Development of a LAMP assay to detect Group B Streptococcus

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Background

Noenatal infections are most commonly caused by Group B Streptococcus (Streptococcus agalactiae) (1). These infections can be classified as either early on-set or late-onset disease.  Early onset infection (EOI) occurs during the first week of life with late onset infection (LOI) occurring between one week and three months. To date, two major prevention strategies have been adopted in many countries (2). The first of these is based on risk stratification of women at time of delivery, with those presenting with risk factors for transmission administered antibiotics. The second approach is a screening approach of pregnant women, where prophylaxis is offered to those identified as carriers.  This approach is also supported by the Centers for Disease Control and Prevention (CDC). To determine carrier status, the CDC recommends culture of GBS from swabs from pregnant women. Some laboratories also carry out the CAMP test to differentiate GBS from other streptococci. This test requires two consecutive overnight cultures. It is clear therefore that development of tests using molecular technologies which could be used in a screening scenario and would shorten the time to result would be of significant benefit to the clinician in making a decision on whether or not to administer antibiotics.

Technical

We describe here the use of a loop-mediated isothermal amplification (LAMP) (3) on the LightCycler™ 480 to detect GBS in vaginal swabs from pregnant women. Specificity and limit of detection of the assay were confirmed. 124 clinical samples were tested directly without any pre-enrichment or nucleic acid extract steps significantly reducing the assay turnaround time.  The test could be an option for a screening diagnostic given that it is a rapid, sensitive and specific test for GBS.

Results

The assay was 100% specific for the organism of interest. 10 GBS isolates, 12 closely related Streptococci and  62 other organisms commonly found at the site of infection were included in the specificity testing. The Limit of Detection of the assay was 18 cell equivalents with a probability of greater than or equal to 95% by Probit analysis. 124 clinical samples were tested directly from swab with no pre-enrichment or extraction

Clinical Performance RPA assay
Positive Negative %
Positive 85(TP) 0 (FP) 100% (PPV)
Negative 1 (RN) 38 (TN) 97.37 (NPV)
% 98.84 Sensitivity 100 Specificity

 

 

 

Conclusion

This LAMP assay could be an option for a screening diagnostic given that it is a rapid, sensitive and specific test for GBS offer the clinician an alternative to culture based methods for rapid results, ultimately positively impacting on patient care.

1.Stoll BJ, Hansen NI, Sanchez PJ et al. Early onset neonatal sepsis: the burden of group B streptococcal and E. coli disease continues. Peadiatrics 2011;817-26).

2.Bekker V, Bijlsma MW, van de Beek D, Kuijpers TW van der Ende A. Incidence of invasive group B streptpcoccla disease and pathogen geneotype distribution in newborn babies in the Netherlands over 25 years: a nationwide surveillance study. Lancet Infect Dis 2014:14:1083-89).

3.Notomi, T, Okayama, H., Masubuchi H et al 2000. Loop-mediated isothermal amplification of DNA. Nucleic Acids Res: 28;vE63.)

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