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ORIGINAL ARTICLE  
Year : 2012  |  Volume : 4  |  Issue : 1  |  Page : 38-42
Assessment of Helicobacter pylori prevalence by scorpion real-time PCR in chronic tonsillitis patients


1 Cell and Molecular Research Center, School of Medicine, Qazvin University of Medical Sciences, Qazvin, Iran
2 Department of Microbiology, School of Medicine, Qazvin University of Medical Sciences, Qazvin, Iran
3 Department of Otolaryngology, School of Medicine, Qazvin University of Medical Sciences, Qazvin, Iran
4 Department of Genetics, School of Medicine, Qazvin University of Medical Sciences, Qazvin, Iran

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Date of Web Publication13-Mar-2012
 

   Abstract 

Background: Occasionally, bacteria or viruses enter the tonsils and these organs become overwhelmed by bacterial or viral infection leading to inflammation. Some studies confirmed the presence of Helicobacter pylori in tonsillar specimens of patients suffering from chronic tonsillitis and some others did not. The difference in results in various studies might be due to different laboratory methods. The aim of this study was to investigate the presence of H. pylori Deoxynucleic acid (DNA) in archival tonsillar tissues of patients with chronic tonsillitis by a rapid, sensitive, and specific technique of Scorpion real-time polymerase chain reaction (PCR). Materials and Methods: Scorpion real-time PCR and modified McMullen's staining was performed on 103 archival paraffin-embedded tonsillar samples collected from patients with chronic tonsillitis following tonsillectomy operation. Results: Our findings showed that H Cell and Molecular Research Center. pylori DNA was present in 21.35% of total specimens by using Scorpion real-time PCR. Modified McMullen's staining of paraffin-embedded sections was positive in 19 patients. Out of our 103 samples, 50 samples showed positive a rapid urease test whereas 53 samples demonstrated negative results, 20 produced positive PCR results, and 83 were negative for H. pylori. There was no significant relationship between the presence of H. pylori, sex, age, and place of residence. Conclusion: Although the existence of H. pylori in tonsillar tissue samples of patients with chronic tonsillitis is controversial, however, our results showed that in our studied specimens, a significant number of patients with chronic tonsillitis had H. pylori colonization.

Keywords: Helicobacter pylori , PCR, Rapid urease test, Real-time, Scorpion, Tonsillitis

How to cite this article:
Farivar T N, Pahlevan A A, Johari P, Safdarian F, Mehr M A, Najafipour R, Ahmadpour F. Assessment of Helicobacter pylori prevalence by scorpion real-time PCR in chronic tonsillitis patients. J Global Infect Dis 2012;4:38-42

How to cite this URL:
Farivar T N, Pahlevan A A, Johari P, Safdarian F, Mehr M A, Najafipour R, Ahmadpour F. Assessment of Helicobacter pylori prevalence by scorpion real-time PCR in chronic tonsillitis patients. J Global Infect Dis [serial online] 2012 [cited 2018 Dec 10];4:38-42. Available from: http://www.jgid.org/text.asp?2012/4/1/38/93760



   Introduction Top


Gastric mucosa extends to the upper aero-digestive tract mucosa and maintains its continuity. In several different studies, the colonization of Helicobacter pylori in locations beyond the gastrointestinal cavity such as adenotonsillar tissues [1],[2] and nasal and sinus mucosa of patients [2],[3] has been reported.

The diagnosis of chronic tonsillitis is mainly done by history and clinical examination [4] and these diagnostic criteria are not enough for diagnosis of H. pylori in related tonsillar clinical specimens and may lead to misdiagnosis of the illness. On the other hand, bacterial infections of the tonsils and adenoids are treated with various antibiotics and surgical removal is considered in situations resistant to medical therapy or in frequently recurrent infections. As H. pylori does not respond to routine antibacterial drugs that are used for chronic tonsillitis, the determination of the prevalence of H. pylori in tonsillar biopsy specimens of patients suffering from chronic tonsillitis may play a role in decision making in treatment, and with evaluation, the extent of the presence of H. pylori in these tonsillar specimens, and with proper antibiotic therapy against H. pylori, even some of the unnecessary tonsillectomy operations may be prevented.

There are studies in which the apparent existence of H. pylori in these tissue samples was confirmed [5],[6],[7] nevertheless, some other studies failed to show the presence of H. pylori in tonsillar materials examined. [8],[9],[10],[11] The difference in results in various studies might be due to different laboratory methods. [6]

There are several different methods for the detection of H. pylori in clinical specimens; among those the culture of organisms, histopathlogy, polymerase chain reaction (PCR), and real-time PCR are the most widely used techniques. Real-time PCR is considered as the most rapid, sensitive, and accurate laboratory method for the detection of H. pylori.

Several real-time PCR assays for the identification of H. pylori have been designed. The Scorpion primer technology allows the precise discrimination between different alleles of a target nucleotide in a single-step process. [12],[13],[14],[15] Using Scorpion primers, the sequence-specific priming and PCR product detection are achieved through the application of a single oligonucleotide.

The ability of the detection of the Scorpion real-time PCR assay not only for H. pylori but also for other infectious agents is the same as real-time PCR and is better than PCR-RFLP. On the other hand, the used probe in the Scorpion assay does not hydrolyze during the PCR process but in real-time PCR the probe hydrolyzes and so the efficacy of PCR in these methods is not the same.

Based on the reports of few studies with contradictory results, [1],[2],[5],[6],[7],[8],[9],[10],[12],[13],[16],[17],[18],[19] this study was designed to detect the colonization of H. pylori in the biopsy specimens in paraffin-embedded tonsillar tissues of patients suffering from chronic tonsillitis.


   Materials and Methods Top


Patients and methods

This retrospective cross-sectional study was performed on 103 paraffin-embedded tonsillar specimens of patients who underwent adenotonsillectomy operation at the ENT ward of Ghods Hospital (Qazvin University of Medical Sciences, Qazvin, Iran) during 2010. All patients had common indications for adenotonsillectomy including recurrent tonsillitis, chronic tonsillitis, and/or adenoiditis. None of the patients had received any antibiotic or gastric acid-lowering drugs during the last 2 weeks prior to surgery. All parents and/or patients agreed with surgery and the study proposal.

Clinical samples

Initially, the paraffin-embedded tonsillar tissues were sectioned into 50-μm slices followed by DNA extraction using the DNA preparation protocol obtained from Section of Cancer Genomics, Genetics Branch, NCI, National Institutes of Health.

Modified McMullen's staining of tonsillar biopsy

The paraffin-embedded sections was dewaxed, dehydrated, and covered by carbolfuchsin for 2 min. The sections were rinsed and stained in malachite green for 2 min. Then slides were rinsed in tap water and air dried. With this procedure, gull-shaped H. pylori were stained in magenta against light green tonsillar tissue.

Rapid urease test

The rapid urease test was performed using a commercial RUT kit obtained from Cham Enzyme (Tehran, Iran). For positive control, a standard strain of H. pylori ATCC 26695 was used and distilled water employed as negative control.

Conventional PCR

DNA amplification was performed using the H. pylori detection kit (Cinnagen, Iran) in a final volume of 30 μl with the cycling program of the PCR instrument (Applied Biosystem, USA) consisting of a cycle of 72°C/30 s, 50°C/20 s, and 94°C/45 s followed by 30 cycles of 72°C/30 s, 50°C/20 s, and 94°C/20 s. Then 10 μl of the amplified sample was directly electrophoresed on 1.5% agarose gel. The presence of a 492-bp DNA fragment was indicative of a positive reaction.

Scorpion real-time

Scorpion real-time PCR was performed using ABI Prism 7500 Sequence Detection System (Applied Biosystem). The tails of the Scorpion primers were designed as previously described by Burucoa. [14] All primers and probes used were synthesized by Metabion(Germany; [Table 1]). The total volume of the real-time PCR was 20 μl containing 5 μl of DNA from a clinical sample or bacterial isolate, 0.1 μM of the oligonucleotide primer 23SF2, and 0.08 μM of 23SScWT, while the total volume of 20 μl was achieved by the addition of distilled water. The cycling conditions were adjusted in a way suitable to be used by ABI Prism 7500 Sequence Detection System (Applied Biosystem) with an initial denaturation at 95°C for 45 s, 50 cycles at 95°C for 15 s, 55°C for 34 s, and 72°C for 20 s. The acquisition of a signal was performed at 57°C during each cycle. A negative control for Scorpion real-time PCR was obtained by the observation of no amplification signal except internal amplification by adding DDW instead of DNA to the prepared real-time PCR master mix. A positive control for Scorpion PCR was constituted by using extracted DNA from H. pylori ATCC 26695. Positive extraction control was performed for each biopsy specimen in a separate tube in a final volume of 25 μl with Premix Ex Taq (TaKaRa, Shiga, Japan), 5 μl of extracted DNA from the biopsy specimen, 0.25 μM of BGLO1 and BGLO2 primers, and ×0.5 Sybergreen 1 (Sigma-Aldrich,Germany). The cycling program was 1 cycle at 95°C for 10 s and 40 cycles at 95°C for 5 s, 55°C for 34 s, and 72°C for 10 s.{Table 1}

Statistical analysis

Data were analyzed using SPSS 11.5 software with 95% confidence intervals (95% CI). Also chi square and paired t-tests were used for statistical analysis.


   Results Top


In this survey, the prevalence of H. pylori infection in tonsils of patients with chronic tonsillitis was determined using the data obtained by the following procedures:

Modified McMullen's staining of tonsilar biopsy: Out of 103 samples from patients with clinical symptoms of chronic tonsillitis, 50 samples showed positive RUT whereas 53 samples demonstrated negative results.

Detection of H. pylori by rapid urease test: Out of 103 samples from patients with clinical symptoms of chronic tonsillitis, 50 samples showed positive RUT whereas 53 samples demonstrated negative results.

Detection of H. pylori by conventional PCR: When conventional PCR was used to detect H. pylori in biopsy specimens, of total 103 samples, 20 produced positive PCR results and 83 were negative for H. pylori. Since the present study was performed on paraffinized biopsies, there was no possibility of testing the samples by culture and recommended routine diagnostic tests.

Detection of H. pylori by Scorpion real-time PCR: Initially, Scorpion real-time PCR was performed on H. pylori ATCC 26695. Of total 103 samples, 21 biopsies were found to be positive for H. pylori by Scorpion real-time PCR whereas 82 samples showed negative results. Among samples with a positive Scorpion real-time PCR test, 17 samples were also RUT positive and 4 samples had RUT negative test results.

Scorpion real-time PCR on DNA extracts of H. pylori ATCC 26695 as control strains produced expected signals. The standard curve used to calculate the concentration of H. pylori DNA in unknown samples is presented in [Figure 1]a. The linear regression coefficient was 0.996 and the efficiency of PCR 99%. [Figure 1]b shows the fluorescent curves of the standard serial dilution from 1 × 10 5 to 1 × 10 2 . The electrophoresis pattern of Scorpion real-time PCR products in agarose gel is shown in [Figure 2]. A 140 bp fragment in the peptidyl transferase gene of 23S rRNA showed a successful amplification. Of the total patients (103 cases) with tonsillitis who entered the study, 22 cases (21.35%) were found to have H. pylori DNA in their tonsillectomy specimens by the Scorpion real-time PCR method. Our study showed that there is no significant association between the sex, age, marital status, history of gastric involvement, gastric disease, and the presence of bacterium. Also no relationship was established between the sex, family income, and accommodation variables and the colonization of H. pylori in tonsil tissues (data not shown).
Figure 1: (a) Standard curve used to calculate the concentration of H. pylori DNA in unknown samples. The linear regression coefficient is 0.996 and the efficiency of PCR is 99%; (b) fluorescent curves of the standard dilution series. From the left to the right, 105, 104, 103, 102, and the negative control is presented by the horizontal curve

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Figure 2: The PCR product was electrophoresed by agarose gel electrophoresis and stained with ethidium bromide. Lane 1: Helicobacter pylori ATCC 26695 as positive control; lane 2: negative control; lane 3: patient's positive sample; lane 4: 100 bp DNA ladder

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   Discussion Top


There are many arguments about different aspects of the association between H. pylori and chronic tonsillitis. Kusano and colleagues [15] in their recent study reported that the tonsillar H. pylori is unable to grow in any known culture media; however, Pavlic et al.[7] in their publication claimed of successful culture of H. pylori obtained from chronic tonsillitis and tonsillar cancer specimens.

This controversial issue is also present when it comes to the application of a molecular technique such as PCR. Eygor et al.[12] and Di Bonaventura et al.[10] reported of their failure in detecting H. pylori in tonsillar samples of patients by PCR (UreC and Urec2 primers) but Pavlic et al.[7] and Bulut et al.[13] both reported of the applicability of PCR in the detection of H. pylori in tonsillar specimens of their patients.

Zahedi et al. showed that 70 patients (73.7%) of their total 95 patients who underwent tonsillectomy had a positive anti-H. pylori IgG antibody in their sera, and the results of the rapid urease test on adenotonsil samples indicated that 42.1% of the specimens were positive for H. pylori.[6] Hence, it was concluded that the tonsils and adenoid tissues are the candidate places for the growth of H. pylori. In another study by Jabbari et al., of total 285 patients, 39.6% were positive for H. pylori in histopathologic examination, with 40 patients with positive RUT and 15 patients with a positive serum IgG anti-H. pylori level. In 40 patients, the results for both histopathology and RUT were positive although in 172 patients the findings associated with histopathology and RUT were negative and they deduced that H. pylori is present in the tonsillar tissue but the RUT is insensitive for the diagnosis of H. pylori in the tonsillar tissue, indicating that the tonsillar tissue might have a role in being a reservoir of H. pylori in children. [9]

Consistent with results mentioned earlier, Minocha et al.,[5] reported of reduced colonization of H. pylori infection in years following surgical adenotonsillectomy and decreased risk of H. pylori infection in gastric mucosa leading to hypothesize that the tonsil is a reservoir for H. pylori. In two studies by Jelavic et al.[8] and Skinner et al., [13] it was shown that the tonsils are not an important reservoir for H. pylori transmission in children; however, Unver et al.[1] reported of a different finding indicating the presence of H. pylori positivity in patients who underwent adenotonsillectomy. In a study by Bulut et al., [20] a relationship between H. pylori infection and adenotonsillar hypertrophy was found. Likewise, Cirak et al.[2] reported of the presence of cagA in H. pylori-positive tonsil and adenoid tissues.

In our study, Scorpion real-time PCR was positive in 21.35% of cases, a finding consistent with the results reported by Unver et al., [1] Cirak et al., [2] Minocha et al., [5] Zahedi et al., [6] Pavlic et al., [7] and Bulut et al., [20] yet in disagreement with findings of several other studies described by Jelvavic et al., [8] Jabbari et al., [9] Di Bonaventura et al., [10] Eygor et al., [12] Skinner et al.[13]

The differences in results of various studies might be due to diverse laboratory methods; [21],[22] however, the high sensitivity and specificity of Scorpion real-time PCR compared to other laboratory techniques, in particular when combined with the application of 23S rRNA of bacterial genome as the target for amplification, makes the authors of the present research more confident over the results obtained through Scorpion real-time PCR testing of tonsillar tissues samples.


   Conclusion Top


Our study showed that a significant number of patients with chronic tonsillitis may have H. pylori colonization. On the other hand, according to findings of numerous studies, it is clear that the H. pylori resistance against clarithromycin, a key component of triple drug treatment protocol to eradicate this organism, is considerably high. This drug resistance against clarithromycin is regarded as the major risk factor for treatment failure leading to a decrease of higher than 70% in the efficacy of the first-line drugs. So, the modification of the antibiotic regime against H. pylori might be considered.


   Acknowledgment Top


The authors thank Moavenat Pajouheshi of Qazvin University of Medical Sciences, Qazvin, Iran, who supported this work.

 
   References Top

1.Unver S, Kubilay U, Sezen OS, Coskuner T. Investigation of Helicobacter pylori colonization in adenotonsillectomy specimens by means of the CLO test. Laryngoscope 2001;111:2183-6.  Back to cited text no. 1
    
2.Cirak MY, Ozdek A, Yilmaz D, Bayiz U, Samim E, Turet S. Detection of Helicobacter pylori and its CagA gene in tonsil and adenoid tissues by PCR. Arch Otolaryngol Head Neck Surg 2003;129:1225-9.  Back to cited text no. 2
    
3.Mravak-Stipetic M, Gall-Troselj K, Lukac J, Kusiæ Z, Paveliæ K, Paveliæ J. Detection of Helicobacter pylori in various oral lesions by nested polymerase chain reaction (PCR). J Oral Pathol Med 1998;27:1-3.  Back to cited text no. 3
    
4.Kurien M, Stanis A, Job A, Brahmadathan K, Thomas throat swab in the chronic tonsillitis: How reliable and valid is it? Singapore Med J 2000;41:324-6.  Back to cited text no. 4
    
5.Minocha A, Raczkowski CA, Richards RJ. Is a history of tonsillectomy associated witha decreased risk of Helicobacter pylori infection? J Clin gastroenterol, 1997;25:580-2.  Back to cited text no. 5
    
6.Zahedi MJ, Darvish Moghadam S, Ahmadi Mosavi M, Mirshekari T. Helicobacter pylori colonization in biopsies of the adenotonsillectomy specimens. Am J ApplSci 2009;6:2050-3.  Back to cited text no. 6
    
7.Pavlík E, Lukes P, Potuzníková B, Astl J, Hrdá P, Soucek A, et al. Helicobacter pylori isolated from patients with tonsillar cancer or tonsillitis chronica could be of different genotype compared to isolates from gastrointestinal tract. Folia Microbiol 2007;52:91-4.  Back to cited text no. 7
    
8.Jelavic b, Bevanda M, Ostojic M. Tonsillar colonization is unlikely to play important role in Helicobacter pylori infection in children. Int J Pediatr Otorhinolaryngol 2007;71:585-90.  Back to cited text no. 8
    
9.Jabbari Moghaddam Y, Rafeey M, Radfar R. Comparative assessment of Helicobacter pylori colonization in children tonsillar tissues Int J pediatric Otorhinolaryngology 2009;73:1199-201.  Back to cited text no. 9
    
10.Dibonaventura G, Neri M, Nerietal G. Do tonsils represent an extra gastric reservoir for Helicobacter pylori infection. J Infect 2001;42:221-2.   Back to cited text no. 10
    
11.Ng CT, Gilchrist CA, Lane A, Roy S, Haque R, Houpt ER. Multiplex real-time PCR assay using scorpion probes and DNA capture for genotype-specific detection of Giardia lamblia on fecal samples. J Clin Microbiol 2005;43:1256-60.  Back to cited text no. 11
    
12.Eyigor M, Eyigor H, Gultekin B, Aydin N. Detection of Helicobacter pylori in adenotonsiller tissue specimens by rapid urease test and polymerase chain reaction. Eur Arch Otorhinolaryngol 2009;266:1611-3.  Back to cited text no. 12
    
13.Skinner LJ, Winter Dc, Curran AJ, Barnes C, Kennedy S, Maguire AJ, et al. Helicobacter pylori and tonsillectomy. Clin otolaryngol Allied Sci 2001;26:505-9.  Back to cited text no. 13
    
14.Burucoa C, Garnier M, Silvain C, FauchèreJL Quadruplex real-time PCR assay using allele-specific scorpion primers for detection of mutations conferring clarithromycin resistance to Helicobacter pylori. J Clin Microbio 2008;46:2320-6.  Back to cited text no. 14
    
15.Kusano K, Inokuchi A, Fujimoto K, Miyamoto H, Tokunaga O, Kuratomi Y, et al. Coccoid Helicobacter pylori exists in the palatine tonsils of patients with IgA nephropathy. J Gastroenterol 2010;45:406-12.  Back to cited text no. 15
    
16.Gião MS, Azevedo NF, Wilks SA, Vieira MJ, Keevil CW. Persistence of Helicobacter pylori in heterotrophic drinking-water Biofilms. Appl Environ Microbiol 2008;74:5898-904.  Back to cited text no. 16
    
17.Aladag I, Bulut Y, Guven M, Eyibilen A, Yelken K. Seroprevalence of Helicobacter pylori infection in patients with chronic nonspecific pharyngitis: Preliminary study. J Laryngol Otol 2008;122:61-4.  Back to cited text no. 17
    
18.Schwartz K, Monsur J, Northrup J, West P, Neale AV. Pharyngitis clinical prediction rules: Effect of inter observer agreement: A Metro Net study. J Clin Epidemiol 2004;57:142-6.  Back to cited text no. 18
    
19.Xia QF, Xu SX, Wang DS, Wen YA, Qin X, Qian SY, et al. Development of a novel quantitative real-time assay using duplex scorpion primer for detection of Chlamydia trachomatis. Exp Mol Pathol 2007;83:119-24.  Back to cited text no. 19
    
20.Bulut Y,Agacayak A,Karlidag T,Torman ZA,Yilmaz M. Association of cagA + Helicobacter pylori with adenotonsillar hypertrophy. Tohoko J Exp Med 2006;209:229-33.  Back to cited text no. 20
    
21.Song Q, Lange T, Spahr A, Adler G, Bode G. Characteristic distribution pattern of Helicobacter pylori in dental plaque and saliva detected with nested PCR. J Med Microbiol 2000;49:349-53.  Back to cited text no. 21
    
22.Khademi B, Niknejad N, Gandomi B, Yeganeh F. Comparison of Helicobacter pylori colonization on the tonsillar surface versus tonsillar core tissue as determined by the CLO test. Ear Nose Throat J 2007;86:498-501.  Back to cited text no. 22
    

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Correspondence Address:
R Najafipour
Department of Genetics, School of Medicine, Qazvin University of Medical Sciences, Qazvin
Iran
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0974-777X.93760

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