Need for species-specific detection for the diagnosis of amoebiasis in ...

Scandinavian Journal of Infectious Diseases

ISSN: 0036-5548 (Print) 1651-1980 (Online) Journal homepage: http://www.tandfonline.com/loi/infd19

Need for species-specific detection for the diagnosis of amoebiasis in a non-endemic setting Gitte N. Hartmeyer, Silje V. Høgh, Ming Chen, Hanne Holt, Marianne N. Skov & Michael Kemp To cite this article: Gitte N. Hartmeyer, Silje V. Høgh, Ming Chen, Hanne Holt, Marianne N. Skov & Michael Kemp (2013) Need for species-specific detection for the diagnosis of amoebiasis in a non-endemic setting, Scandinavian Journal of Infectious Diseases, 45:11, 868-871, DOI: 10.3109/00365548.2013.816440 To link to this article: http://dx.doi.org/10.3109/00365548.2013.816440

Published online: 01 Aug 2013.

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Date: 03 May 2016, At: 23:50

Scandinavian Journal of Infectious Diseases, 2013; 45: 868–871

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Need for species-specific detection for the diagnosis of amoebiasis in a non-endemic setting

Downloaded by [University of Southern Denmark] at 23:50 03 May 2016

GITTE N. HARTMEYER1,2, SILJE V. HØGH1, MING CHEN3, HANNE HOLT1, MARIANNE N. SKOV1 & MICHAEL KEMP1 From the 1Department of Clinical Microbiology, Odense University Hospital, Odense, 2Slagelse Hospital, Slagelse, and 3Hospital of Southern Jutland, Sønderborg, Denmark

Abstract The diagnosis of amoebiasis caused by Entamoeba histolytica is traditionally based on microscopy. However, the specificity of this method may be questioned, especially in areas where infections by E. histolytica are rare. In the present study, a species-specific real-time PCR was used for the identification of the morphologically similar species E. histolytica and Entamoeba dispar. Out of 15 microscopy-positive stool samples, all were negative for E. histolytica and positive for E. dispar. In 2 cases, a suspicion of amoebic liver abscesses was confirmed by detection of E. histolytica DNA in stored sample material. Microscopy alone is clearly insufficient for the detection of E. histolytica in a setting where this parasite is rare. Microscopy-positive stool samples should be further tested by species-specific tests to distinguish E. histolytica from the non-pathogenic parasite E. dispar. On specific suspicion of amoebiasis, such as the suspicion of amoebic liver abscesses, species-specific tests can be applied even after storage.

Keywords: Entamoeba, real-time PCR, microscopy

Introduction Amoebiasis is caused by the parasite Entamoeba histolytica. Amoebiasis presents as diarrhoea (amoebic dysentery) or as a liver abscess. Amoebic liver abscesses are probably caused exclusively by E. histolytica. It is a rare infection in Denmark, only occasionally imported from endemic areas. Most cases of amoebiasis seen on microscopy are asymptomatic; in a minority of cases, amoebic dysentery is seen, and very rarely metastatic amoebic abscesses occur in the liver [1,2]. The diagnosis is traditionally made by microscopy of stool samples, microscopy of material from liver abscesses, or by serological tests. Other methods have been used, such as the testing of separated purified cyst fractions obtained from faeces, using specific DNA probes in a dot-blot test [3]. This is a relatively time-consuming method and is not used routinely in the laboratory. Microscopy does have some limitations. Sensitivity is poor and the method does not allow distinction between the pathogenic

species E. histolytica and the non-pathogenic species Entamoeba dispar and Entamoeba moshkovskii. Even in areas endemic for amoebiasis, there is a high rate of false-positive microscopy results due to misidentification of cysts from these Entamoeba species [4–7]. Furthermore, parasites in the trophozoite stage present in fresh stool and in liver abscesses die quickly and become undetectable by microscopy when they are out of the body. They may be found by microscopy in fresh sample material kept warm during examination. Due to the rareness of amoebiasis in Denmark, there is generally a low level of suspicion for this disease, even in the presence of a relevant travel history [8]. Therefore, sample material is usually treated by standard procedures that do not allow subsequent detection of trophozoites by microscopy. Due to the known limitations of microscopy for Entamoeba species, we established real-time PCR assays for E. histolytica and E. dispar. No assay was made for E. moshkovskii, as this parasite is not

Correspondence: G. N. Hartmeyer, Department of Clinical Microbiology, Odense University Hospital, J.B. Winsløwsvej 21, 2, DK-5000 Odense, Denmark. Tel: ⫹ 45 6541 1347/⫹ 45 3125 6474. Fax: ⫹ 45 6541 4785. E-mail: [email protected]; [email protected] (Received 17 April 2013 ; accepted 12 June 2013) ISSN 0036-5548 print/ISSN 1651-1980 online © 2013 Informa Healthcare DOI: 10.3109/00365548.2013.816440

Species-specific detection for amoebiasis reported in the region [9]. The assays were used for the identification of Entamoeba species reported as E. histolytica after detection by microscopy of stool samples, and for the demonstration of E. histolytica as the cause of liver abscesses in material not initially examined for this pathogen.

All data concerning samples processed at the Department of Clinical Microbiology of Odense University Hospital since 2006 are stored in an electronic laboratory information system. The number of samples and persons tested during the period 2006 to 2013, and the number of reports of the detection of E. histolytica, were extracted from the system.

rRNA) gene. The E. histolytica/E. dispar-specific primers, forward primer Ehd-239F (5′-ATTGTCG TGGCATCCTAACTCA-3′) and reverse primer Ehd-88R (5′-GCGGACGGCTCATTATAACA-3′), amplified a fragment inside the SSU rRNA gene [9]. The probes E. histolytica 5′-FAM-CATTGAATG AATTGGCCATT-MGB-3′ and E. dispar 5′-VIC C T TAC ATA A AT T G G C C AC T T T- M G B - 3 ′ detected E. histolytica- and E. dispar-specific amplification, respectively [11]. The 25-μl reactions contained 1 ⫻TaqMan Fast Universal PCR Master Mix, 2 ⫻ No AmpErase UNG (Applied Biosystems), 1000 nM of the primers Ehd-239F and Ehd-88R, 200 nM of the probes E. histolytica and E. dispar, and 5 μl DNA eluate. The real-time PCR was performed using a 7500 Fast Real Time PCR System (Applied Biosystems). The PCR programme was 95°C for 20 s, followed by 45 cycles of 95°C for 3 s and 60°C for 30 s.

Patients and sample material

Results

During the period June 2010 to January 2013, we collected 13 faecal samples from 13 patients who had been found positive for E. histolytica by microscopy. We collected a further 2 microscopy-positive stool samples from Slagelse Hospital. All stool samples were kept at ⫺ 80°C until analysed by PCR. Pus from 2 liver abscesses was collected and investigated by PCR after a negative bacterial culture result. In both cases the suspicion of amoebic liver abscesses was established days after primary sample handling for bacterial culture had taken place, and fresh warm sample material had not been examined by microscopy.

Microscopy

Materials and methods Extraction of data from the laboratory system


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Data extracted from the laboratory information system for the period January 2006 to January 2013 showed 40,763 stool samples from 16,704 individuals analysed by microscopy. Samples from 48 patients were reported to be positive for E. histolytica. This corresponds to an average of 6.9 cases/y, or an incidence of 287.4 cases/100,000 examined patients. Real-time PCR assays

Microscopic examination for the presence of ova and cysts was routinely performed by examination of iodine-stained wet-mount preparations after formalin–ethyl acetate concentration, at a magnification of ⫻ 400 [10].

Stool samples from 15 patients reported as positive for E. histolytica after routine microscopy examination were tested by the species-specific real-time PCR assays. They were all positive in the assay for E. dispar and negative in the E. histolytica-specific assay. Material from the 2 liver abscesses was positive for E. histolytica and negative for E. dispar when tested by real-time PCR.

DNA extraction

Discussion

Prior to DNA extraction, a cotton swab was submerged into the stool sample and resuspended in 4 ml physiological NaCl solution. Automated DNA extraction was performed using the NucliSENS easyMAG system (bioMérieux, France) in accordance with the manufacturer’s instructions.

In the present study, real-time PCR assays were used to identify Entamoeba species seen on microscopy of stool samples and to detect E. histolytica in liver abscesses. E. dispar was detected by the realtime PCR in all 15 microscopy-positive stool samples, whereas E. histolytica was not detected in any of them. As E. dispar is considered non-pathogenic and not a cause of amoebiasis, this finding clearly indicates that microscopy alone is insufficient to make a definite diagnosis of amoebiasis. Samples found positive for E. histolytica by microscopy

Microscopy for intestinal parasites

Real-time PCR Samples were subjected to a rapid real-time PCR targeting the small subunit ribosomal RNA (SSU


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should be examined by a species-specific assay before any final conclusion is drawn, since treatment and further investigations of the individual patient rely on an exact diagnosis. False-positive reports of E. histolytica may lead to unnecessary treatment and a delay in the diagnosis of other causes of the symptoms. The species-specific real-time PCR assays proved particularly useful for the diagnosis of amoebic liver abscesses. Patients with liver abscesses usually do not shed parasites in their stool, and suspected cases may only be confirmed by serology if fresh warm abscess material is not examined immediately after sampling. However studies have shown that serology for E. histolytica has been absent in acute illness with a diagnosis of abscess, and therefore could give a false-negative result in these cases [2]. In one of the present cases, the suspicion arose when an experienced clinical microbiologist noticed the typical macroscopic appearance of the material after it had been kept for several days in the laboratory for bacterial culture. Amoebiasis is not an individually notifiable disease in Denmark and data on the occurrence are not available. The microscopy-based estimated incidence of 287.4 cases/100,000 examined patients may represent over-diagnosis, and amoebiasis may be much rarer in Denmark than currently believed. On the other hand, patients shedding trophozoites of E. histolytica are not identified by microscopic examination of the stool, which may contribute to under-estimating of the occurrence of the disease. Studies on the usefulness of species-specific confirmation of microscopy have been carried out in other non-endemic countries. In Sweden, Lebbad and Svärd [12] found 167 positive for E. dispar and only 10 positive for E. histolytica when examining samples positive by microscopy. A Spanish study of species distribution showed 10 cases of E. histolytica and 117 cases of E. dispar among immigrants [13]. The great majority of amoebiasis in travellers, inhabitants of tropical countries, and men who have sex with men (MSM) is shown to be E. dispar when a positive sample is tested by species-specific PCR [1,2,14,15]. Most studies on E. histolytica using PCR have been done on microscopy-positive samples [2,15], therefore studies using species-specific, microscopy-independent assays for detection are clearly warranted for the estimation of the true frequency of occurrence of amoebiasis in Denmark. In conclusion, we present data showing that parasites identified as the pathogenic species E. histolytica by standard microscopy of stool samples are actually often the non-pathogenic species E. dispar, and that patients are misdiagnosed with amoebiasis. In addition the species-specific real-time PCR assays

proved useful in the diagnosis of amoebic liver abscesses, even when examination of fresh warm sample material had not been done.

Acknowledgements The authors thank the laboratory staff of the sections for parasitology, and the clinical microbiology departments of Slagelse Hospital and Odense University Hospital for their help in collecting the stool samples. Declaration of interest: No conflict of interest or funding.

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