Pakistan Journal of Medical Sciences

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ISSN 1681-715X

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ORIGINAL ARTICLE

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Volume 24

July - September 2008

Number  4


 

Abstract
PDF of this Article

Nested PCR based diagnosis of Salmonella enterica
serovar Paratyphi A directly from blood samples

Aamir Ali1, Abdul Haque2, Yasra Sarwar3, Asma Haque4, Mashkoor Mohsin5,
Amna Afzal6, Tayyaba Iftikhar7, Ayesha Tariq8

ABSTRACT

Objective: Development of a rapid, reliable PCR - based method for molecular identification of Salmonella enterica serovar Paratyphi A directly from blood samples.

Methodology: S. Paratyphi A isolates were used for regular PCR targeting specific region of fliC-a gene. New primers were designed and conditions were optimized for a nested PCR that could be directly applicable on blood samples. The procedure was tested on 70 blood samples from suspected cases of typhoidal infection and comparison made with blood culture.

Results: Blood culture was able to diagnose only four patients as infected with S. Paratyphi A. Regular PCR was unable to detect S. Paratyphi A directly from blood where as nested PCR detected S. Paratyphi A in blood of thirteen patients.

Conclusions: S. Paratyphi A, which is emerging as a major pathogen can be detected with better sensitivity by nested PCR as compared with blood culture.

KEY WORDS: Salmonella Paratyphi A, Nested PCR, Paratyphoid Diagnosis.

Pak J Med Sci    July - September 2008    Vol. 24 No. 4    545-549

How to cite this article:

Ali A, Haque A, Sarwar Y, Haque A, Mohsin M, Afzal A, et al. Nested PCR based diagnosis of Salmonella enterica serovar Paratyphi A directly from blood samples. Pak J Med Sci 2008;24(4):545-9.


1. Aamir Ali, D.V. M., M. Phil. (Biotech)
2. Abdul Haque, M. Sc. (Med. Micro), Ph. D. (Med Mol. Biol)
3. Asma Haque, M. Sc. (Chem), M. Phil. (Chem)
4. Yasra Sarwar, M. Phil. (Biotech)
5. Mashkoor Mohsin, M. Phil. (Biotech)
6. Amna Afzal, M. Sc. (Biochem)
7. Tayyaba Iftikhar, M. Sc. (Zoology)
8. Ayesha Tariq, MBBS, M. Phil. (Biotech)
1-8: Health Biotechnology Division,
National Institute for Biotechnology and
Genetic Engineering (NIBGE),
Jhang Road, Faisalabad, Pakistan.

Correspondence

Dr. Abdul Haque
Principal Scientific Officer, Health Biotechnology Division,
National Institute for Biotechnology and
Genetic Engineering (NIBGE), P.O.Box 577, Jhang Road,
Faisalabad, Pakistan.
E-mail: ahaq_nibge@yahoo.com
abdulhaq@nibge.org

* Received for Publication: March 20, 2008
* Revision Received: May 23, 2008
* Revision Accepted: May 24, 2008


INTRODUCTION

Although S. Typhi (Salmonella enterica serovar Typhi) is still the major cause of typhoidal infection, S. Paratyphi A is fast emerging as a major pathogen in developing countries especially in Indian subcontinent.1 It has a disease presentation highly similar to that of typhoid fever but it seems to follow a distinct route of transmission: whereas typhoid fever is spread predominantly within the household, paratyphoid fever is mainly transmitted outside the patient’s home.2 Clinically, in general, patients with S. Paratyphi A enteric fever have more rose spots than patients with typhoid fever.3 In laboratory, the diagnosis of paratyphoid fever depends on demonstrating the pathogen in blood, bone marrow, stool or urine cultures. However, bacteriological methods are time consuming and usually require 5-11 days.4 Additionally, in developing countries like Pakistan sensitivity of blood culture is lowered due to irrational use of antibiotics. The Widal test; a serologic test has a number of limitations including failure to diagnose S. Paratyphi A infection.5,6

Polymerase chain reaction (PCR), in addition to analysis of foods, has also been successfully applied to the detection and identification of pathogenic organisms in clinical and environmental samples.7 It has been successfully used for diagnosis of S. Typhi and proved superior to conventional methods.8 A similar approach for diagnosis of S. Paratyphi A can be of great help.

Different genes have been targeted to detect S. Paratyphi A in water, milk, blood (after spiking with bacteria) and fecal samples but none of them is reported as applicable on patient’s blood samples.9,10 A very specific portion of fliC-a gene, present only in S. Paratyphi A and not in S. Typhi was targeted to identify purified colonies of S. Paratyphi A.11 However, this test involves blood culturing and isolation of bacteria that limits its sensitivity as blood culture is positive in only 50% cases at most.12 In order to make this technique directly applicable to patient’s blood samples with imperative advantages of PCR including better sensitivity and specificity, we designed new internal primers of fliC-a gene, developed a nested PCR and optimized it so that it can be successfully applied on blood samples and used for rapid diagnosis of paratyphoid fever.

METHODOLOGY

Five purified S. Paratyphi A isolates were taken from NIBGE (National Institute for Biotechnology and Genetic Engineering, Faisalabad, Pakistan) stock cultures. Other related bacteria including S. Typhi, Escherichia coli, Proteus vulgaris, Pseudomonas aeruginosa, Klebsiella aerogenes, Aerobacter spp., and Micrococcus were used as negative controls. The stocks were kept at -20 ºC in TSB (Tryptic soy broth, Merck, Germany) containing 10% dimethyl sulfoxide. For use, these stocks were revived in TSB and cultivated on MacConkey agar overnight to ascertain purity. Isolated colonies were proceeded with to act as controls.

Blood samples from seventy patients, admitted to different hospitals in Faisalabad, Pakistan, having fever for more than three days with enlarged spleen, headache, malaise, abdominal discomfort, and/or agitation, were taken (3ml each) in tubes containing anticoagulant (20mM potassium EDTA), stored at 4oC and processed for PCR within two days of collection. For blood culture, 2ml of blood was inoculated into a culture bottle containing 16ml of TSB with 0.02% SPS (sodium polyanethanol sulfonate) and incubated at 37oC for 72 hours. Subculturing was done on MacConkey agar and after overnight incubation, isolated colourless, smooth colonies with 2-3mm diameter were selected for further investigation. Biochemical identification was done by inoculation into Triple Sugar Iron (TSI) medium (Merck, Germany) and results were interpreted according to manufacturer’s guidelines. All isolates after biochemical identification were subjected to regular and nested PCR for confirmation.

The genomic DNA from overnight grown culture (3ml each) of purified colonies was extracted by conventional phenol-chloroform method.13 whereas DNAs from 200µl of each of blood samples were extracted by genomic DNA extraction kit K0512 (Fermentas, USA). The integrity of extracted DNAs was checked by 1% agarose gel electrophoresis. For regular PCR, primers targeting specific region of fliC-a gene of S. Paratyphi A were used, 11 whereas primers for second round (nested) PCR were designed from the nucleotide sequence database (Genbank [X03393]). Both primer sets were supplied by Sigma (Dorset, United Kingdom) and the sequences are shown in Table-I.

The sensitivity of the technique was optimized to be directly applicable on blood samples by procedures as reported for S. Typhi.8 Each 50µL reaction mixture for regular PCR, in addition to 10µL of template, contained 1.5mM MgCl2, 50nmol of each dNTP, 40pM of each primer and 2U of Taq polymerase (Fermentas, Maryland, USA). The thermal cycler (MasterCycler; Eppendorf, Hamburg, Germany) conditions for 30 cycles were as follows; denaturation at 940C for one minute, annealing at 480C for one minute and extension at 720C for 1.5 minutes. For nested PCR, each 50µL reaction mixture, in addition to 10µL template, contained 1.5mM MgCl2, 50nmol of each dNTP, 40pM of each primer and 2U of Taq polymerase (Fermentas, USA). The thermal cycler conditions for 30 cycles were as follows; denaturation at 940C for one minute, annealing at 55 0C for one minute and extension at 720C for 1.5 minutes. The amplification products of both regular and nested PCRs were electrophoresed on 2% agarose gel, stained, and photographed by UV transilluminator Eagle Eye (Stratagene, California, USA).

RESULTS

Regular PCR of stock cultures and blood culture isolates of S. Paratyphi A amplified a specific region of 329 base pairs (bp) of fliC-a gene as previously reported.11 Optimization of conditions for newly designed primers for nested PCR of these isolates resulted in successful amplification of a 259 bp region of fliC-a gene in all S. Paratyphi A isolates [Figure-1]. No amplification was obtained using extracted DNAs of the related bacteria S. Typhi, Escherichia coli, Proteus vulgaris, Pseudomonas aeruginosa, Klebsiella aerogenes, Aerobacter spp., and Micrococcus as templates

From seventy blood samples, blood culture was positive for S. Paratyphi A in only four cases. Regular PCR was unable to detect any Salmonella directly from blood samples. However, nested PCR detected thirteen blood samples as infected with S. Paratyphi A by amplification of fliC-a gene (259 bp) [Figure-2].

DISCUSSION

Enteric fever refers to a systemic infection caused by typhoidal Salmonella. Classic enteric fever is caused by S. Typhi and a similar but relatively less severe syndrome is caused by S. Paratyphi A.14 Up to half of the cases of enteric fever are considered to be due to S. Paratyphi A instead of S. Typhi that declares S. Paratyphi A as a emerging cause of enteric fever in Indian subcontinent including India, Pakistan, China, Nepal, Vietnam and Indonesia.15 There is no statistically significant difference in patient symptoms and rate of complications between infections due to S. Typhi or S. Paratyphi A.16

For an effective treatment of S. Paratyphi A infection, its early and precise diagnosis is important. It would prevent not only complications like haemorrhage and perforation but also the spread of infection in poor hygienic conditions.17 PCR in one variant or another is being used increasingly in routine diagnostic laboratory settings to overcome the problems of poor sensitivity and specificity of conventional diagnostic methods.18 S. Paratyphi A has already been targeted in water, milk, spiked blood and fecal samples by PCR9,10 but not directly in clinical blood samples. Regular PCR targeting specific fliC-a gene (phase-1 flagellin; H:a) of S. Paratyphi A was used with a limited scope of identifying purified bacterial colonies only.11 We tried to apply this technique of detection of S. Paratyphi A directly on blood samples but failed. To overcome this problem, we designed internal primers for the second round (nested) PCR. After optimizing the conditions, nested PCR was applied to seventy clinical blood samples and it successfully identified thirteen patients infected with S. Paratyphi A. Specificity of the test was validated as no amplification was found in case of negative controls using DNAs of related bacteria. Blood culture was able to detect only four cases as S. Paratyphi A infections. These four cases were also found positive by nested PCR. The poor sensitivity of blood culture is due to multiple factors including host’s immune response system19 that becomes even worse if the patient has already taken some antibiotic drugs which is common practice in this region.

We conclude that this PCR-based detection of S. Paratyphi A from blood samples is more sensitive than blood culture. Of course there is additional advantage of a quick result in one to two days as compared with nearly one week required for blood culture.

ACKNOWLEDGMENTS

The research facilities for this work were provided by the National Institute for Biotechnology and Genetic Engineering (NIBGE), Faisalabad, Pakistan and the funds were provided by Higher Education Commission (HEC) of Pakistan.

REFERENCES

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2. Vollaard AM, Ali S, van Asten HA. Risk factors for typhoid and paratyphoid fever in Jakarta, Indonesia. JAMA 2004;291:2607-15.

3. Belilos E, Tu RP, Sacks-Berg A. Rose spots associated with enteric fever secondary to Shizella sonnei. Int J Dermatol 1988;27:402-3.

4. United States Department of Agriculture Animal and Plant Health Inspection Service. The national poultry improvement plan. Subpart B. Bacteriological examination procedure 147-11. Laboratory procedure recommended for the bacteriological examination of Salmonella, United States Department of Agriculture, Washington, DC 1996;14-9.

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12. Guerra-Caceres JG, Gotuzzo-Herencia E, Crosby-Dagnino E, Miro-Quesada M, Carrillo-Parodi C. Diagnostic value of bone marrow culture in typhoid fever. Trans R Soc Trop Med Hyg 1979;73:680-3.

13. Sambrook J, Fritsch EF, Maniatis T. Molecular cloning: a laboratory manual. 2nd ed. New York: Cold Spring Harbor Laboratory Press, 1989.

14. Shlim DR, Schwartz E, Eaton M. Clinical importance of Salmonella paratyphi A infection to enteric fever. J Travel Med 1995;2:165-8.

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16. Maskey AP, Day JN, Phung QT, Thwaites GE, Campbell JI, Zimmerman M, et al. Salmonella enterica serovar Paratyphi A and S. enterica serovar Typhi cause indistinguishable clinical syndromes in Kathmandu, Nepal. Clin Infect Dis 2006;42:1247-53.

17. Luxemburger C, Chau MC, Mai NL, Wain J, Tran TH, Simpson JA, et al. Risk factors for typhoid fever in the Mekong delta, southern Viet Nam: a case-control study. Trans R Soc Trop Med Hyg 2001;95:19-23.

18. Emmanuel PJ. Polymerase chain reaction from bench to bedside: Applications for infectious disease. J Fla Med Assoc 1993;80:627-30.

19. Escamilla J, Floretz-Ugarte H, Kilpatrick ME. Evaluation of blood clot cultures for isolation of Salmonella typhi, Salmonella paratyphi A and Brucella melitensis. J Clin Microbiol 1986;24:388-90.


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