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REAL-TIME PCR FOR THE DETECTION OF PROTOZOA IN FAECES
Original Article
DETECTION OF GIARDIA LAMBLIA, CRYPTOSPORIDIUM SPP. AND ENTAMOEBA HISTOLYTICA IN CLINICAL STOOL SAMPLES BY USING MULTIPLEX REAL-TIME PCR AFTER AUTOMATED DNA ISOLATION Van Lint P1, Rossen JW2, Vermeiren S1, Ver Elst K1, Weekx S1, Van Schaeren J1, 3, Jeurissen A1, 3 1
Laboratory of Molecular Diagnostics, GZA St. Augustinus, Wilrijk, Belgium, 2Laboratory of Medical Microbiology and Immunology, St. Elisabeth Hospital, Tilburg, the Netherlands and 3 Laboratory of Microbiology, GZA St. Augustinus, Wilrijk, Belgium Correspondence and offprint requests to: Philippe Van Lint, E-mail: [email protected]
ABSTRACT Diagnosis of intestinal parasites in stool samples is generally still carried out by microscopy; however, this technique is known to suffer from a low sensitivity and is unable to discriminate between certain protozoa. In order to overcome these limitations, a real-time multiplex PCR was evaluated as an alternative approach for diagnosing Giardia lamblia, Cryptosporidium spp. and Entamoeba histolytica in stool samples. Therefore, a total of 631 faecal samples were analysed both by microscopy as well as by real-time PCR following automated DNA extraction. Results showed that real-time PCR exhibited sensitivity and specificity of both 100%, whereas traditional microscopy exhibited sensitivity and specificity of 37.5% and 99.8% respectively. As real-time PCR provides simple, sensitive and specific detection of these three important pathogenic protozoan parasites, this technique, rather than microscopy, has become our diagnostic method of choice for the detection of enteric protozoan parasites for the majority of patients. Key words: Real-time PCR, Taqman, microscopy, protozoa, gastro-enteritis
INTRODUCTION Diarrhoea is a major health problem worldwide, and in most cases the aetiologies of diarrhoea are related to viruses,
Acta Clinica Belgica, 2013; 68-3
bacteria, and parasites (1). The intestinal parasites with the highest prevalence worldwide are Giardia lamblia and Cryptosporidium spp. (2). G. lamblia (synonyms: G. intestinalis and G. duodenalis) infections are very common throughout the world and are considered one of the main causes of gastrointestinal complaints in industrialised countries (3). Cryptosporidium spp., with C. parvum and C. hominis being the main causes of cryptosporidiosis in humans, has been recognized as the cause of large water and foodborne outbreaks of gastro-enteritis (4). E. histolytica infections, which are more rare, are associated with a high morbidity and mortality by causing amoebic colitis, amoebic dysentery, and amoebic liver abscess (5). Furthermore, diagnosis of this species is hampered by the fact that E. histolytica is morphologically identical to the nonpathogenic species E. dispar, a harmless commensal. Therefore diagnosis cannot be made by microscopy alone (6). G. lamblia, Cryptosporidium spp., and E. histolytica are three diarrhoea causing intestinal protozoa with often similar clinical presentation. Accurate and fast diagnosis of intestinal protozoa is important in the decision of a treatment plan, as well as for early detection of outbreaks (3). Due to its simplicity and ability to detect all kinds of parasites, microscopy is the classical method of choice for diagnosing parasitic infections. However, microscopy has its limitations. Some protozoa are difficult to differentiate and many protozoa are only present in small quantities in faeces samples; thus, quality of the microscopic examination is highly dependent on the skills of the laboratory technician. In order to implement a more reliable test, a real time PCR was developed for the simultaneous detection of G. lamblia, Cryptosporidium spp., and E. histolytica and its performance was compared to conventional microscopy.
doi: 10.2143/ACB.3170
REAL-TIME PCR FOR THE DETECTION OF PROTOZOA IN FAECES
MATERIALS AND METHODS Controls and samples
Faecal samples (n = 631) submitted to the department of Microbiology of the GZA Hospitals in Antwerp (Belgium) for microscopic examination for parasitic enteric pathogens between October 2010 and June 2011 were included in the study. On arrival, unpreserved faecal samples were stored at 4°C.
Microscopy
Microscopic examination for the presence of ova and cysts was performed routinely by examination of iodine-wet mounts after formol-ether concentration with a magnification of X400 according to standard procedures. Auramine staining was performed on direct smears and after formolether concentration for the detection of Cryptosporidium spp.
DNA isolation
Within one week after sampling, faecal samples were pretreated for DNA extraction. In short, a double volume of buffer AVL (Qiagen, Hilden, Germany) was added to 300- 600 mg faecal material (w/v) and the sample was disrupted by shaking for 5 minutes on a disruptor Genie (Scientific Industries, Bohemia, NY). Suspensions were then centrifuged for 2 min at 16.100xg. Supernatans was transferred to a fresh tube and stored at
REAL-TIME PCR FOR THE DETECTION OF PROTOZOA IN FAECES
Original Article
DETECTION OF GIARDIA LAMBLIA, CRYPTOSPORIDIUM SPP. AND ENTAMOEBA HISTOLYTICA IN CLINICAL STOOL SAMPLES BY USING MULTIPLEX REAL-TIME PCR AFTER AUTOMATED DNA ISOLATION Van Lint P1, Rossen JW2, Vermeiren S1, Ver Elst K1, Weekx S1, Van Schaeren J1, 3, Jeurissen A1, 3 1
Laboratory of Molecular Diagnostics, GZA St. Augustinus, Wilrijk, Belgium, 2Laboratory of Medical Microbiology and Immunology, St. Elisabeth Hospital, Tilburg, the Netherlands and 3 Laboratory of Microbiology, GZA St. Augustinus, Wilrijk, Belgium Correspondence and offprint requests to: Philippe Van Lint, E-mail: [email protected]
ABSTRACT Diagnosis of intestinal parasites in stool samples is generally still carried out by microscopy; however, this technique is known to suffer from a low sensitivity and is unable to discriminate between certain protozoa. In order to overcome these limitations, a real-time multiplex PCR was evaluated as an alternative approach for diagnosing Giardia lamblia, Cryptosporidium spp. and Entamoeba histolytica in stool samples. Therefore, a total of 631 faecal samples were analysed both by microscopy as well as by real-time PCR following automated DNA extraction. Results showed that real-time PCR exhibited sensitivity and specificity of both 100%, whereas traditional microscopy exhibited sensitivity and specificity of 37.5% and 99.8% respectively. As real-time PCR provides simple, sensitive and specific detection of these three important pathogenic protozoan parasites, this technique, rather than microscopy, has become our diagnostic method of choice for the detection of enteric protozoan parasites for the majority of patients. Key words: Real-time PCR, Taqman, microscopy, protozoa, gastro-enteritis
INTRODUCTION Diarrhoea is a major health problem worldwide, and in most cases the aetiologies of diarrhoea are related to viruses,
Acta Clinica Belgica, 2013; 68-3
bacteria, and parasites (1). The intestinal parasites with the highest prevalence worldwide are Giardia lamblia and Cryptosporidium spp. (2). G. lamblia (synonyms: G. intestinalis and G. duodenalis) infections are very common throughout the world and are considered one of the main causes of gastrointestinal complaints in industrialised countries (3). Cryptosporidium spp., with C. parvum and C. hominis being the main causes of cryptosporidiosis in humans, has been recognized as the cause of large water and foodborne outbreaks of gastro-enteritis (4). E. histolytica infections, which are more rare, are associated with a high morbidity and mortality by causing amoebic colitis, amoebic dysentery, and amoebic liver abscess (5). Furthermore, diagnosis of this species is hampered by the fact that E. histolytica is morphologically identical to the nonpathogenic species E. dispar, a harmless commensal. Therefore diagnosis cannot be made by microscopy alone (6). G. lamblia, Cryptosporidium spp., and E. histolytica are three diarrhoea causing intestinal protozoa with often similar clinical presentation. Accurate and fast diagnosis of intestinal protozoa is important in the decision of a treatment plan, as well as for early detection of outbreaks (3). Due to its simplicity and ability to detect all kinds of parasites, microscopy is the classical method of choice for diagnosing parasitic infections. However, microscopy has its limitations. Some protozoa are difficult to differentiate and many protozoa are only present in small quantities in faeces samples; thus, quality of the microscopic examination is highly dependent on the skills of the laboratory technician. In order to implement a more reliable test, a real time PCR was developed for the simultaneous detection of G. lamblia, Cryptosporidium spp., and E. histolytica and its performance was compared to conventional microscopy.
doi: 10.2143/ACB.3170
REAL-TIME PCR FOR THE DETECTION OF PROTOZOA IN FAECES
MATERIALS AND METHODS Controls and samples
Faecal samples (n = 631) submitted to the department of Microbiology of the GZA Hospitals in Antwerp (Belgium) for microscopic examination for parasitic enteric pathogens between October 2010 and June 2011 were included in the study. On arrival, unpreserved faecal samples were stored at 4°C.
Microscopy
Microscopic examination for the presence of ova and cysts was performed routinely by examination of iodine-wet mounts after formol-ether concentration with a magnification of X400 according to standard procedures. Auramine staining was performed on direct smears and after formolether concentration for the detection of Cryptosporidium spp.
DNA isolation
Within one week after sampling, faecal samples were pretreated for DNA extraction. In short, a double volume of buffer AVL (Qiagen, Hilden, Germany) was added to 300- 600 mg faecal material (w/v) and the sample was disrupted by shaking for 5 minutes on a disruptor Genie (Scientific Industries, Bohemia, NY). Suspensions were then centrifuged for 2 min at 16.100xg. Supernatans was transferred to a fresh tube and stored at
