• Specificity studies were performed using a panel of 10 Group B Streptococcus agalactiae strains, 11 of the most commonly occurring Streptococcus genus strains and 60 other organisms commonly found in the human genital tract. • One hundred and twenty four (124) vaginal swabs sampled from pregnant women were tested with the real-time LAMP assay and compared to a real- time PCR assay (5). Neonatal infections are most commonly caused by Group B Streptococcus GBS 10 (Streptococcus agalactiae) (1). These infections can be classified as either early on-set • Of the 124 vaginal swabs, 85 tested positive for Group B Streptococcus and & or late-onset disease. Early onset infection (EOI) occurs during the first week of life 39 tested negative. with late onset infection (LOI) occurring between one week and three months. It is Strep11 thought that early onset disease develops in the foetus after aspiration of infected • Comparable results were achieved by both the real-time PCR and real –time amniotic fluid (2). Late onset infection is less well understood, and while passage LAMP assays. Results are listed in Table 4. Also shown in the table are through the birth canal is an obvious route of infection, it is also thought that community previously obtained microbiological culture data for the isolation and sources are involved (2). Other identification of GBS. 60 One approach used to identify potential for infection is a screening of pregnant women, where prophylaxis is offered to those identified as carriers (3). This approach is also • Figure 1: Breakdown of specificity panels investigated during the development of the Table 2: Results of GBS testing of vaginal swabs. supported by the Centres for Disease Control and Prevention (CDC). To determine GBS LAMP assay carrier status, the CDC recommends culture of GBS from vaginal/rectal swabs from Figure 2: Process for testing of clinical samples in the LAMP reaction pregnant women. Some laboratories also carry out the CAMP test to differentiate GBS Streptococcus Genital Panel - species associated with human infection and species associated species Panel ! with genital tract environment ! from other streptococci. This test requires two consecutive overnight cultures. Microbiological S. anginosus ! A c i n e t o b a c t e r Bifidobacterium breve! Lactobacillus Proteus vulgaris! Vaginal Swabs! Real-time LAMP! Real-time PCR! Development of tests using molecular technologies for use in a screening scenario baumanii! fermentum! Culture! would shorten the time to result and assist the clinician in making a decision on whether or not to administer antibiotics. S. dysgalactiae ! A c i n e t o b a c t e r C a m p y l o b a c t e r Lactobacillus iners! Pseudomonas aeruginosa! calcoaceticus! concisus ! GBS positive! 85! 85! 85! We describe here the feasibility of using loop-mediated isothermal amplification (LAMP) S. gordonii ! A l c a l i g e n e s C a m p y l o b a c t e r L a c t o b a c i l l u s Pseudomonas putida! faecalis! hominis ! jensenii! (4) directly from crude lysate to detect GBS in vaginal swabs from pregnant women. S. intermedius ! A l c a l i g e n e s Campylobacter jejuni! Lactobacillus oris! Serratia marcescens! GBS negative! 39! 39! 39! Specificity and limit of detection of the assay are described. In total 124 clinical samples faecalis subsp. were tested directly without any pre-enrichment or nucleic acid extract steps faecalis! significantly reducing the assay turnaround time. The test reported here could be an S. mitis ! A n a e r o c o c c u s Citrobacter freundii! L a c t o b a c i l l u s Staphylococcus aureus! vaginalis vaginalis option for a screening diagnostic given that it is a rapid, sensitive and specific test for ! ! • The GBS LAMP assay specifically detects Group B Streptococcus. S. mutans ! Atopobium vaginae! C o r y n e b a c t e r i u m Mobiluncus curtisii! S t a p h y l o c o c c u s • All 10 S. agalactiae serotypes were detected by the LAMP assay and no cross-reaction GBS . genitalium ! epidermidis! occurred with any non-GBS species tested (Figure 3). S. oralis ! Bacteroides bivius! C o r y n e b a c t e r i u m Moraxella S t a p h y l o c o c c u s genitalium! ( M o r a x e l l a ) intermedius! osloensis! B S. parasanguinis ! B a c t e r o i d e s Enterobacter cloacae! Morganella Ureaplasma urealyticum! A disiens! morganii! S. pneumoniae ! B a c t e r o i d e s Enterococcus faecalis! Mycoplasma Veillonella parvula! • This GBS LAMP test is a valuable method for the rapid and specific detection of GBS in melaninogenica! hominis! pregnant women and offers advantages over other established non-isothermal methods • LAMP primers were designed using the online Primer Explorer version 4 tool (http:// S. pyogenes ! Bacteroides oralis! Enterococcus Peptococcus niger ! Veillonella parvula subsp. such as reduced temperature requirements, less sophisticated equipment, highly specific primerexplorer.jp/elamp4.0.0/index.html) to specifically target Group B Streptococcus. faecium Atypica! S. salivarius ! Bacteroides ovatus! Escherichia coli! P o r p h y r o m o n a s Candida albicans! amplification using crude lysate. No DNA extraction is required for the test described here. • The LAMP reaction consisted of the following components per 20µL reaction: LAMP parvulus! mastermix 12.5µL (Isoplex DNA kit Mast France), BIP 1µL (1.6µM final), FIP 1µL ! It shows potential for application in clinical settings with the capability of identifying GBS in a ! B a c t e r o i d e s F u s o b a c t e r i u m P o r p h y r o m o n a s Gardnerella vaginalis! (1.6µM final), F3 Primer 1µL (0.2µM), B3 Primer 1µL (0.2µM), loop primers 1&2 thetaiotaomicron! nucleatum! asaccharolytica! turnaround time of approximately 40 minutes. 0.625µL each (final 1µM), Bst polymerase 1µL (Isoplex DNA kit Mast), Picogreen ! B a c t e r o i d e s Fusobacterium varium! Prevotella bivia! Trichomonas vaginalis! (Quant-iT Invitrogen) 1µL, water (0.25µL). Thermocycling conditions consisted of 30 uniformis! B a c t e r o i d e s Klebsiella pneumoniae ! Propionibacterium Neisseria gonorrhoeae! cycles of 62°C for 60s, 1 cycle of 95°C for 2 mins and 1 cycle of 40°C 10 sec. vulgates! acnes! 1. Stoll BJ, Hansen NI, Sanchez PJ et al. Early onset neonatal sepsis: the burden of group B streptococcal and E. coli • All reactions were carried out on the LightCycler 480 ™(Roche). Bifidobacterium L a c t o b a c i l l u s Proteus mirabilis! Giardia lamblia! Figure 3: Amplification curve demonstrating A, specific detection of GBS from a specificity disease continues. Peadiatrics 2011;817. • The LAMP amplification reaction was performed on the Roche LightCycler 480 using bifidum! acidophilus! panel of 11 non-GBS species and B, specific detection of GBS from a panel of 60 species 2. Schuchat A. Epidemiology of grouop B streptococcal disease in the United States: shifting paradigms. Clin Microbiol. Mast Isoplex® Nucleic Acid Amplification reagents. PicoGreen™ was included in the taken from the sample environment. Rev 1998; 11:497. 3. Bekker V, Bijlsma MW, van de Beek D, Kuijpers TW van der Ende A. Incidence of invasive group B streptpcoccla reaction to allow real-time monitoring of the LAMP reaction. Table 1: Organisms used in specificity testing of GBS LAMP assay. disease and pathogen geneotype distribution in newborn babies in the Netherlands over 25 years: a nationwide surveillance study. Lancet Infect Dis 2014:14:1083. 4. Notomi, T, Okayama, H., Masubuchi H et al 2000. Loop-mediated isothermal amplification of DNA. Nucleic Acids Res: 28;vE63. The Limit of Detection of the assay was determined by Probit analysis of data from three 5. Wernecke M., Mullen C., Sharma V., Morrison J., Barry T., Maher M., Smith T. Evaluation of a novel real-time PCR test independent experiments showing the average limit of detection to be 10.03 genome copies based on the ssrA gene for the identification of group B streptococci in vaginal swabs. BMC Infect. Dis. 2009 9:148 (95% probability). .