1.

Title of the proposed research project:

Development of a novel and rapid diagnostic technique depending on Volatile Organic
Compounds from exhaled breath of COVID-19 patients

2. Rationale (up to 350 words):

Novel coronavirus disease 2019/SARS-CoV-2, which is causing COVID-19 pandemic, reported around 180
and 30 million population infected worldwide and in India, respectively (WHO, 2021) till June 24, 2021.
COVID-19 is an acute respiratory disease that can lead to fatal respiratory infections. To date, no specific
antiviral treatment for COVID-19 has been approved by the Food and Drug Administration (FDA). For the
management of COVID-19 outbreaks, testing is still the basis for successful coordination of clinical and public
health responses. In the market several tests are available, required the use of correct specimen type and
laboratory-testing technique in the right clinical scenario could be challenging for non-specialists. In addition,
RT-qPCR is the gold standard method for the diagnosis COVID-19 worldwide, detecting SARS-CoV-2 RNA
in nasopharyngeal and/or oral swabs. Results from RT-PCR using primers targeting different viral genome
parts can be affected by viral RNA sequence variation. In addition, false-negative results may occur because
of viral evolution (Shen, et al, 2020) or from errors in swab sampling or laboratory processing, or because the
virus is not yet/no longer present in the oropharynx (Tang et al, 2020). Other limitations of RT-qPCR tests
include sample storage, low-quality nucleic acid purification, cost, wait times (Smyrlaki, et al, 2020), runs the
risk of reduced specificity cross-contamination (Udugama et al, 2020).

Analysis of factors associated early diagnosis in COVID-2019 are mandatory in the present scenario. Previous
porcine and human studies have identified breath biochemistry of volatile organic compounds (VOCs)
derangements in acute respiratory distress syndrome (ARDS) that resulted in elevated exhaled ethanol signals
(Meidert et al, 2021, Dorota et al, 2020). Methanol and ketones have also been proposed as markers for
gastrointestinal inflammation, and ketosis, respectively. There is some evidence that viral infection leads to
the increase of production of fatty acids, alkanes, and alkanes related products. For instance, 2,3-butandione,
aldehydes, 2,8-dimethyl-undecane and n-propyl acetate have all been correlated with viral infection. Currently,
the literature points to markers of oxidative stress (nitric oxide, aldehydes etc) being the most useful in the
determination of viral infection. Wang et al, confirmed VOCs as a biomarkers for lung cancer in their study
based on 23 VOCs through which they easily discriminate lung cancer patients from controls with 96.47%
sensitivity and 97.47% specificity (Wang et al, 2012).To keep the things in mind the present study is designed
with the aim of rapid detection of COVID-19 suspects though their breath, breath VOCs would be a great step
forward for diagnostic purposes.

3. Novelty/Innovation (up to 100 words):

The benefit of detection VOCs such as aldehydes can be found in breath in just minutes after their formation
in tissues and elevating level of aldehyde has no relation with the age or gender of the patients. GC-TOF-MS
system to monitor breath VOCs in ventilated and non-ventilated patients with COVID-19. The direct and fast
measurement of so many breath VOCs at the same time allows us to study the progression of the disease
and the response to therapeutic strategies. This would be the first study in MP as well in India that could help
to test the COVID-19 suspects.
 Expected outcomes

The rapid detection of COVID-19-specific breath VOCs would be a great step forward for diagnostic purposes
and a spectrum of VOCs could be used for monitoring disease progression or response to conventional or
investigational drugs. Breath-analysis is attractive because it offers-point of care location, rapid results without
dependency on reagents, non-invasive sampling with a low biosecurity burden, and usability in a world-wide
range of scenarios, including low-resourced environments such as community or primary care settings. Since
therapies for COVID-19 are experimental at this stage, directly monitoring the effect of a therapeutic drug
speeds up the development of an efficient treatment for COVID-19. In addition, early diagnosis of COVID-19
will help to start treatment timely.

4. Project Description (up to 700 words): (Describe study setting, study design, sampling strategy, sample
size, feasibility, expected outcomes, inter-department/inter-institutional collaborations if any, timelines,
budget)
Objectives:

1. To evaluate the level of volatile organic compounds from exhaled breath of COVID-19 patients and
healthy individuals.
2. To determine the patient's likely disease trajectory in terms of recovery versus progression to
respiratory and or multi-organ failure.
3. To validate the technique by RT-qPCR for various performance characteristics including sensitivity,
specificity, PPV, NPV, limit of detection and reproducibility etc.

Experimental Design:

Sample collection

Exhaled breath samples and nasopharyngeal / oropharyngeal swabs samples (n=500) will be collected from
COVID-19 patients and healthy individuals. Samples will be collected from the patients at the time of
diagnosis (same day when RT-qPCR found positive) day zero (day 0), during (day 7 and day 14) and after recovery
from illness at 8-12 weeks (at day 60-120). Samples are coming for COVID-19 testing at VRDL, GRMC, Gwalior
will be chosen for the analysis after taking the consent of the patients and healthy volunteers.

Objective 1:

Our primary aim is to collect breath samples from COVID-19 positive patients at the time of diagnosis, during and
after recovery using the same patient as his/her own control to identify VOCs specific to SARS-COV-2 viral
infection. Breath samples will be collected as soon as the patient will diagnosis of COVID-19 by RT-qPCR in
nasopharyngeal/throat swab. A second breath sample will be obtained one week later. A third sample will be taken
to 8-12 weeks after recovery from the illness with a negative COVID-19 RT-PCR test. VOCs in exhaled breath
will be measured by gas chromatography-mass spectrometry (GC-TOF-MS or GCxGC-TOFMS) at the VRDL,
GRMC, Gwalior (MP) India.
Objective 2:

VOC profiles from the symptomatic phase and recovery phase will be compared to determine if there are unique
VOCs associated with COVID-19 infection. Comparison of VOC profiles between those who recover and those
who progressed will provide information on potential prognostic features.

References
 Udugama, B. et al. Diagnosing COVID-19: the disease and tools for detection. ACS Nano 14, 3822–3835 (2020).
 Shen, Z. et al. Genomic diversity of severe acute respiratory syndromecoronavirus 2 in patients with coronavirus disease 2019. Clin.
Infect. Dis. 71, 713–720 (2020).
 Smyrlaki, I. et al. Massive and rapid COVID-19 testing is feasible by extraction-free SARS-CoV-2 RT-PCR. Nat. Commun. 11, 4812
(2020).
 Meidert, A.S. et al. Exhaled Breath and Oxygenator Sweep Gas Propionaldehyde in Acute Respiratory Distress Syndrome. Molecules.
26(1),145 (2021). https://doi.org/10.3390/molecules26010145
 Dorota, M.R. et al. Diagnosis of COVID-19 by analysis of breath with gas chromatographyion mobility spectrometry - a feasibility
study. The Lancet- EClinicalMedicine. 27, 100609 (2020). https://doi.org/10.1016/j.eclinm.2020.100609
Time line (objective wise)

Objectives Work plan Time-lines (in months)

6 12 18
Objective 1 Sample collection, Analysis of VOCs in exhaled breath by GC-MS
Objective 2 1.Validate recovery versus progression to respiratory and or
multi-organ failure
2. Validate the technique and measure the different performance
characteristics including sensitivity, specificity, PPV, NPV, limit of
detection and reproducibility
Final evaluating the results achieved, writing of the manuscript
and reports

Budget details

S.No. Budget Requirements 1st year 2nd year (for Total (Rs.)
6 months)
1 Staff
Scientist 'C' 55440X12 = 57660X6 = 1011240/-
(Non-medical)-1 665280/- 345960/-
Rs. 51000/month
(corresponding to PB-3 Rs. 15,600 – 39,100 + Grade
Pay Rs. 6600) (with 10% increment each year)
2 Recurring 1350000/- 150000/- 1500000/-
(supplies, materials, consumables, etc.)
3 Non-recurring (Equipments) (GC-MS/MS) 6000000 - 6000000
3 Travel 40000/- 10000/- 50000/-
4 Contingencies 150000/- 50000/- 200000/-
5 Overhead charges (3%) 64958/- 16378/- 81336/-
Total 8270238/- 572338/- 8842576/-
5. Strength of PI:
PI details:
Dr. Vaibhav Misra, MBBS, MD (Microbiology)
Professor in Microbiology Department,
Gajra Raja Medical College, Gwalior (M.P.)
DOB: 24/04/1974
Email id: drvaibhavmisra74@gmail.com
Mobile no: 8370004411
Academic qualification:
Degree Subject Year University/Institution
MBBS All subjects 2000 Rani Durgavati Vishwavidyalaya,
Jabalpur/Netaji Subhash Chandra Bose
Medical College, Jabalpur
MD Microbiology 2005 University of Mumbai/ Lokmanya Tilak
Municipal Medical College, Sion,
Mumbai

Research Experience:
Designation Department Institution From To Total
Asst. Professor Microbiology G.R. Medical 25.05.2002 26.06.2010 05(y)01(m)
College, Gwalior
Assoc. Professor Microbiology G.R. Medical 26.06.2010 13.03.2019 08(y)08(m)
College, Gwalior
Professor Microbiology G.R. Medical 13.03.2018 Till Date 03(y)3(m)
College, Gwalior
Publications:
1. Verma R, Misra V et al., Potential of Selected Indian Herbs for COVID-19. Current Traditional Medicine; 2020;
http://doi.org/10.2174/2215083806999201125122620.
2. Jain AK, Misra V et al., Speciation, Biofilm Formation and Antifungal Susceptibility of Candida Isolates from
Clinically Diagnosed Patient of UTI in a Tertiary Care Hospital. J Assoc Physicians India; 2019; 67: 42-45.
3. Misra V et al., Isolation and identification of Candida species in the patients of UTI. Journal of Evolution of Medical
and Dental Sciences; 2016; 5:4532-4536.
4. Misra V et al., Incidence of neonatal fungaemia in a tertiary care hospital in north India. Journal of Evidence Based
Medicine and Healthcare; 2016; 3:2326-2331.
5. Sahai S, Mishra V et al., Viral Hepatitis in Pregnancy A study of its Effect on Maternal and Foetal Outcome; 2015;
63:28-33.
6. Kothari S, Mishra V et al., Third generation cephalosporin-resistance in Klebsiella pneumoniae isolates: an
emerging threat. International journal of Basic & Clinical Pharmacology; 2013; 2:56-60.
7. Kothari S, Mishra V et al., Antimicrobial activity and phytochemical screening of serial extract from leaves of
Aegle marmelos (linn.). Acta Poloniae Pharmaceutica; 2011; 11:687-692.

Co-PI details:
Dr. Rishika Khetan, MBBS
Demonstrator in Microbiology Department,
Gajra Raja Medical College, Gwalior (M.P.)
DOB: 09/12/1976
Email id: rishikabansalgwl@gmail.com
Mobile no: 9826262641
 Academic qualification:
Degree Subject Year University/Institution
MBBS All subjects 2000 Rajasthan University/ SMS Medical
College, Jaipur

Research Experience:
Designation Department Institution From To Total
Demonstrator Microbiology G.R. Medical 30.09.2004 Till Date 16 (y)09(m)
College, Gwalior

6. Institutional Support:
Equipment available in the sponsoring departments and laboratories
Generic Name of the Equipment Company (quantity) Year of Remarks including
purchase accessories available and
current usage of equipment
Real-Time PCR QuantStudio-5, Applied 2020, 2021 In use
Biosystems (4)
Bio-Rad CFX-96 (2) 2020, 2021
Roche Diagnostics (1) 2021
PCR Cabinate/Work Station BIOBASE (2) 2019 In use
BIOSAFTY CABINATE Type B2 Microfilt (3), BIOBASE (1) 2019, 2020 In use
Automated DNA/RNA/protein Thermoscientific (2) 2020 In use
Extraction Machine
Automated nucleic acid extraction Genolution (1) 2021 In use
machine
Milli Q water plant Millipore (1) 2020 In use
Spectophotometer Eppendorf (1) 2019 In use
Plasma Bath & Water Bath Terumo Penpol (1) 2020 In use
Deep freezers (-20°C)
 Terumo (1), Western (1) 2020 In use
Refrigerator Panasonic (3) 2020 In use
Incubator Remi (2) 2020 In use
Refrigerated Centrifuge Eppendorf (3) 2020 In use
QBD2 DRY BLOCK HEATER Grant (1) 2020 In use
Vertical Electric Autoclave Surgicoin (1) 2018 In use
ELISA reader Meril (1) 2020 In use
ELISA washer Meril (1) 2020 In use
Flow Cytometer (BD BD Biosciences (1) 2018 In use
FACSCANTO-II)
Fluorescent Microscope Leica (1) 2019 In use

Ethical
Ethical approval will be taken from Institutional Human Ethics Committee (IHEC) before initiating the research project. It is being
submitted for IHEC clearance.
DSIR certificate
DSIR certificate is in under-process.