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Detection of Salmonella in Blood by PCR using iroB gene.

ABSTRACT: BACKGROUND:Salmonella, a genus of more than 2500 serological variants (serovars), includes many organisms that can cause human disease. Enteric fever remains an important public health problem in developing countries. Non typhoidal Salmonella generally produce a self limited gastroenteritis in healthy individuals whereas in extremes of age and immunocompromised cause severe fatal disease. The protean manifestations make this disease a true diagnostic challenge. AIM:The present study was carried out to optimize PCR for detecting the Salmonella genus using iroB gene and evaluate its use in the rapid diagnosis of typhoid and non typhoidal salmonellosis. MATERIALS AND METHODS:The study was carried out between August 2009 and July 2011 on blood samples from patients attending JIPMER hospital, Pondicherry, India with clinical suspicion of enteric fever and salmonellosis. Whole blood was used DNA extraction and conventional PCR done with iroB and fliC primers. Blood culture and Widal test were performed for all the patients. STATISTICAL ANALYSIS:Performed using Fischer's exact test with Graphpad Instat 3. RESULTS:PCR results were compared with blood culture. Sensitivity and specificity of PCR with fliC gene are 95.6% and 93.3% respectively. Sensitivity and specificity of PCR with iroB gene are 96.6% and 93.3% respectively CONCLUSION:With iroB gene, additional cases of Salmonella Paratyphi A and non typhoidal Salmonella were detected when compared to fliC gene.

SUBMITTER: Ganesan V

PROVIDER: S-EPMC4290232 | biostudies-literature | 2014 Nov

REPOSITORIES: biostudies-literature

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Detection of Salmonella in Blood by PCR using iroB gene.

Ganesan Vithiya V Harish Belgode Narasimha BN Menezes Godfred Antony GA Parija Subash Chandra SC

Journal of clinical and diagnostic research : JCDR 20141120 11

<h4>Background</h4>Salmonella, a genus of more than 2500 serological variants (serovars), includes many organisms that can cause human disease. Enteric fever remains an important public health problem in developing countries. Non typhoidal Salmonella generally produce a self limited gastroenteritis in healthy individuals whereas in extremes of age and immunocompromised cause severe fatal disease. The protean manifestations make this disease a true diagnostic challenge.<h4>Aim</h4>The present stu ...[more]

PMID: 25584214

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Project description:BackgroundSalmonella Typhi causes an estimated 21 million new cases of typhoid fever and 216,000 deaths every year. Blood culture is currently the gold standard for diagnosis of typhoid fever, but it is time-consuming and takes several days for isolation and identification of causative organisms. It is then too late to initiate proper antibiotic therapy. Serological tests have very low sensitivity and specificity, and no practical value in endemic areas. As early diagnosis of the disease and prompt treatment are essential for optimal management, especially in children, a rapid sensitive detection method for typhoid fever is urgently needed. Although PCR is sensitive and rapid, initial research indicated similar sensitivity to blood culture and lower specificity. We developed a fast and highly sensitive blood culture PCR method for detection of Salmonella Typhi, allowing same-day initiation of treatment after accurate diagnosis of typhoid.MethodsAn ox bile tryptone soy broth was optimized for blood culture, which allows the complete lysis of blood cells to release intracellular bacteria without inhibiting the growth of Salmonella Typhi. Using the optimised broth Salmonella Typhi bacteria in artificial blood samples were enriched in blood culture and then detected by a PCR targeting the fliC-d gene of Salmonella Typhi.ResultsTests demonstrated that 2.4% ox bile in blood culture not only lyzes blood cells completely within 1.5 hours so that the intracellular bacteria could be released, but also has no inhibiting effect on the growth of Salmonella Typhi. Three hour enrichment of Salmonella Typhi in tryptone soya broth containing 2.4% ox bile could increase the bacterial number from 0.75 CFU per millilitre of blood which is similar to clinical typhoid samples to the level which regular PCR can detect. The whole blood culture PCR assay takes less than 8 hours to complete rather than several days for conventional blood culture.ConclusionsThis novel blood culture PCR method is superior in speed and sensitivity to both conventional blood culture and PCR assays. Its use in clinical diagnosis may allow early detection of the causative organism and facilitate initiation of prompt treatment among patients with typhoid fever.

Project description:BackgroundEnteric fever remains an important cause of morbidity in many low-income countries and Salmonella Paratyphi A has emerged as the aetiological agent in an increasing proportion of cases. Lack of adequate diagnostics hinders early diagnosis and prompt treatment of both typhoid and paratyphoid but development of assays to identify paratyphoid has been particularly neglected. Here we describe the development of a rapid and sensitive blood culture PCR method for detection of Salmonella Paratyphi A from blood, potentially allowing for appropriate diagnosis and antimicrobial treatment to be initiated on the same day.MethodsVenous blood samples from volunteers experimentally challenged orally with Salmonella Paratyphi A, who subsequently developed paratyphoid, were taken on the day of diagnosis; 10 ml for quantitative blood culture and automated blood culture, and 5 ml for blood culture PCR. In the latter assay, bacteria were grown in tryptone soy broth containing 2.4% ox bile and micrococcal nuclease for 5 hours (37°C) before bacterial DNA was isolated for PCR detection targeting the fliC-a gene of Salmonella Paratyphi A.ResultsAn optimized broth containing 2.4% ox bile and micrococcal nuclease, as well as a PCR test was developed for a blood culture PCR assay of Salmonella Paratyphi A. The volunteers diagnosed with paratyphoid had a median bacterial burden of 1 (range 0.1-6.9) CFU/ml blood. All the blood culture PCR positive cases where a positive bacterial growth was shown by quantitative blood culture had a bacterial burden of ≥ 0.3 CFU/ ml blood. The blood culture PCR assay identified an equal number of positive cases as automated blood culture at higher bacterial loads (≥0.3 CFU/ml blood), but utilized only half the volume of specimens.ConclusionsThe blood culture PCR method for detection of Salmonella Paratyphi A can be completed within 9 hours and offers the potential for same-day diagnosis of enteric fever. Using 5 ml blood, it exhibited a lower limit of detection equal to 0.3 CFU/ml blood, and it performed at least as well as automated blood culture at higher bacterial loads (≥0.3 CFU/ml blood) of clinical specimens despite using half the volume of blood. The findings warrant its further study in endemic populations with a potential use as a novel diagnostic which fills the present gap of paratyphoid diagnostics.

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Detection of Salmonella in Blood by PCR using iroB gene.

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Detection of Salmonella in Blood by PCR using iroB gene.

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