FIELD OF INVENTION:
The present invention relates to the field of medical diagnostics of bacterial infections. The invention provides a rapid and efficient method to isolate bacteria from whole blood and simultaneously culture them for further diagnostic tests.
BACKGROUND OF INVENTION:
Specific and accurate Identification of the bacteria is a very crucial step in disease diagnosis and treatment. There are a number of culture-dependent and independent methods available for isolation and identification of pathogens in clinical samples such as ELISA, FISH, Magicplex etc.
Isolation and culture are vital steps and majority of pathogenic bacteria have been grown on artificially provided media and hence today, blood culture is considered as the gold standard for isolation, identification, and diagnosis of bloodstream pathogens. Though the Turn Around Time for culture dependent procedures is typically 24-72 hrs, this is mainly because of the complicated pre-analytical processing, the requirement of relatively large numbers of viable organisms, limited organism spectrum, analytical variability and/or cost. It is clearly depicted that though the rapid procedures for pathogen identification and antimicrobial susceptibility/resistance profiling are available (for example FISH, PCR and qPCR), the culturing of pathogen to attain detectable density of bacteria has been the major bottleneck in the process as it takes significant time (at-least 6 hrs for liquid culture and 36 hrs for solid culture).
The ultimate time to result guiding treatment choices causes delay and less than optimal treatment outcome and causes emergence of rapid antimicrobial resistance. However, regardless of these disadvantages, culture is still the gold standard for evaluating/isolating bacterial pathogens, as it allows carrying out antibiotic susceptibility testing, antigenic & genetic studies, experimental models etc.
DETAILS OF THE INVENTION:
During antimicrobial susceptibility testing, sensitivity of blood culture is impacted by the pause from blood taken to transfer sample to blood culture bottles and into the apparatus and further delay is caused by the need to grow bacteria in growth media over extended period of time to yield single colonies for Identification and susceptibility testing. In the case of severe infections, such as sepsis, percentage of recovery gets negatively affected (by -7.6%) every hour if

the patient is not treated with appropriate antibiotics immediately and hence these assays are not the appropriate diagnostics for the purpose.
Shortening the time to isolate and culturing of bacteria will facilitate proper diagnosis of the disease. Thus guided clinical decision making and this will contribute to the reduced burden of hospitalization time and cost that will result in the decline of mortality rate.
Consequently, improved rapid methods for isolation and culture of bacteria are essential for prompt diagnosis of the disease and rapid treatment. Therefore, in this proposal, we aim to develop a rapid, simple and affordable device that will allow isolation of bacteria from whole blood sample. The enriched media provided in the device will enhance their growth in order to achieve required cell-density in culture cartridge in a shorter period of time. These cultures could be utilized for further diagnostic procedures for identification of bacteria and/or susceptibility profiling purposes.
In this innovation, we hypothesized that every cell has a specific size thus we can discriminate bacteria from mammalian cells using their cell size as a biomarker. Therefore, we propose to use filters of different pore sizes to separate bacteria and other components of blood. As shown in the figure, the device is proposed to have series of filters which are as given below
□ filter-1 (size 10|im): separate neutrophils, eosinophils, basophils and
monocytes
D filter-2 (size 6|im): separate erythrocyte and lymphocyte
□ filter-3 (size 0.5|im): separate bacteria and platelets
Thus, serial filtration process will allow isolating and concentrating bacteria in a defined compartment in the cartridge and remaining cell-free sample will be collected in attached sample tank for further usage. This chamber of the cartridge will also have crystallized form of enriched growth media to facilitate the growth of bacteria in short period of time, once appropriately incubated. These cultured bacteria can be further utilized for pathogen identification and/or antimicrobial susceptibility testing.
Despite efforts to reduce the chances of contamination during blood transfer and culturing process, contamination of blood cultures continues to be a significant clinical problem. Whereas this device is simple and syringe compatible

thereby minimize the contamination risk and simultaneously culturing will be done in the device itself, reduce the requirement of additional culture accessories and procedure.
The device would have following advantageous characteristics over existing solutions
• Rapid isolation and culture of pathogenic bacteria; less TAT (<90 min)
• Spectrophotometer compatible for easy & digital read-out
• Syringe compatible, biosafe
• Sample fractions compatible for follow-on assays/molecular assay
• Easy sample/device transport
• Low-resource requirement
• Affordable (approximately 50-150 INR)
SUMMARY OF INVENTION:
The present invention provides a novel device for easily and rapidly isolating and culturing of bacteria from whole blood. The device could be utilized to facilitate rapid identification and analysis for bacterial specimens in the clinical samples. The process would require <1 hours. The inventors more specifically propose to isolate bacteria from sample and allow them to grow/synchronize for a short-period of time (<90 min) and use them for further diagnostic procedures.
DESCRIPTION OF THE ACCOMPANYING DRAWINGS:
Figure 1. Shows initial protype: A. Complete device; B. description of different sizes filters; C. Cartidge and D. Cell free sample tank, according to an embodimemt of the present invention.
DETAIL DESCRIPTION OF INVENTION:
The present invention provides more particularly a rapid and affordable method for isolation and culturing of bacteria for clinical samples. The method involves separation of different blood components with the help of different filters present in cartilage and culturing of bacteria in isolated chamber provided with growth medium.
It will be apparent to those skilled in the art that various modifications and variations can be made in the device and methods of the present invention without departing from the spirit or scope of the invention. Thus, it is intended that the present invention cover the modifications and variations of this invention provided they come within the scope of the appended claims and their

equivalents. Additionally, the following examples are appended for the purpose of illustrating the claimed invention, and should not be construed so as to limit the scope of the claimed invention.

We Claim:
1. A device capable of rapidly isolating and culturing bacteria from whole
blood sample therein, comprising:
(a) a tank for collecting sample
(b) a syringe compatible sample inlet and
(c) a syringe with filters.

2. A device as claimed in claim 1, wherein tank for collecting sample includes a cork plug for facilitating air exchange.
3. A device as claimed in claim 1, wherein syringe with filters includes preferably 3 filters.
4. A device as claimed in claiml and claim 3, wherein filters are of size 1 -10 |im, 2-6 |am and 3 - 0.5 |xm.
5. A device as claimed in claim 1, wherein the device requires less than one hour to isolate and culture the bacteria from whole blood cells.
6. A device as claimed in claiml and claim 3, wherein filter 1 separates neutrophils, eosinophils, basophils and monocytes.
7. A device as claimed in claiml and claim 3, wherein filter 2 separates erythrocyte and lymphocyte.
8. A device as claimed in claiml and claim 3, wherein filter 3 separates bacteria and platelets.