Indian Journal of Medical Microbiology Medknow Publications on behalf of Indian Association of Medical Microbiology ISSN: 0255-0857 EISSN: 1998-3646 Vol. 29, Num. 2, 2011, pp. 141-146

Indian Journal of Medical Microbiology, Vol. 29, No. 2, April-June, 2011, pp. 141-146

Original Article

Evaluation of small-subunit rRNA touchdown polymerase chain reaction for direct detection of Entamoeba histolytica in human pus samples from patients with amoebic liver abscess

P Singh¹, BR Mirdha¹, V Ahuja², S Singh¹

¹ Department of Microbiology, All India Institute of Medical Sciences, Ansari Nagar, New Delhi - 110 029, India
² Department of Gastroenterology and Human Nutrition Unit, All India Institute of Medical Sciences, Ansari Nagar, New Delhi - 110 029, India

Correspondence Address:B R Mirdha Department of Microbiology, All India Institute of Medical Sciences, Ansari Nagar, New Delhi - 110 029 India mirdhabr@hotmail.com

Date of Submission: 19-Aug-2010
Date of Acceptance: 23-Mar-2011

Code Number: mb11033

PMID: 21654108

DOI: 10.4103/0255-0857.81795

Abstract

Keywords: Amoebic liver abscess, polymerase chain reaction, small-subunit rRNA, touchdown PCR

Introduction

Hepatic amoebiasis caused by Entamoeba histolytica is often diagnosed by suggestive clinical symptomatology, radiological investigations and positive serology. The diagnosis of hepatic amoebiasis is most commonly attempted by direct microscopy, in vitro culture of a pus sample and serological tests. The sensitivity of microscopy is poor and is often confound with false-positive results due to misidentification of macrophages as trophozoites, polymorphonuclear cells as cyst and other Entamoeba species. ^[1] In vitro culture of E. histolytica is technically difficult and often not rewarding. ^[2] Detection of E. histolytica antibodies in patient′s sera with a clinical diagnosis of amoebic colitis and ameobic liver abscess (ALA) is an adjunctive test. However, the interpretation of antibody test is often inconclusive in an endemic area. ^[3] Such discrepancies underscore the need for more sensitive, accurate and faster methods for the diagnosis of ALA. Several investigators have reported the successful application of conventional polymerase chain reaction (PCR) for the diagnosis of aomebiasis. ^[4],[5],[6] However, the conventional PCR assay is often encountered with technical problems of standardization, mispriming and production of non-specific products, followed by detection of amplified products. In contrast, touchdown PCR (TD-PCR) offers a simple and rapid means to optimize PCR with increased specificity, sensitivity and yield without the need of lengthy optimization and/or redesigning of primers. It also abrogates mispriming and production of non-specific PCR products. ^[7] We have optimized a novel TD-PCR targeting small-subunit rRNA (SSU rRNA) and compared its results with conventional PCR to diagnose ALA.

Material and Methods

The present prospective study was conducted in the Department of Microbiology at our tertiary care referral and teaching hospital. The study protocol was approved by the Institutional ethics committee (IEC). Sixty-seven (n = 67) patients were recruited over a period of 22 months with a clinical diagnosis of ALA. Clinical suspicion of ALA was made on the basis of clinical findings such as pain in the right upper quadrant of abdomen, tenderness in the right hypochondrium and hepatomegaly with or without fever. Corroborative radiological findings and supportive haematological and microbiological findings were also included. ^[8] From each patient, 5-7 ml of blood was collected aseptically in a collection tube without anticoagulant. Three milliliters to 5 ml of liver pus was aspirated under aseptic conditions under ultrasound guidance and transported to the laboratory as soon as possible. All clinical samples were stored at 4°C in order to maintain the structural integrity of the cellular components, until processed.

Microscopic examination of clinical specimens

Direct saline wet mounts from the pus samples were examined under the microscope for demonstration of trophozoites of E. histolytica.

Anti-E.histolytica immunoglobulin G antibody detection by enzyme-linked immunosorbent assay

Serum from blood samples was used for performing the serologic test using a commercially available kit [RIDASCREEN E. histolytica IgG (1721); R-Biopharm, Darmstadt, Germany].

Culture of pus samples

An in vitro culture was performed by inoculating the clinical samples into the National Institute of Health (NIH) medium. ^[9]

DNA extraction

DNA extractions from pus samples were carried out using a commercially available REDExtract-N-Amp DNA extraction kit (Sigma, St. Louis, MO, USA) according to the manufacturer′s instructions. Briefly, 100 μl of the extraction solution and 25 μl of the tissue preparation solution were pipetted out into a microcentrifuge tube. 10 of the sample was added to the solution and mixed thoroughly by vortexing followed by incubation at room temperature for 10 min and further incubation at 95°C for 3 min. Finally, 100 μl of the neutralization solution was added and mixed by vortexing. The neutralized sample extract was stored at 4°C or tested immediately using PCR. Strains of E. histolytica isolated earlier from stool samples and confirmed positive by both microscopy and by species-specific PCR and maintained in NIH medium served as the positive control for the PCR assay.

Polymerase chain reaction

Amplification of the SSU rRNA gene was carried out by using a published primers ^[10] (GenBank accession no. X64142). The forward primer sequence (EntaF) was derived from the central region of the SSU rRNA gene that is conserved for the genus Entamoeba, whereas the reverse primer is specific for E. histolytica. Primers were designed from signature sequences on the repetitive SSU rRNA sequences that are specific to E. histolytica.

Forward primer P1 5′ - ATG CAC GAG AGC GAA AGC AT- 3′

Reverse primer P2 5′ - GAT CTA GAA ACA ATG CTT CTC T-3

The size of the amplified product was 166 base pair (bp). The specificity of the primers was tested by subjecting them to BLAST.

Conventional polymerase chain reaction

For performing the PCR assay, the reaction mixture contained 10 μl of REDExtract-N-Amp PCR reaction mixture, 0.4 μl each of forward and reverse primer and 4 μl of tissue extract (DNA). Doubled-distilled PCR-grade water was added to the above mixture to make the total volume up to 20 μl. The PCR was performed by using an initial denaturation at 94°C for 5 min followed by 35 cycles of amplification at 94°C for 1 min, 65°C for 1 min and 72°C for 1 min in an ABI 2720 thermocycler (Applied Biosystem, Foster City, CA, USA).

Touchdown polymerase chain reaction

The reaction mixture contained 5.2 μl of distilled water, 10 μl of REDExtract-N-Amp PCR reaction mixture, 0.4 μl each of forward and reverses primer and 4 μl of the tissue extract (DNA) for a total volume of 20 μl.

TD-PCR was performed using an initial pre-heating step of 10 min at 95°C and the TD was of 15 s at 94°C. The annealing step was performed at 62°C for 30 s and the temperature was further decreased to 52°C per cycle during the first 10 cycles and then treated for 15 s at 72°C. The subsequent 30 cycles were performed for 15 s at 92°C, 30 s at 52°C and 15 s at 72°C. Finally, an extension period of 5 min at 72°C was performed in an ABI 2720 thermocycler.

To avoid contamination, the reagent preparation, DNA extraction and amplification were performed in separate areas/rooms within the laboratory using different sets of micropipettes as well as filter tips. PCR mixtures and the extraction steps were prepared in a laminar flow cabinet. To further exclude possible contaminations, negative control (ultra-pure distilled water) was included in each PCR reaction/experiment.

Sensitivity and specificity of the touchdown polymerase chain reaction

Sensitivity of the TD-PCR was tested using different dilutions of the positive DNA in the ratio of 1:1, 1:5, 1:25, 1:125 and 1:625 in triplicate. Specificity of the PCR assay was determined by performing PCR against a panel of genomic DNA extracted from pus samples obtained from cases other than amoebic abscess. Pus samples obtained from a case of brain abscess due to Bacteroides fragilis and another from a suppurative lymphadenitis caused by Staphylococcus aureus were tested to rule out any non-specific amplification. No cross-amplification was observed.

Amplified products were directly loaded onto the 1.5% agarose gels without the addition of any gel-loading dye and the bands were detected and photographed under UV light.

Results

A total of 67 subjects were diagnosed with ALA in the present study.

Results of microscopic examination, anti-amoebic antibody detection by enzyme-linked immunosorbent assay, culture, polymerase chain reaction and touchdown polymerase chain reaction

None of the samples showed the presence of motile trophozoites of E. histolytica either by microscopy or by in vitro culture. A total of 51 (76.11%, 51/67) patients were found to be positive for anti-E.histolytica IgG antibody by enzyme-linked immunosorbent assay (ELISA). Bacteriological cultures were also performed in all pus samples (both aerobic and anaerobic culture). Only two pus samples grew bacteria on aerobic culture. The bacteria were identified as Staphylococcus epidermidis and aerobic spore bearers. ^[11] Both these bacteria are skin commensals and represent the probable contamination of pus samples by skin flora, and were not truly pathogenic to cause liver abscess. With the help of conventional PCR [Figure - 1] and [Figure - 2], 55 (82.08%, 55/67) patients were positive for E. histolytica. All 67 clinical samples aspirated from patients were further subjected to a TD-PCR assay by using the same set of primers as used in conventional PCR [Figure - 3] and [Figure - 4]. By using the TD-PCR assay, three additional positive cases that were missed by conventional PCR were detected [Table - 1a] and [Table - 1b]. Thus, 58 (86.5%, 58/67) patients were found to be positive by TD-PCR. This was also able to detect DNA up to a 1:125 dilution of the positive control, thereby showing a greater sensitivity besides no non-specific amplifications [Figure - 5].

Discussion

Diagnosis of hepatic amoebiasis is generally endeavored by direct microscopy, in vitro culture of a pus sample and serological tests. The former two tests are not sensitive and the latter one is not definitive. Lack of sensitivity of microscopic examination to detect and differentiate the species of Entamoeba has made serological assays an adjunct tool for the diagnosis of invasive amoebiasis. However, studies have demonstrated the limitations of serological tests to diagnose ALA in the areas of endemicity. The use of conventional PCR has proved to be a better diagnostic tool for the detection of E. histolytica in terms of sensitivity and specificity. The success of the PCR results relies mainly upon the quality of the extracted DNA being used; therefore, optimization of the DNA extraction procedure is a critical step for the success of the PCR assay. ^[11] In the past, the isolation of DNA directly from samples was technically demanding and laborious. In recent times,simple and effective methods for the isolation of parasite-specific DNA has enhanced the sensitivity of PCR assay. However, many of these methods include multiple steps that are time-consuming and expensive. Hence, only a limited number of samples can be processed at a given point of time. But, the RED Extract-N-Amp PCR kit that was used to extract DNA from clinical samples have provided many advantages, ranging from low sample usage to less time consumption, i.e. only 15 min.

Furthermore, it is evident from the published literature that the conventional single-round PCR usually suffers with problems of standardization (including testing various concentrations of the reaction components such as Mg ²⁺ , dNTPs, primers and template), mispriming and production of non-specific products followed by difficulty in detection of amplified products, especially when amplifying products from genomic DNA. In the standardization procedure, the annealing temperature of the reaction is one of the most important parameters. A rapid way to determine the ideal annealing temperature is to use a temperature gradient across the thermal cycler block for the annealing step but, without a gradient block, annealing temperature optimization can be a lengthy process, requiring repeated runs to test each different annealing temperature. Nevertheless, there are methods that can be used to increase the specificity of the reaction without actually determining the optimal annealing temperature. On the other hand, TD-PCR offers a simple and rapid means to optimize the PCR assay, resulting in increased specificity, sensitivity and better yield of amplified products. TD-PCR is a method of polymerase chain reaction by which primers will avoid amplifying a non-specific sequence and the temperature at which primers anneal during a cycle of PCR determines the specificity of annealing. The melting point of the primer sets the upper limit on annealing temperature. At temperatures just below this point, only very specific base pairing between the primers and the template occur. At lower temperatures, the primers bind less specifically. Non-specific primer binding obscures the PCR results as the non-specific sequences to which primers anneal in the early steps of amplification will "swamp out" any specific sequences. This happens because of the exponential nature of the polymerase amplification. The earliest steps of a TD-PCR cycle have high annealing temperatures. The annealing temperature is decreased in increments for every subsequent set of cycles. The number of individual cycles and increments of temperature decrease can be adjusted accordingly. The primer will anneal at the highest temperature, which is least-permissive of non-specific binding. Thus, the first sequence amplified is the one between the regions of greatest primer specificity and is often most likely the sequence of interest. These fragments are further amplified during the subsequent rounds at lower temperatures, and will "out compete" with the non-specific sequences to which the primers may bind at these lower temperatures. If the primer, during the higher-temperature phases, binds to the sequence of interest, subsequent rounds of PCR can be performed upon the product to further amplify the above fragments. It is likely that TD-PCR would also be beneficial when the melting temperature of the two primers is different. TD-PCR has found wide applicability in standard PCR protocols and single-nucleotide polymorphism screening.

Other authors like You et al, reported the applicability of TD-PCR for detection of mutations in genes related to drug resistance in Mycobacterium leprae isolates from leprosy patients in Korea. ^[12] Similarly, Duckworth et al. proposed the utility of TD-PCR for increased sensitive and specific mutation detection in patients with mantle cell lymphoma. ^[13] Likewise, Piraee et al. projected the use of TD-PCR for more specific and sensitive amplification of the halogenase gene from Streptomyces venezuelae.^[14] Another investigator, Zumarraga et al. had also developed a rapid and sensitive TD-PCR for the detection of Mycobacterium bovis in bovine samples by targeting the IS6110 element. ^[15] Fietto et al, described the construction of cDNA libraries using TD-PCR ^[16] and Horra et al, also compared single and TD-PCR protocols for detecting Pneumocystis jirovecii DNA in paraffin-embedded lung tissue samples and suggested the enhancing effect of TD-PCR. ^[17]

We have standardized TD-PCR and compared its results with conventional PCR using the same set of primers that hybridizes with the SSU rRNA gene and generates a product size of 166 bp. With this TD-PCR assay, it was possible to detect E. histolytica DNA in a total of 58 (86.56%, 58/67) patients as compared with 55 (82%, 55/67) with conventional PCR. Moreover, the length of the TD-PCR assay was shortened to 2 h instead of 3.5-4 h that was noted in the conventional PCR. TD-PCR did not amplify any of the pus samples that were other than ALA. It also gave negative results with DNA extracted from different bacteria such as Escherichia coli, Staphylococcus aureus, Bacteroides fragilis, Enterobacter cloacae and Klebsiella pneumoniae, which are some of the common causes of pyogenic liver abscess. The overall sensitivity and specificity of the PCR assay was found to be 86.56% and 100%, respectively. The goal of the study was to improve the amplification sensitivity using TD-PCR.

When we did the cost analysis for both the serological and molecular methods, we found that the cost for ELISA, conventional PCR and TD-PCR were more or less same ($ 4/Rs. 200 per sample). However, TD-PCR had a relatively better sensitivity and specificity as compared with the other test. We were unable to detect E. histolytica-specific DNA in nine samples (n = 9) by TD-PCR, which could possibly be either due to the presence of some inhibitors such as blood that sometimes contaminate pus samples during aspiration or due to other unknown inhibitors present in the pus.

In conclusion, we have developed a sensitive and specific TD-PCR and our findings are in concordance with the published literature. Therefore, the present study proposes the use of a TD-PCR assay targeting the SSU rRNA gene for effective detection of E. histolytica DNA directly from the liver pus sample.

Acknowledgement

This study was supported by a financial grant sanctioned to Dr B R Mirdha, corresponding author, by the Indian Council of Medical Research (ICMR), Department of Health Research, Ministry of Health and Family Welfare, Government of India.

References

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