Selenite enrichment broth to improve the sensitivity in molecular diagnostics of Salmonella

Boer, Maria Dullaert-de; Boer, Richard F. de; Lameijer, Annel; Sterne, Elles; Skidmore, Ben; Suijkerbuijk, Anita; Heck, Max; Zanden, Adri G. M. van der

doi:10.1016/j.mimet.2018.12.018

Cited by 9 publications

(6 citation statements)

References 21 publications

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“…Comparing the limits of detection of different Salmonella isolate conditions demonstrated that selenite pre-culture achieves a significantly lower limit of detection (1 CFU/ml) as opposed to direct isolates (10 CFU/ml) and Salmonella-spiked stool (10 CFU/ml). Selenite F broth is a selective broth that suppresses the growth of fecal coliforms and streptococci in order to optimize Salmonella growth 42 . The LOD achieved after sub-culturing samples in Selenite enrichment broth agrees with results demonstrated by other studies including a study done by Boer et al who demonstrated that sub-culturing samples in Selenite F broth promotes the recovery of Salmonella in stool samples and improves sensitivity if samples are subsequently tested using molecular methods like PCR 42,43 .…”

Section: Discussionmentioning

confidence: 99%

Performance of molecular methods for the detection of Salmonella in human stool specimens

Chirambo¹,

Nyirenda²,

Jambo³

et al. 2020

Wellcome Open Res

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Background: The relationship between asymptomatic Salmonella exposure within the gastrointestinal tract and Salmonella bacteraemia is poorly understood, in part due to the low sensitivity of stool culture, and the lack of validated molecular diagnostic tests for the detection of Salmonella in stool. The study aimed to determine a reliable molecular diagnostic test for Salmonella in stool specimens. Methods: We optimized an in-house monoplex real time polymerase chain reaction (PCR) for the detection of Salmonella TTR and InvA genes in stool by including a selenite broth pre-culture step for Salmonella before DNA extraction, and validated their specificity against other local common pathogens. Then we assessed their performance against a well-validated multiplex PCR targeting the same TTR and InvA genes, and against stool culture using clinical stool specimens collected from a cohort of 50 asymptomatic healthy Malawian children that were sampled at 1-month intervals over a period of 12 months. We employed a latent Markov model to estimate the specificities and sensitivities of PCR methods. Results: TTR and InvA primers were both able to detect all the different Salmonella serovars tested, and had superior limits of detection if DNA was extracted after selenite pre-culture. TTR sensitivity and specificity for monoplex-PCR were (99.53%, 95.46%) and for multiplex-PCR (90.30%, 99.30%) respectively. InvA specificity and specificity for using monoplex-PCR was (95.06%, 90.31%) and multiplex-PCRs (89.41%, 98.00%) respectively. Sensitivity and specificity for standard stool culture were 62.88% and 99.99% respectively. Culture showed the highest PPV (99.73%) and mono-TTR had the highest NPV (99.67%). Conclusion: Test methods demonstrated high concordance although stool culture and monoplexed TTR primers had superior specificity and sensitivity respectively. The use of selenite pre-enrichment step increased Salmonella detection rate. Taken together, molecular detection methods used here could be used to reveal the true extent of both asymptomatic and symptomatic Salmonella exposure events.

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Section: Discussionmentioning

confidence: 99%

Performance of molecular methods for the detection of Salmonella in human stool specimens

Chirambo¹,

Nyirenda²,

Jambo³

et al. 2020

Wellcome Open Res

View full text Add to dashboard Cite

show abstract

“…Comparing the limits of detection of different Salmonella isolate conditions demonstrated that selenite pre-culture achieves a significantly lower limit of detection (1 CFU/ml) as opposed to direct isolates (10 CFU/ml) and Salmonella -spiked stool (10 CFU/ml). Selenite F broth is a selective broth that suppresses fecal coliforms and streptococci growth to optimise Salmonella growth 46 . The LOD achieved after sub-culturing samples in Selenite enrichment broth agrees with results demonstrated by other studies, including a study done by Boer et al, who showed that sub-culturing samples in Selenite F broth promotes the recovery of Salmonella in stool samples and improves sensitivity if samples are subsequently tested using molecular methods like PCR 46 , 47 .…”

Section: Discussionmentioning

confidence: 99%

Performance of molecular methods for the detection of Salmonella in human stool specimens

et al. 2021

View full text Add to dashboard Cite

Background: The relationship between asymptomatic Salmonella exposure within the gastrointestinal tract and Salmonella bacteraemia is poorly understood, in part due to the low sensitivity of stool culture and the lack of validated molecular diagnostic tests for the detection of Salmonella in the stool. The study aimed to determine a reliable molecular diagnostic test for Salmonella in stool specimens. Methods: We optimised an in-house monoplex real-time polymerase chain reaction (PCR) for the detection of Salmonella ttr and InvA genes in stool by including a selenite broth pre-culture step for Salmonella before DNA extraction and validated their specificity against other local common pathogens. Then we assessed their performance against a well-validated multiplex PCR targeting the same ttr and InvA genes and against stool culture using clinical stool specimens collected from a cohort of 50 asymptomatic healthy Malawian children that were sampled at 1-month intervals over 12 months. We employed a latent Markov model to estimate the specificities and sensitivities of PCR methods. Results: Ttr and InvA primers were both able to detect all the different Salmonella serovars tested and had superior limits of detection when DNA was extracted after selenite pre-culture. Ttr sensitivity and specificity for monoplex-PCR were (99.53%, 95.46%) and for multiplex-PCR (90.30%, 99.30%) respectively. InvA specificity and specificity for using monoplex-PCR was (95.06%, 90.31%) and multiplex-PCRs (89.41%, 98.00%) respectively. Sensitivity and specificity for standard stool culture were 62.88% and 99.99%, respectively. Culture showed the highest PPV (99.73%), and monoplex-ttr had the highest NPV (99.67%). Conclusion: Test methods demonstrated high concordance, although stool culture and monoplexed ttr primers had superior specificity and sensitivity, respectively. The use of selenite pre-enrichment step increased Salmonella detection rate. Taken together, molecular detection methods used here could be used to reveal the true extent of both asymptomatic and symptomatic Salmonella exposure events.

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“…In total, 329 isolates from environmental and food samples, containing 126 serovars belonging to all subspecies of Salmonella were identified using invA (Malorny et al, 2007;Kasturi and Drgon, 2017). The ttr locus has also been used as an essential molecular marker for reducing false-positive results (Ssemanda et al, 2018;Boer et al, 2019).…”

Section: Discussionmentioning

confidence: 99%

Validation of Droplet Digital Polymerase Chain Reaction for Salmonella spp. Quantification

Villamil

Calderón

Arias³

et al. 2020

Front. Microbiol.

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Salmonellosis is a foodborne disease caused by Salmonella spp. Although cell culture is the gold standard for its identification, validated molecular methods are becoming an alternative, because of their rapidity, selectivity, and specificity. A simplex and duplex droplet digital polymerase chain reaction (ddPCR)-based method for the identification and quantification of Salmonella using ttr, invA, hilA, spaQ, and siiA gene sequences was validated. The method has high specificity, working interval between 8 and 8,000 cp/µL in ddPCR reaction, a limit of detection of 0.5 copies/µL, and precision ranging between 5 and 10% measured as a repeatability standard deviation. The relative standard measurement uncertainty was between 2 and 12%. This tool will improve food safety in national consumption products and will increase the competitiveness in agricultural product trade.

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Selenite enrichment broth to improve the sensitivity in molecular diagnostics of Salmonella

Cited by 9 publications

References 21 publications

Performance of molecular methods for the detection of Salmonella in human stool specimens

Performance of molecular methods for the detection of Salmonella in human stool specimens

Performance of molecular methods for the detection of Salmonella in human stool specimens

Validation of Droplet Digital Polymerase Chain Reaction for Salmonella spp. Quantification

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