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J Pediatr Infect Dis 2021; 16(01): 018-025
DOI: 10.1055/s-0040-1719164
Original Article

Detection of Streptococcus pneumoniae in Urine by Loop-Mediated Isothermal Amplification

María Dolores Cima-Cabal 
1   Escuela Superior de Ingeniería y Tecnología (ESIT), Universidad Internacional de La Rioja (UNIR), Logroño, Spain
,
Emma Vázquez-Espinosa
2   Departamentode Neumología, Hospital Universitario de La Princesa, Madrid, Spain
,
Fernando Vazquez 
3   Servicio de Microbiología, Hospital Universitario Central de Asturias, Oviedo, Spain
4   Departamento de Biología Funcional, Área de Microbiología, Universidad de Oviedo, Oviedo, Spain
5   Fundación de Investigación Oftalmológica, Instituto Oftalmológico Fernández-Vega, Oviedo, Spain
6   Instituto de Investigación Sanitaria del Principado de Asturias (ISPA), Oviedo, Spain
,
María del Mar García-Suárez 
1   Escuela Superior de Ingeniería y Tecnología (ESIT), Universidad Internacional de La Rioja (UNIR), Logroño, Spain
› Author Affiliations Funding This research was funded by University of Oviedo (UNOV-08-MB-3) and Universidad Internacional de La Rioja (UNIR).
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Abstract

Objective To assess the loop-mediated isothermal amplification (LAMP) to detect cell-free DNA from Streptococcus pneumoniae in urine samples from children with pneumococcal pneumonia.

Methods LAMP reactions using four primers (backward inner primer, forward inner primer, B3, and F3) targeting conserved regions of the S. pneumoniae ply gene and DNA from the recombinant plasmid pTrc99A-ply were optimized for temperature (65°C) and MgSO4 concentration (8 mM) conditions. Urine samples from 71 patients with symptoms of pneumonia and from 17 healthy children were tested side by side using the isothermal methodology LAMP and the commercial urinary antigen test, BinaxNOW S. pneumoniae assay. Percentages of sensitivity, specificity, positive predictive value (PPV), negative predictive value, and positive (LR) were calculated to compare both tests.

Results The specificity of the LAMP reaction was confirmed against several species of bacteria and yeast that can cause pneumonia or urine infections. The suitability of the LAMP assay was evaluated in urine samples from 71 patients and 17 healthy children. All patients (100%) with confirmed pneumococcal pneumonia were positive for the LAMP assay. Among patients with possible/probable pneumonia, 74.1% were identified as positive using the LAMP test. Notably, a higher specificity (95.4%), PPV (94.1%) and positive LR (21.7) were found compared with the urinary antigen test.

Conclusion The presence of S. pneumoniae cell-free DNA in urine samples of pediatric patients can be used as a specific diagnostic biomarker for community-acquired pneumonia by using the LAMP methodology.

Keywords

community-acquired pneumonia - Streptococcus pneumoniae - LAMP - urine - DNA - children


Publication History

Received: 05 May 2020

Accepted: 30 September 2020

Article published online:
23 November 2020

© 2020. Thieme. All rights reserved.

Georg Thieme Verlag KG
Rüdigerstraße 14, 70469 Stuttgart, Germany

 

  • References

  • 1 World Health Organization, Estimated Hib and pneumococcal deaths for children under 5 years of age, Technical Report 2012, World Health Organization, Immunization, Vaccines and Biologicals. 2008
  • 2 Rodrigues CMC, Groves H. Community-acquired pneumonia in children: the challenges of microbiological diagnosis. J Clin Microbiol 2018; 56 (03) e01318-e17
    PubMedGoogle Scholar
  • 3 Scott JA, Wonodi C, Moïsi JC. Pneumonia Methods Working Group. et al. The definition of pneumonia, the assessment of severity, and clinical standardization in the Pneumonia Etiology Research for Child Health study. Clin Infect Dis 2012; 54 (Suppl. 02) S109-S116
    CrossrefPubMedGoogle Scholar
  • 4 Howie SRC, Morris GAJ, Tokarz R. et al. Etiology of severe childhood pneumonia in the Gambia, West Africa, determined by conventional and molecular microbiological analyses of lung and pleural aspirate samples. Clin Infect Dis 2014; 59 (05) 682-685
    CrossrefPubMedGoogle Scholar
  • 5 Harris M, Clark J, Coote N. British Thoracic Society Standards of Care Committee. et al. British Thoracic Society guidelines for the management of community acquired pneumonia in children: update 2011. Thorax 2011; 66 (Suppl. 02) ii1-ii23
    CrossrefPubMedGoogle Scholar
  • 6 Domínguez J, Blanco S, Rodrigo C. et al. Usefulness of urinary antigen detection by an immunochromatographic test for diagnosis of pneumococcal pneumonia in children. J Clin Microbiol 2003; 41 (05) 2161-2163
    CrossrefPubMedGoogle Scholar
  • 7 Navarro D, García-Maset L, Gimeno C, Escribano A, García-de-Lomas J. Spanish Pneumococcal Infection Study Network. Performance of the Binax NOW Streptococcus pneumoniae urinary antigen assay for diagnosis of pneumonia in children with underlying pulmonary diseases in the absence of acute pneumococcal infection. J Clin Microbiol 2004; 42 (10) 4853-4855
    CrossrefPubMedGoogle Scholar
  • 8 Jefferies JMC, Tocheva AS, Rubery H. et al. Identification of novel pneumolysin alleles from paediatric carriage isolates of Streptococcus pneumoniae . J Med Microbiol 2010; 59 (Pt 7): 808-814
    CrossrefPubMedGoogle Scholar
  • 9 Niemz A, Ferguson TM, Boyle DS. Point-of-care nucleic acid testing for infectious diseases. Trends Biotechnol 2011; 29 (05) 240-250
    CrossrefPubMedGoogle Scholar
  • 10 Mori Y, Notomi T. Loop-mediated isothermal amplification (LAMP): a rapid, accurate, and cost-effective diagnostic method for infectious diseases. J Infect Chemother 2009; 15 (02) 62-69
    CrossrefPubMedGoogle Scholar
  • 11 Andresen D, von Nickisch-Rosenegk M, Bier FF. Helicase-dependent amplification: use in OnChip amplification and potential for point-of-care diagnostics. Expert Rev Mol Diagn 2009; 9 (07) 645-650
    CrossrefPubMedGoogle Scholar
  • 12 Notomi T, Okayama H, Masubuchi H. et al. Loop-mediated isothermal amplification of DNA. Nucleic Acids Res 2000; 28 (12) E63
    CrossrefPubMedGoogle Scholar
  • 13 Kaneko H, Kawana T, Fukushima E, Suzutani T. Tolerance of loop-mediated isothermal amplification to a culture medium and biological substances. J Biochem Biophys Methods 2007; 70 (03) 499-501
    CrossrefPubMedGoogle Scholar
  • 14 Soares-Santos V, Pardo I, Ferrer S. Direct and rapid detection and quantification of Oenococcusoeni cells in wine by cells-LAMP and cells-qLAMP. Front Microbiol 2018; 9: 1945
    CrossrefPubMedGoogle Scholar
  • 15 Wachiralurpan S, Sriyapai T, Areekit S. et al. Rapid colorimetric assay for detection of Listeria monocytogenes in food samples using LAMP formation of DNA concatemers and gold nanoparticle-DNA probe complex. Front Chem 2018; 6: 90
    CrossrefPubMedGoogle Scholar
  • 16 Khan M, Wang R, Li B, Liu P, Weng Q, Chen Q. Comparative evaluation of the LAMP assay and PCR-based assays for the rapid detection of Alternaria solani . Front Microbiol 2018; 9: 2089
    CrossrefPubMedGoogle Scholar
  • 17 Wong YP, Othman S, Lau YL, Radu S, Chee HY. Loop-mediated isothermal amplification (LAMP): a versatile technique for detection of micro-organisms. J Appl Microbiol 2018; 124 (03) 626-643
    CrossrefPubMedGoogle Scholar
  • 18 De Schutter I, Vergison A, Tuerlinckx D. et al. Pneumococcal aetiology and serotype distribution in paediatric community-acquired pneumonia. PLoS One 2014; 9 (02) e89013
    CrossrefPubMedGoogle Scholar
  • 19 Baron EJ, Pfaller MA, Tenover FC. et al. In: Murray PR. ed. Manual of Clinical Microbiology. 7th ed.. Washington, DC: ASM Press; 1999
  • 20 del Mar García-Suárez M, Cima-Cabal MD, Villaverde R. et al. Performance of a pneumolysin enzyme-linked immunosorbent assay for diagnosis of pneumococcal infections. J Clin Microbiol 2007; 45 (11) 3549-3554
    CrossrefPubMedGoogle Scholar
  • 21 Cima-Cabal MD, Vázquez F, de los Toyos JR, Méndez FJ. Rapid and reliable identification of Streptococcus pneumoniae isolates by pneumolysin-mediated agglutination. J Clin Microbiol 1999; 37 (06) 1964-1966
    CrossrefPubMedGoogle Scholar
  • 22 Wilson EB. Probable inference, the law of succession, and statistical inference. JASA 1927; 22: 209-212
    CrossrefPubMedGoogle Scholar
  • 23 von Baum H, Welte T, Marre R, Suttorp N, Ewig S. CAPNETZ study group. Community-acquired pneumonia through Enterobacteriaceae and Pseudomonas aeruginosa: diagnosis, incidence and predictors. Eur Respir J 2010; 35 (03) 598-605
    CrossrefPubMedGoogle Scholar
  • 24 Kaplan V, Angus DC, Griffin MF, Clermont G, Scott Watson R, Linde-Zwirble WT. Hospitalized community-acquired pneumonia in the elderly: age- and sex-related patterns of care and outcome in the United States. Am J Respir Crit Care Med 2002; 165 (06) 766-772
    CrossrefPubMedGoogle Scholar
  • 25 Machado D, Hoffmann J, Moroso M. et al. RSV infection in human macrophages promotes CXCL10/IP-10 expression during bacterial co-infection. Int J Mol Sci 2017; 18 (12) 2654
    CrossrefPubMedGoogle Scholar
  • 26 Ieven M, Coenen S, Loens K. GRACE consortium. et al. Aetiology of lower respiratory tract infection in adults in primary care: a prospective study in 11 European countries. Clin Microbiol Infect 2018; 24 (11) 1158-1163
    CrossrefPubMedGoogle Scholar
  • 27 Xia Y, Guo XG, Zhou S. Rapid detection of Streptococcus pneumoniae by real-time fluorescence loop-mediated isothermal amplification. J Thorac Dis 2014; 6 (09) 1193-1199
    PubMedGoogle Scholar
  • 28 Seki M, Yamashita Y, Torigoe H, Tsuda H, Sato S, Maeno M. Loop-mediated isothermal amplification method targeting the lytA gene for detection of Streptococcus pneumoniae . J Clin Microbiol 2005; 43 (04) 1581-1586
    CrossrefPubMedGoogle Scholar
  • 29 Kim DW, Kilgore PE, Kim EJ. et al. The enhanced pneumococcal LAMP assay: a clinical tool for the diagnosis of meningitis due to Streptococcus pneumoniae . PLoS One 2012; 7 (08) e42954
    CrossrefPubMedGoogle Scholar
  • 30 Peters RP, de Boer RF, Schuurman T. et al. Streptococcus pneumoniae DNA load in blood as a marker of infection in patients with community-acquired pneumonia. J Clin Microbiol 2009; 47 (10) 3308-3312
    CrossrefPubMedGoogle Scholar
  • 31 Loonen AJM, Kesarsing C, Kusters R, Hilbink M, Wever PC, van den Brule AJC. High pneumococcal DNA load, procalcitonin and suPAR levels correlate to severe disease development in patients with pneumococcal pneumonia. Eur J Clin Microbiol Infect Dis 2017; 36 (09) 1541-1547
    CrossrefPubMedGoogle Scholar
  • 32 Vinkeles Melchers NV, van Dam GJ, Shaproski D. et al. Diagnostic performance of Schistosoma real-time PCR in urine samples from Kenyan children infected with Schistosoma haematobium: day-to-day variation and follow-up after praziquantel treatment. PLoS Negl Trop Dis 2014; 8 (04) e2807
    CrossrefPubMedGoogle Scholar
  • 33 Vaccaro L, Izquierdo F, Magnet A. et al. First case of legionnaire's disease caused by Legionella anisa in Spain and the limitations on the diagnosis of Legionella non-pneumophila infections. PLoS One 2016; 11 (07) e0159726
    CrossrefPubMedGoogle Scholar
  • 34 Jain S, Su YH, Su YP. et al. Characterization of the hepatitis B virus DNA detected in urine of chronic hepatitis B patients. BMC Gastroenterol 2018; 18 (01) 40
    CrossrefPubMedGoogle Scholar
  • 35 Calvert AE, Biggerstaff BJ, Tanner NA, Lauterbach M, Lanciotti RS. Rapid colorimetric detection of Zika virus from serum and urine specimens by reverse transcription loop-mediated isothermal amplification (RT-LAMP). PLoS One 2017; 12 (09) e0185340
    CrossrefPubMedGoogle Scholar
  • 36 Murdoch DR, Anderson TP, Beynon KA. et al. Evaluation of a PCR assay for detection of Streptococcus pneumoniae in respiratory and nonrespiratory samples from adults with community-acquired pneumonia. J Clin Microbiol 2003; 41 (01) 63-66
    CrossrefPubMedGoogle Scholar
  • 37 Liu Z, Zhang P, Tang S. et al. Urine real-time polymerase chain reaction detection for children virus pneumonia with acute human cytomegalovirus infection. BMC Infect Dis 2014; 14: 245
    CrossrefPubMedGoogle Scholar
  • 38 Csoma E, Mészáros B, Asztalos L, Gergely L. WU and KI polyomaviruses in respiratory, blood and urine samples from renal transplant patients. J Clin Virol 2015; 64: 28-33
    CrossrefPubMedGoogle Scholar
  • 39 Das BK, Suri S, Nath G, Prasad R. Urine nested polymerase chain reaction in neonatal septicemia. J Trop Pediatr 2015; 61 (04) 295-300
    CrossrefPubMedGoogle Scholar
  • 40 Robinson JL, Meier K, Lee BE, Larke B. Could urine be useful for the diagnosis of Chlamydia trachomatis pneumonia in infancy?. Diagn Microbiol Infect Dis 2014; 79 (03) 308-309
    CrossrefPubMedGoogle Scholar
  • 41 Lawn SD, Kerkhoff AD, Burton R. et al. Rapid microbiological screening for tuberculosis in HIV-positive patients on the first day of acute hospital admission by systematic testing of urine samples using Xpert MTB/RIF: a prospective cohort in South Africa. BMC Med 2015; 13: 192
    CrossrefPubMedGoogle Scholar
  • 42 Ibrahim DD, Ousmane S, Moumouni K, Mahamane AE. Measurement of pneumococcal IgG antibody, carriage and infection with Streptococcus pneumoniae in children under five years of age with acute respiratory infection in Niger. J Infect Public Health 2017; 10 (05) 657-660
    CrossrefPubMedGoogle Scholar
  • 43 Johnston C, Hinds J, Smith A, van der Linden M, Van Eldere J, Mitchell TJ. Detection of large numbers of pneumococcal virulence genes in streptococci of the mitis group. J Clin Microbiol 2010; 48 (08) 2762-2769
    CrossrefPubMedGoogle Scholar