(P454) Towards point-of-care test development for Bacterial Vaginosis (BV), a polymicrobial infection

Wednesday, July 26, 2023

16:00 – 18:00 CST

Primary Presenter(s)

Seema Sood, MD

Professor
All India Institute of Medical Sciences, New Delhi, India, India

Co-author(s)

RM

Rajni Mala

Delhi, India
SM

Shilpi Malhotra

Research Associate
All India Institute of Medical Sciences, United States
SG

Somesh Gupta, MD

All India Institute of Medical Sciences, New Delhi, India, India

Background: Bacterial Vaginosis (BV) is the most common cause of vaginal discharge (VD) in women of reproductive age group. It is marked by displacement of beneficial Lactobacillus spp. by polymicrobial flora. It is associated with significant reproductive morbidity and increased susceptibility to STIs. Diagnosis of BV in resource-constraint setting is challenging due to poor laboratory infrastructure. Reliance is placed on syndromic case management (SCM), which leads to overtreatment. The clinical (Amsel’s) & microbiological criteria (Nugent’s scoring) have limitations and the commercially available POCT has poor sensitivity in Indian settings. In view of the above, there is need for innovation. Therefore, our study aims at developing a recognition molecule using aptamer technology against sialidases of BV associated bacteria. Such a molecule should exclude sialidases produced by other pathogens, e.g., Trichomonas vaginalis, also an important cause of VD in women.

Methods: A ssDNA library of 10¹⁴unique sequences was designed and SELEX (Systematic Evolution of Ligands by EXponential enrichment) technique was used to select aptamers against catalytic domains of sialidases of BV pathogens, i.e., G. vaginalis, Bacteroides fragilis and P. bivia. Further, counter selection was performed with sialidases of T. vaginalis. This was followed by cloning in pGMET vector and sequencing. The dissociation constants (Kd) of the selected aptamers were determined by non-linear regression analysis. Further, characterization was performed using online tool Clustal Omega and Mfold. Proof of principle was established by gold nanoparticle (GNP) assay.

Results: A total of 15 aptamers have been successfully generated using SELEX. Dissociation constants (Kd) of all the aptamers were in low nanomolar range i.e., 2.5 nM to 196 nM. There were no conserved sequences or motifs among the aptamers indicating all are unique. The results of GNP assay showed that the Aptamer S7 was highly specific for sialidases produced by BV pathogens and it does not recognize the sialidases produced by T. vaginalis.

Conclusion: We have identified highly specific aptamers for detection of BV associated bacteria. The selected aptamers possess potential for use in POCT development for BV diagnosis. (Indian Patent Application No. 202211029191)