Simultaneous and high sensitive detection of Salmonella typhi and Salmonella paratyphi a in human clinical blood samples using an affordable and portable device
- 98 Downloads
Enteric fever is one of the leading causes of infection and subsequent fatality (greater than 1.8 million) (WHO 2018), especially in the developing countries due to contaminated water and food inter twinned with unhygienic practices. Clinical gold standard technique of culture-based method followed by biochemical tests demand 72+ hours for diagnosis while newly developed techniques (like PCR, RT-PCR, DNA microarray etc.) suffer from high limit of detection or involve high-cost infrastructure or both. In this work, a quick and highly specific method, SMOL was established for simultaneous detection of Salmonella paratyphi A and Salmonella typhi in clinical blood samples. SMOL consists of (i) pre-concentration of S. typhi and S. paratyphi A cells using magnetic nanoparticles followed by (ii) cell lysis and DNA extraction (iii) amplification of select nucleic acids by LAMP technique and (iv) detection of amplified nucleic acids using an affordable portable device (costs less than $70). To identify the viability of target cells at lower concentrations, the samples were processed at two different time periods of t = 0 and t = 4 h. Primers specific for the SPA2539 gene in S. paratyphi A and STY2879 gene in S. typhi were used for LAMP. Within 6 h SMOL was able to detect positive and negative samples from 55 human clinical blood culture samples and detect the viability of the cells. The results were concordant with culture and biochemical tests as well as by qPCR. Statistical power analysis yielded 100%. SMOL results were concordant with culture and biochemical tests as well as by qPCR. The sensitive and affordable system SMOL will be effective for poor resource settings.
KeywordsSalmonella typhi Salmonella paratyphi A LAMP Diagnosis Cross-reactivity Device
The authors would like to acknowledge the financial support from the Department of Science and Technology (YSS/2014/000880, and IDP/MED/05/2014), Indo-German Science and Technology Centre (IGSTC/Call 2014/Sound4All/24/2015-16), Naval research board (NRB/4003/PG/359), BIRAC, Department of Biotechnology (BIRAC/BT/AIR0275/PACE-12/17).
- S.A. Besuschio, M. Llano Murcia, A.F. Benatar, S. Monnerat, I. Cruz Mata, A. Picado de Puig, et al., Analytical sensitivity and specificity of a loop-mediated isothermal amplification (LAMP) kit prototype for detection of Trypanosoma cruzi DNA in human blood samples. PLoS Negl. Trop. Dis. 11(7) (2017). https://doi.org/10.1371/journal.pntd.0005779 CrossRefGoogle Scholar
- CLSI document M47-A, Principles and procedures for blood cultures; approved guideline. Clinical and Laboratory Standards Institute. (2007)Google Scholar
- P.B. Crichton, Enterobacteriaceae: Escherichia, Klebsiella, proteus and other genera. Mackie and McCartney Practical Medical Microbiology 14, 361–384 (1996)Google Scholar
- S. Dahiya, P. Sharma, B. Kumari, S. Pandey, R. Malik, N. Manral, et al., Characterisation of antimicrobial resistance in Salmonellae during 2014–2015 from four centres across India: An ICMR antimicrobial resistance surveillance network report. Indian J. Med. Microbiol. 35(1), 61–68 (2017)CrossRefGoogle Scholar
- S. Dutta, S. Das, U. Mitra, P. Jain, I. Roy, S.S. Ganguly, et al., Antimicrobial resistance, virulence profiles and molecular subtypes of Salmonella enterica serovars Typhi and Paratyphi A blood isolates from Kolkata, India during 2009-2013. PLoS ONE 9(8) (2014). https://doi.org/10.1371/journal.pone.0101347 CrossRefGoogle Scholar
- V. Gupta, J. Kaur, J. Chander, et al., An increase in enteric fever cases due to Salmonella Paratyphi A in & around Chandigarh. Indian J. Med. Res. 129(1), 95–98 (2009)Google Scholar
- A. Kaur, A. Kapil, R. Elangovan, S. Jha, D. Kalyanasundaram, Highly-sensitive detection of Salmonella typhi in clinical blood samples by magnetic nanoparticle-based enrichment and in-situ measurement of isothermal amplification of nucleic acids. PLoS One 13(3) (2018b). https://doi.org/10.1371/journal.pone.0194817 CrossRefGoogle Scholar
- Y.T. Kim, Y. Chen, J.Y. Choi, W.-J. Kim, H.-M. Dae, J. Jung, T.S. Seo, Integrated microdevice of reverse transcription-polymerase chain reaction with colorimetric immunochromatographic detection for rapid gene expression analysis of influenza A H1N1 virus. Biosens. Bioelectron. 33(1), 88–94 (2012)CrossRefGoogle Scholar
- W. Mokhtari, S. Nsaibia, D. Majouri, A. Ben Hassen, A. Gharbi, M. Aouni, Detection and characterization of Shigella species isolated from food and human stool samples in Nabeul, Tunisia, by molecular methods and culture techniques. J. Appl. Microbiol. 113(1), 209–222 (2012). https://doi.org/10.1111/j.1365-2672.2012.05324.x CrossRefGoogle Scholar
- M. Mukhtar, S.S. Ali, S.A. Boshara, A. Albertini, S. Monnerat, P. Bessell, et al., Sensitive and less invasive confirmatory diagnosis of visceral leishmaniasis in Sudan using loop-mediated isothermal amplification (LAMP). PLoS Negl. Trop. Dis. 12(2) (2018). https://doi.org/10.1371/journal.pntd.0006264 CrossRefGoogle Scholar
- Wan, L., Gao, J., Chen, T., Dong, C., Li, H., Wen, Y. Z., … Martins, R. P. LampPort: a handheld digital microfluidic device for loop-mediated isothermal amplification (LAMP). Biomed. Microdevices https://doi.org/10.1007/s10544-018-0354-9 (2019)