Novel Strategies to Improve STI Screening

Seema SoodM.D., MNAMS

Professor of MicrobiologyAll India Institute of Medical Sciences, New Delhi

Background

STIs are a significant public health problem

Profound impact on sexual and reproductive health of adults

Newborn & child health

Early diagnosis is crucial

WHO Estimates: Curable STIs in 2012

Source: WHO/RHR/16.09, WHO 2016

Early Detection

Screening

Diagnosis

Is the best opportunity for effective treatment and for preventing further transmission

Huge variation in availability of tests

Screening remains rare in resource-limited settings

Syndromic Management

Advantages

Disadvantages

Over time, it is important to move from syndromic to etiological approach

Source: WHO/RHR/16.09, WHO 2016

Point of care tests

Conventional

• Gram staining

• KOH mount

• Tzanck Smear

• Wet mount

• RPR

• Dark ground microscopy

Newer

• Immune-chromatography

• Optical immunoassay,

• Latex agglutination

WHO-ASSURED Criteria

• Affordable A• SpecificS• SensitiveS• User-friendly U• Rapid and robustR• Equipment-free E• Delivered - to end usersD

Bacterial STIs

Disease burden disproportionately affect women

Emergence & spread of AMR in NG is a global concern

Diagnostic tests with high sensitivity & specificity are

available

POCTs: syphilis

Point of Care Patient Testing

Available POCTs for detection of CT or NG have low accuracy or require expensive equipment

NearPOCT: GeneXpert (Cepheid, Sunnyvale, CA, USA)

Urgent need for improved assays

New technologies

Biosensor Technology

Emerging technology in Diagnostics

Provides rapid, accurate diagnosis

Offers a low detection limit

Compact instrumentation compatible with portable devices

Potential to be used as an integral component in POC test (POCT) devices

Singh R, Mukherjee MA, Sood S et al. Sensors & Actuators: B: Chemical, 2014:197: 385-404.

Principle of Biosensor

Figure. (a) Schematic of a biosensor

NPG Asia Materials (2011) 3, 17–24; doi:10.1038/asiamat.2010.137

Electrochemical DNA Biosensor(opa-gene)

Methylene Blue was used as hybridization monitor agent

Decrease in signal intensity was observed for dsDNA

Optimization of working conditions was done using synthetic DNA

Probe sequences

Sequence NameLength

(bases)Sequence

Probe 195’S-S-C6-CGGTGCTTCATCACCTTAG

3'

Complementary

sequence19 5' CTAAGGTGATGAAGCACCG 3'

One-base

mismatch19 5' CTAAGTTGATGAAGCACCG 3'

Non-

complementary19 5' ACGGTTCACGCGGTACTTA 3'

PATENT: International Application no. PCT/IN2010/000457

Overview

CV, DPV,

SEM

Preparation of

a Solid Support

Immobilization

of Probe DNA

Biosensor

Characterization

Characterization

Target DNA

Electrochemical

Signal Detection

Response

Characteristics

CV, DPV, SEMProbe DNA

Synthetic DNA

PCR Amplicons

Clinical samplesCV, DPV

In-house PCR

Gene Forward primer Reverse primer PCR conditions Amplicon

size

opa

gene

5’CGGTGCTTCA

TCACCTTAG 3'

5'GGATTCATTTT

CGGCTCCTT 3'

94C for 45 s

56C for 45 s 30cycles

72C for 45 s

188 bps

Verma R, Sood S. Ind J Med Res 2016; 34(4): 560-01Verma R, Sood S*, Bala M et al. Epidemiol Infect 2012; 140(11): 2110-16

PCR (Female Patients)

Methods Microscop

y

GC

Culture

PCR

Number of

Patients

1 (250) 1 (250) 17 (250)

Sood S, Verma R, Mir S et al. Ind J Med Res 2014; 140: 649-52

16 False

Negatives

Selection of suitable matrix for Biosensor

Fabrication was attempted using different matrices

Gold was selected for further studies

The characteristics that met approval Sensitivity

Reproducibility

Stability

• Biosensors and Bioelectronics 2009; 24(7): 2232-38

• J Mol Recognit 2010. 23(5):472-9

• Thin Solid Films 2010; 519:1135–40

• J Biotech, 2010; 150: 357-65

• Biosensors and Bioelectronics, 2011; 26: 2967-74

• Bioelectrochemistry, 2012; 86: 30-37

• Microchimica Acta, 2012; 177(1-2): 201-10

Screen-printed gold electrodes

procured from DropSens, SpainWorking Electrode

Reference Electrode (Ag)

Counter Electrode (Pt)

ConnectorElectrochemical Analyzer

Setup

Fabrication of Biosensor

Verma R, Sood S, Singh R, et al. Diagn Microbiol Infect Dis. 2014; 78: 16-23

-0.5 -0.4 -0.3 -0.2 -0.1 0.0

0.0

2.0µ

4.0µ

6.0µ

8.0µ

10.0µ

12.0µ

14.0µ

16.0µ

Comp DNA

Probe DNA

OBM DNA

Non-Comp DNA

curr

en

t (

A)

Potential (V)

Synthetic

DNA

Prob

e

OBM Comp

.

Non-

comp

.

Percenta

ge

Decrease

0 -38.9 40 0

Figure. Redox peaks of MB with synthetic DNA

Table. Percentage decrease of current-

synthetic DNA with respect to probe

Studies with Synthetic DNA

Detection Limit with PCR amplicons

Detection Limit: 0.23 attogm/µl

-0.50 -0.45 -0.40 -0.35 -0.30 -0.25 -0.20 -0.15 -0.10

0.0

2.0µ

4.0µ

6.0µ

8.0µ

10.0µ

12.0µ

14.0µ

16.0µ

10-12

M

10-18

M

10-20

M (0.23 attogm/ul)

10-22

M

10-15

M

10-9 M

Probe DNA

cu

rre

nt

(A

)

Potential (V)

PCR amplicons

Biosensor: Evaluation

Study on PCR amplicons: 26 positive and 16 negative patient samples

Study on clinical samples: 10 positives, 10 negatives

ROC Analysis

Interpretation: Area under curve (accuracy)

0.90-1: Excellent

0.80-0.90: Good

0.70-0.80: Fair

0.60-0.70: Poor

0.50-0.60: Fail

Verma R, Sood S, Singh R, et al. Diagn Microbiol Infect Dis. 2014; 78: 16-23

Performance characteristics

Sood S, Verma R, Singh R, et al. P4-S1.02. Sex Transm Infect. 2011, 87: A307Verma R, Sood S, Singh R, et al. Diagn Microbiol Infect Dis. 2014; 78: 16-23

DNA from negative patient samples

-400 -300 -200

0

10

20

30

40

50

60

4395

12520

14536

20680

Probe

4297

16728

19497

11484

16859

8931

Negative patient samples (DNA)

Cu

rre

nt

(uA

)

Voltage (mV)

Verma R & Sood S Ind J Med Microbiol, 2016; 34(2): 139-145.

DNA from positive patient samples

-0.4 -0.3 -0.2 -0.1

0.0

2.0µ

4.0µ

6.0µ

8.0µ

10.0µ

12.0µ

14.0µ

16.0µ

DNA from Patient samples (1:10 diln)

4575

9048

15399

28430

11991

4575 dil

28488

30025

Probe

Cu

rre

nt

(uA

)

Voltage (v)

-0.4 -0.3 -0.2

0.0

2.0µ

4.0µ

6.0µ

8.0µ

10.0µ

12.0µ

14.0µ

DNA from Patient samples (further diluted 1:5 times)

11991

27818

28430

28488

4728

904830025

14273

A/737

15399

4575

Probe

Cu

rre

nt

(uA

)

Voltage (V)

Verma R & Sood S Ind J Med Microbiol, 2016; 34(2): 139-145.

Progress made so far ....

Coupling of DNA biosensor with PCR provides a sensitive method for specific detection of NG

Integration of biosensors with microfluidics

Direct detection from clinical samples: needs further study

Priority Populations

Sex workers

MSMs

IDUs

Other vulnerable people

Communication technologies

e-health (electronic-health)

STD Tracker

m-health (mobile-health)

Challenges

Prevalence of disease influences PPV of the tests

There is the potential that organisms can evolve to evade detection by NAATs

nvCT

NG isolates lacking porA pseudogene

There are no assays that detect gonococcal AMR

Sample preparation

Conclusion

There have been significant changes in the field of testing for STIs

Novel testing technologies combined with modern communication systems will have clinical & public health impact

Acknowledgement

Sincere thanks to DBT, GOI for financial assistance (BT/PR7667/MED/14/1057 & T/PR12530/MED/32/385/2015

Prof. (Dr.) BD Malhotra Deptt. Of Biotechnology DTU, Delhi

Prof.(Dr.) V.K. Sharma Deptt. of Dermatology &

Venereology, AIIMS Prof (Dr.) Somesh Gupta Deptt. of Dermatology &

Venereology, AIIMS

Dr. Sumana Scientist E, NPL, New Delhi Dr. Rachna Verma

& Dr. Renu Singh

Thank You