EVALUATION OF DRIED BLOOD SPOTS (DBS) FOR THE DIAGNOSIS OF SCRUB TYPHUS: A COMPARATIVE STUDY WITH TRADITIONAL SERUM METHOD

Abstract

This invention describes a novel diagnostic method for detecting Scrub typhus caused by Orientia tsutsugamushi that employs Dried Blood Spots (DBS) in conjunction with EnzymeLinked .lmmunosorbent Assay (ELISA). The procedure is less invasive than typical serum collection techniques, making sample collection, storage, and transport easier, especially in resource-limited or distant situations. In this novel method, whole blood is collected via finger prick onto specialized filter paper, which is subsequently dried and preserved. The dried samples are then eluted to remove antibodies specific to Orientia tsutsugamushi, which aids in accurate diagnosis. Clinical testing shows that this DBS-based approach has I 00% sensitivity, specificity, positive predictive value, and negative predictive value, which matches the performance of traditional serum-based assays. The idea not only improves diagnostic accessibility, but also enables quick and effective Scrub typhus treatment, increasing patient outcomes and public health responses in endemic areas. This technology represents substantial improvements in the field of infectious disease diagnostics, resulting in improved disease control and management strategies.

Specification

FIELD OF THE INVENTION:.
The invention relates to diagnostic methods and technologies for infectious diseases, with
a specific focus on the speedy and reliable detection of Scrub typhus caused by Orientia
tsutsugamushi. This invention relates to advancements in serological testing using Dried Blood
Spots (DBS) and Enzyme-Linked Immunosorbent Assay (ELISA) procedures. The idea seeks to
address the obstacles associated with existing serum collection methods, improving accessibility,
efficiency, and practicality in a variety of healthcare settings, particularly in resource-limited or
rural places where Scrub typhus is prevalent. This diagnostic technique helps public health by
allowing for more rapid diagnosis and treatment of scrub typhus, thereby improving patient
outcomes and disease management measures.



BACKGROUND OF THE INVENTION:
The innovation is based on the increasing global frequency of scrub typhus, a deadly
infectious disease caused by the bacteria Orientia tsutsugamushi. This disease is transmitted by
the biting of infected chiggers, resulting in a variety of clinical symptoms ranging from mild
fever to severe multi-organ failure. The quick and accurate diagnosis of Scrub typhus is critical
for effective treatment, which often· includes antibiotics like doxycycline or azithromycin.
Traditionally, diagnostic procedures have relied on serological tests, such as indirect
immunofluorescence assays and ELISA, which need serum samples collected via venipuncture.
However, these traditional approaches provide significant logistical obstacles, especially in
distant or resource-constrained regions where access to healthcare institutions may be limited.
Issues such as the requirement for freezing during transportation, the complexity of sample
collection, and the possibility of sample deterioration impede effective disease surveillance and
early diagnosis. DBS offer a viable alternative to typical serum collection methods. DBS enables
easy sample collection with minimum blood volume, which can be stored at ambient
temperatures without the need for sophisticated logistics. This approach is especially useful in
endemic areas where Scrub typhus is prevalent since it streamlines the diagnosis process and
improves accessibility for both patients and healthcare providers. Despite the benefits of DBS,
there has been little research on their efficacy in diagnosing Scrub typhus when compared to
conventional serum-based approaches. The invention aims to close this gap by providing a
dependable DBS-based diagnostic approach based on ELISA, with the goal of improving the sensitivity and specificity of Scrub typhus diagnosis while resolving the practical constraints
encountered in endemic· places. This innovation proposes to improve disease management and
control in at-risk groups by using advances in diagnostic technology.
OBJECTIVES OF THE INVENTION:
•:• To develop a DBS-based diagnostic approach for detecting Scrub typhus caused by
Orientia tsutsugamushi that has good sensitivity and specificity when compared to
standard serum-based assays.
•:• To provide a minimally invasive and easy-to-implement diagnostic tool that can be used
in resource-limited or remote settings, thereby increasing access to timely and accurate
Scrub typhus diagnosis.
•:• To use DBS technology, which allows for easier blood collection, storage, and transport
without the need for refrigeration or complex logistics, hence improving disease
surveillance and management.
•:• To improve patient care and treatment outcomes by providing quick detection of scrub
typhus, consequently lowering morbidity and death in endemic areas.
•:• To compare the performance of DBS-based ELISA to standard serum-based techniques,
thereby confirming its reliability as a diagnostic option.
•:• To create a scalable diagnostic system that can be integrated into public health initiatives
for early diagnosis and management of scrub typhus, thereby improving overall disease
control efforts in affected populations.
•:• To create the framework for future research and development of DBS-based diagnostics
for other infectious diseases, hence encouraging the wider use of this technology in the
field of infectious disease diagnosis.


BRIEF DESCRIPTION OF DRAWINGS:
I. Figure I: Flowchart of the Diagnostic Method
This figure illustrates the step-by-step prpcess of the DBS-based diagnostic method for scrub
typhus. It includes the steps of blood collection via finger prick, application onto the DBS card,
drying and storage of the card, extraction of antibodies, and the subsequent ELISA testing.
2. Figure 2: Dried Blood Spot Preparation
This image shows a close-up of how Dried Blood Spots are prepared dn Whatman 903 cards. It
shows the blood spotting procedure, drying conditions, and DBS quality testing.
3. Figure 3: ELISA Process Overview
This figure depicts the ELISA method used to detect antibodies in serum and DBS samples. It
describes how to add certain antigens, incubate the samples, and read the optical density for
analysis purposes.
4. Figure 4: Sensitivity and Specificity Results
This graph displays the statistical analysis findings comparing the sensitivity and specificity of
the DBS-based ELISA approach to traditional serum-based methods, demonstrating diagnostic
accuracy.
5. Figure 5: Clinical Sample Collection Setup
This graph depicts the setup for clinical specimen collection, including the tools and environment
used to collect whole blood samples and prepare DBS.


SUMMARY OF THE INVENTION:
The invention describes a novel diagnostic method for detecting Scrub typhus caused by
Orientia tsutsugamushi that uses DBS in conjunction with an ELISA. This novel methodology
tackles the logistical issues associated with. standard serum collection methods, especially in
resource-constrained and remote situations. The procedure is taking whole blood by a finger prick, transferring it to customized DBS cards, and allowing the samples to dry. After drying, the
cards are stored in ideal conditions to protect the samples' integrity. Antibodies specific to
Orientia tsutsugamushi are recovered from the DBS with a simple buffer solution, and their
presence is detected by ELISA. Clinical trials show that this DBS-based approach has high
sensitivity, specificity, positive predictive value, and negative predictive value, which are
comparable to traditional serum-based assays. This diagnostic strategy improves accessibility
and speeds up the diagnosis process by simplifying sample collection and transportation, thereby
enhancing patient treatment and public health responses in endemic areas. Overall, the idea helps
to advance diagnostic skills for scrub typhus, promote early and effective treatment, and support
larger public health activities targeted at controlling this infectious illness.
DETAILED DESCRIPTION OF THE INVENTION:
The invention relates to a Scrub typhus diagnostic method that uses DBS in combination
with an ELISA to detect Orientia tsutsugamushi. The following is a full discussion of the
invention, including its methods, advantages, and prospective applications.
Methodology
I. Sam pie Collection:
Finger pricks are used to collect whole blood samples from patients suspected of having
scrub typhus. This minimally invasive technique is intended to allow for easy and speedy blood
collection while reducing the discomfort and dangers associated with venipuncture.


Preparation of Dried Blood Spots (DBS):
•:• Blood is promptly put onto Whatman 903 protein saver cards or comparable DBS cards in
50 J.!l aliquots. The blood spots are allowed to cure at room temperature for 2 to 4 hours,
or until totally dry ..
•:• Once dried, the DBS cards are stored in gas-impermeable zipper bags with desiccant
sachets to keep them dry and prevent degradation, with long-term storage at -70°C
advised.



Sample Extraction:
•:• To perform the diagnostic assay, two 5 mm punched-out circles from the DBS are
inserted in Eppendorf tubes containing I 00 fll of an extraction buffer, commonly
phosphate-buffered saline (PBS) with 0.0 I% Tween 20.
•:• The samples are gently shaken at room temperature for I .5 hours to extract antibodies
from the DBS. After incubation, the tubes are centrifuged and the supernatant collected
for examination.
Enzyme-Linked lmmunosorbent Assay (ELISA):
•:• The extracted material is submitted to commercially available ELISA kit (Scrub typhus
lgM ELISA). Orientia tsutsugamushi-specific antigens are added to the wells of an
ELISA plate, followed by sample extracts.
•:• After incubation, suitable secondary antibodies conjugated with an enzyme are added.
The enzyme's substrate is subsequently introduced, and the reaction creates a detectable
signal, which is commonly measured using optical density at specified wavelengths.
Advantages:
Minimally Invasive: Finger pricks for blood collection are less invasive than standard
venipuncture methods, reducing discomfort and risk.
Ease of Transportation: DBS may be stored and transported at room temperature, eliminating
the need for refrigeration and complicated logistics.
Simplified Processing: The method makes it easier to handle samples m resource-limited
environments, allowing for greater access to diagnostic testing .
High Diagnostic Accuracy: Preliminary data show that the DBS-based ELISA has 100%
sensitivity and specificity, confirming its dependability in detecting scrub typhus.

Applications:
Field Diagnostics: The DBS-based approach is ideal for usage in rural places with limited
healthcare access, allowing for prompt diagnosis and treatment commencement.
Surveillance Programs: This strategy can improve public health surveillance efforts for scrub
typhus, assisting with disease tracking and outbreak controL
Research and Development: The methodology can be used to the diagnosis of other infectious
disorders, broadening the scope of DBS technology's diagnostic applications .

Novelty:
•:• The use of finger pricks for blood collection is a less invasive alternative to standard
venipuncture methods, making it more acceptable, especially for vulnerable groups like
children and those in remote places.
•:• DBS technology enables the convenient collection, storage, and transportation of blood
samples without the need for refrigeration. This logistical simplicity is especially useful
in resource-constrained environments where access to healthcare institutions is frequently
restricted.
•:• ln preliminary trials, the approach displayed high sensitivity and specificity (both at
I 00%), establishing it as a viable alternative to traditional serum-based assays. This
precision is essential for the timely identification and efficient treatment of scrub typhus.
•:• The idea expands access to diagnostic testing by making DBS more accessible in
endemic areas, hence boosting public health activities targeted at improving disease
management and surveillance.
Q) •:• The methodology developed in this innovation has the potential to be applied to the
C)
Ill diagnosis of additional infectious diseases, broadening its utility beyond Scrub typhus and
D..
Q) driving advances in global health diagnostics.
•:• The invention addresses the need for effective diagnostic instruments in remote and
underserved locations, improving disease surveillance and outbreak response.
•:• Overall, the use of DBS based ELISA for Scrub typhus diagnosis represents a substantial
development in diagnostic techniques, addressing crucial issues of accessibility, logistics,
and accuracy in infectious disease detection.


Annexure-11
WE CLAIM:
I. A diagnostic method for detecting Scrub typhus, caused by Orientia tsutsugamushi,
comprising:
• Collecting a complete blood sample from a person via finger prick;
• Apply the collected blood on a Dried Blood Spot (DBS) card;
• Allowing the blood to dry on the DBS card;
• Storing the DBS card in conditions suitable for preservation;
• Extracting antibodies from the DBS using an extraction buffer; and
• Using an Enzyme-Linked lmmunosorbent Assay (ELISA) to detect Orientia
tsutsugamushi-specific antibodies in the extracted sample.
2. The method of claim I, wherein the extraction buffer comprises phosphate-buffered saline
(PBS) with 0.0 I% Tween 20.
3. The method of claim I, wherein the DBS card is a Whatman 903 protein saver card or a
similar protein:preserving substrate.
4. The technique described in claim I also includes a phase that analyzes the ELISA data to
ascertain whether Scrub typhus is present in the subject with I 00% diagnostic sensitivity and
specificity.
5. The technique described in claim I, which is applied in environments with limited resources,
improves accessibility to Scrub typhus diagnostic.

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