination of intestinal parasites is usually performed on fixed stools. Numerous groups have tried with variable success molecular methods on this type of sample, and recently, the effect of formalin fixation on PCR was further investigated (17). It was shown that even if its effect on DNA is indirect, concentrations of formalin higher than 1% seemed to inhibit PCR amplification from 4 days of fixation.…”

mentioning

confidence: 99%

Detection and Differentiation of Entamoeba histolytica and Entamoeba dispar Isolates in Clinical Samples by PCR and Enzyme-Linked Immunosorbent Assay

2003

View full text Add to dashboard Cite

Differential diagnosis of Entamoeba histolytica (pathogenic) and Entamoeba dispar (nonpathogenic), which are two morphologically identical species of amebae, is essential both for treatment decision and public health knowledge. The study reported here was designed to choose a reference differentiation technique. Stool samples (n ‫؍‬ 95) were tested by microscopy, TechLab enzyme-linked immunosorbent assays (ELISAs), and an in-house PCR. The target for the PCR amplification was a small region (135 bp) of the SSU rRNA selected to increase the sensitivity of the test. Sixty-eight specimens tested positive by PCR: 2 for E. histolytica and 66 for E. dispar. For detection of E. dispar, ELISA performance was lower than that of microscopy in this reference context, while PCR was much more sensitive than microscopy. Given the low proportion of E. histolytica cases, test performance for this species is difficult to assess. However, for differentiation, PCR performed well on simulated samples, while ELISA gave a discordant result for one of the two samples PCR positive for E. histolytica during the study. This report also confirms that E. dispar infection is significantly higher among travelers and underlines the possibility of acquiring E. histolytica infection in regions that are not areas of endemicity. Because of its lower sensitivity, the interest of ELISA for Entamoeba detection and differentiation in stools seems questionable in nontropical regions. On the other hand, results suggest that PCR should be useful as a reference test for sensitive differentiation of both species and to contribute to physicians' decision in treatment of E. histolytica-or E. dispar-infected patients. Amebiasis is an important parasitic disease in humans (18).Entamoeba histolytica and Entamoeba dispar parasitize approximately 10% of the world population, of which 90% are asymptomatic infections. It is estimated, however, that amebiasis causes up to 110,000 deaths a year (15). While the infectious agent was discovered in 1875 by Fedor A. Lösch and the distinction between E. dispar and E. histolytica was first suspected in 1925 (3), the evidence for the dichotomy in two different species, pathogenic (E. histolytica) and nonpathogenic (E. dispar), is relatively recent (5). However, E. dispar and E. histolytica are morphologically indistinguishable from one another. Isoenzyme analysis is considered the "gold standard" for differentiating E. histolytica and E. dispar, but this method is not currently available and not readily usable for routine diagnosis (16). More recently, several studies have been devoted to the development of new techniques either based on monoclonal antibodies (8, 9, 10, 20, 21) or molecular biology methods (1,2,4,6,19,22,23) to successfully distinguish the two species in human feces. Reliable distinction would have a medical impact as until now, both infections are usually treated, whereas only approximately 10% (pathogenic infections) need to be treated. This proportion drops to much lower levels in developed countries, wh...

show abstract

“…The negative influence of formalin on various stages of intestinal parasites was reported by, e.g. Ramos et al (1999), who showed that preservation of stool samples in 10% buffered formalin for more than 7 days hampered successful PCR amplification of DNA of the trophozoite Entamoeba histolytica . Contrastingly, Paglia and Visca (2004) demonstrated results of a study in which nested PCR, with initial amplification of the 1076-bp fragment of the SSU rRNA gene, had been applied for the specific detection of E. histolytica/Entamoeba dispar in faeces fixed in 10% formalin for 90 days.…”

Section: Discussionmentioning

confidence: 85%

First detection and genotyping of Giardia intestinalis in stool samples collected from children in Ghazni Province, eastern Afghanistan and evaluation of the PCR assay in formalin-fixed specimens

Lass

Karanis²,

Korzeniewski

2017

Parasitol Res

View full text Add to dashboard Cite

It is estimated that faecal-orally transmitted diseases are common in Afghanistan, as a consequence of poor hygienic standards of life and widespread contamination of water and food with both human and animal faeces. However, there is little information in the literature concerning infections caused by intestinal parasites in the Afghan population. In this study, we report the occurrence of Giardia intestinalis assemblages (A and B) in formalin-fixed stool samples collected from 245 Afghan schoolchildren living in Ghazni Province in eastern Afghanistan. Detection of the parasite’s DNA and genotyping was performed using real-time PCR, specific to the β-giardin gene of G. intestinalis. Positive results were recorded in 52 (21.2%) samples. Genotyping was successful in 39 faecal samples and showed the predominance of assemblage B of G. intestinalis in this population (15 assemblage A and 24 assemblage B). Co-infection with both genotypes A and B was detected in four samples. Additionally, we evaluated the effect of 10% buffered formalin fixative on the detection of G. intestinalis DNA using real-time PCR and nested PCR characterised by different lengths of PCR products (74 and 479 bp, respectively). The human faeces containing the Giardia cysts were tested for 16 weeks. Amplification of G. intestinalis DNA with real-time PCR was possible up to 6 weeks of preservation of stool sample in formalin, compared to only 2 weeks with nested PCR. This suggests that real-time PCR is a more suitable tool in cases where stool samples have to be kept in formalin for longer periods of time.

show abstract

The effect of formalin fixation on the polymerase chain reaction characterization of Entamoeba histolytica

Cited by 25 publications

References 9 publications

Laboratory Diagnostic Techniques for Entamoeba Species

Laboratory Diagnostic Techniques for Entamoeba Species

Detection and Differentiation of Entamoeba histolytica and Entamoeba dispar Isolates in Clinical Samples by PCR and Enzyme-Linked Immunosorbent Assay

First detection and genotyping of Giardia intestinalis in stool samples collected from children in Ghazni Province, eastern Afghanistan and evaluation of the PCR assay in formalin-fixed specimens

Contact Info

Product

Resources

About