Dessertation - Early treatment of Bloodstream Infections

Dr. B. Lal Institute of
Biotechnology
(An Exclusive Biotechnology Institute)
Early Treatment of Bloodstream Infections
Thursday, June 20, 2019 1
Submitted by:-
Shradheya R.R. Gupta
B.Sc. Biotechnology III Year
Under the guidance of:-
Dr. Yogesh Kumar Singh
Senior Scientist
Dr. B. Lal Clinical Laboratory

1. Introduction
Bloodstream infection is major cause of mortality in both
developed and developing countries.
Bloodstream infection result in sepsis, which is the major
cause in ICU patients, result in death up to 35-50% due to
late diagnosis.
This requires urgent generation of drug sensitivity reports.

2. Diagnostic Problems
 The manual methods takes
22 hours (≈1 day) for drug sensitivity .
 Delay of drug sensitivity
report lead to inappropriate treatment.
 Drug resistance
(use of broad spectrum antibiotics).
 Damage to gut flora
which increase opportunistic pathogen.
 High mortality rate.
 The automated methods are
available but they are very high in cost.

3. Proposed Solution
We proposed modified protocol for the drug profile of the
organisms.
With this modified protocol we can reduce the duration of
drug sensitivity up to 14 hours.
Which is expected to give result faster as compared to the
current protocol and, at very low cost compare to automated
methods.

4. Work Plan
Samples were screened in Bactec 9050 culture
machine
Positive sample vials were selected
A preliminary report of gram stain and
critical call out for the final report after
processing & sensitivities are done
Negative sample vials were not selected
First report will be provided after 48 hours of
receiving the samples and second report will
be provided after 5 days
Identification of microbe is done by
sample culture and biochemical tests
Positive isolates Standard strains
Antimicrobial susceptibility test is
done on Mueller Hinton Agar of
Inhibition zone is measured in every 4,6,8,10,20 and 22 hours were measured
Result were noted and compared with standard strain

4.1. Sample Culture Observation
1. Growth on Mac Conkey Agar 2. Growth on Blood Agar
4.2. Biochemical Tests Observation
Escherichia coli Pseudomonas
aeruginosa
Staphylococcus aureus Salmonella typhi
LF/NLF LF NLF - NLF
Indole + - - -
Citrate _ + - -
Urease _ + + -
TSI A/A(G) K/NC A/A K/NC*
MR/VP +/- -/- +/-,+ +/-
Catalase + + + +
Cogulase - - + -

4. 3. Antibiotic Sensitivity Observation
3. E.coli standard strain on Mueller-
Hinton agar for disk diffusion method.
4. S.aureus standard strain on Mueller-
Hinton agar for disk diffusion method.
5. Pseudomonas aeruginosa standard strain on
Mueller-Hinton agar for disk diffusion method.

Escherichia coli
Interpretation
[Sensitive(S)]
[Resistant(R)]
Sensitivity
Range(mm)
(equal or more
than)
Antibiotic (No.)
Zones in hours (mm)
4 hours 6 hours 8 hours
10
hours
20
hours
22
hours
Ampicillin 10µg (2) 20 20 20 20 20 20 S 17
Cefepime 30µg (60) 25 25 25 25 25 25 S 25
Levofloxacin 5µg
(47)
30 30 30 30 30 30 S 17
Imipenem 10µg (59) 24 24 24 24 24 24 S 23
Meropenem 10µg
(44)
26 26 26 26 26 26 S 23
Gentamycin 10µg
(19)
19 19 19 19 19 19 S 15
0
5
10
15
20
25
30
35
4 6 8 10 20 24
Ampicillin 10µg (2)
Cefepime 30µg (60)
Levofloxacin 5µg (47)
Imipenem 10µg (59)
Meropenem 10µg (44)
Gentamycin 10µg (19)
4.4. Observation
4.4.1. Standard strain:-

Pseudomonas aeruginosa
Interpretation
[Sensitive(S)]
[Resistant(R)]
Sensitivity
Range(mm)
(equal or more
than)
Antibiotics (No.)
Zones in hours (mm)
10
hours
20
hours
22
hours
Tobramycin 10µg (32) 6 6 20 20 20 20 S 15
Imipenem 10µg (59) 6 6 30 30 30 30 S 19
Ceftazidime 30µg (9) 6 6 18 18 18 18 S 18
Cefepime 30µg (60) 6 6 25 25 25 25 S 25
Piperacillin-
tazobactum 100/10µg
(40)
6 6 22 22 22 22 S 21
Meropenem 10µg (44) 6 6 23 23 23 23 S 19
0
5
10
15
20
25
30
35
4 6 8 10 20 22
Tobramycin 10µg (32)
Imipenem 10µg (59)
Ceftazidime 30µg (9)
Cefepime 30µg (60)
Piperacillin-tazobactum 100/10µg
(40)

Staphylococcus aureus
Interpretation
[Sensitive(S)]
[Resistant(R)]
Sensitivity
Range(mm)
(equal or more
than)
Antibiotics (No.)
Zones in hours (mm)
10
hours
20
hours
22
hours
Penicillin G 10U (49) 6 22 22 22 22 22 S 20
Cefoxine 30µg
(50)
6 32 32 32 32 32 S 22
Azothromycin 15µg
(54)
6 20 20 20 20 20 S 18
Erothomycin 15µg (13) 6 23 23 23 23 23 S 23
Clindamycin 2µg (55) 6 25 25 23 25 25 S 21
Doxycyclin 30µg (53) 6 30 30 30 30 30 S 16
0
5
10
15
20
25
30
35
4 6 8 10 20 24
Penicillin G 10U (49)
Cefoxine 30µg
(50)
Azothromycin 15µg (54)
Erothomycin 15µg (13)
Clindamycin 2µg (55)
Doxycyclin 30µg (53)

Salmonella typhi
Interpretation
[Sensitive(S)]
[Resistant(R)]
Sensitivity
Range(mm)
(equal or more
than)
Antibiotics (No.)
Zones in hours (mm)
10
hours
20
hours
22
hours
Ampicillin 10µg (2) 6 25 25 25 25 25 S 17
Cefepime 30µg (60) 6 30 30 30 30 30 S 25
Levofloxacin 5µg
(47)
6 28 28 28 28 28 S 17
Imipenem 10µg (59) 6 26 26 26 26 26 S 23
Meropenem 10µg
(44)
6 29 29 29 29 29 S 23
Gentamycin 10µg
(19)
6 20 20 20 20 20 S 15
0
5
10
15
20
25
30
35
4 6 8 10 20 24
Cefepime 30µg (60)
Imipenem 10µg (59)

4.4.2. Clinical strains:-
 Total samples collected – 361
 Total positive sample – 62
29 of 62 Gram-negative bacilli (46.77%) and 33 of 62 Gram-positive cocci (53.23%)
were correctly identified.
 Total negative sample – 299
Microorganism Detected Number of Positive Sample
Staphylococcus aureus 31
Salmonella typhi 9
Salmonella paratyphi - A 9
Pseudomonas aeruginosa 7
Klebsilla pneumonia 1
Escherichia coli 2
Enterococus 2
Enterobacter aerogenus 1

List of Positive Samples
Serial No. Patient ID Microorganism detected
1 CDC/172 Staphylococcus aureus
2 RSC/462 Salmonella paratyphi A
3 SMS/331 Staphylococcus aureus
4 PNC/324 Salmonella paratyphi A
6 SMS/719 Enterobacter aerogenus
7 V/2768 Salmonella typhi
8 SCH/49 Staphylococcus aureus
9 SCH/50 Staphylococcus aureus
10 M/3740 Salmonella typhi
11 P/124 Salmonella typhi
13 M/675 Staphylococcus aureus
14 M/1105 Salmonella paratyphi A
15 SCC/79 Salmonella paratyphi A
16 RPC/435 Salmonella paratyphi A
17 P/769 Staphylococcus aureus
22 M/2954 Salmonella paratyphi A
23 JHC/420 Salmonella paratyphi A
25 RSC/1104 Staphylococcus aureus
26 V/2875 Staphylococcus aureus

Serial No. Patient ID Microorganism detected
31 SHC/96 Pseudomonas aeruginosa
33 V/1897 Staphylococcus aureus
35 P/1316 Pseudomonas aeruginosa
36 P/1317 Klebsilla pneumonia
40 SCH/184 Escherichia coli
41 KHC/492 Pseudomonas aeruginosa
42 PNC/33 Salmonella typhi
45 SDC/364 Staphylococcus aureus
47 SHC/201 Staphylococcus aureus
48 HSL/155 Staphylococcus aureus
52 PBC/533 Staphylococcus aureus
54 R/927 Escherichia coli
57 CDC/190 Pseudomonas aeruginosa
58 SHC/367 Salmonella typhi
60 RPC/736 Pseudomonas aeruginosa
61 R/804 Enterococus
62 CDC/99 Enterococus fucaus

01/08/15 V-3126 Salmonella typhi
Interpretation
[Sensitive(S)]
[Resistant(R)]
Sensitivity
Range(mm)
(equal or more
than)
Antibiotics (No.)
Zones in hours (mm)
10
hours
20
hours
22
hours
Ampicillin 10µg (2) 6 22 22 22 22 22 S 17
Cefepime 30µg (60) 6 28 28 28 28 28 S 25
Levofloxacin 5µg
(47)
6 37 37 37 37 37 S 17
Imipenem 10µg (59) 6 26 26 26 26 26 S 23
Meropenem 10µg
(44)
6 31 31 31 31 31 S 23
Gentamycin 10µg
(19)
6 21 21 21 21 21 S 15
0
5
10
15
20
25
30
35
40
4 6 8 10 20 24
Cefepime 30µg (60)
Imipenem 10µg (59)

27/07/15 M/3393 Staphylococcus aureus
Interpretation
[Sensitive(S)]
[Resistant(R)]
Sensitivity
Range(mm)
(equal or more
than)
Antibiotics (No.)
Zones in hours (mm)
10
hours
20
hours
22
hours
Penicillin G 10U (49) 6 10 10 10 10 10 R 20
Cefoxine 30µg (50) 6 29 29 29 29 29 S 22
Azothromycin 15µg
(54)
6 6 6 6 6 6 R 18
Erothomycin 15µg (13) 6 7 7 7 7 7 R 23
Clindamycin 2µg (55) 6 24 24 24 24 24 S 21
Doxycyclin 30µg (53) 6 34 34 34 34 34 S 16
0
5
10
15
20
25
30
35
40
4 6 8 10 20 24
Cefoxine 30µg(50)

03/08/15 M-375 Staphylococcus aureus
Interpretation
[Sensitive(S)]
[Resistant(R)]
Sensitivity
Range(mm)
(equal or more
than)
Antibiotics (No.)
Zones in hours (mm)
10
hours
20
hours
22
hours
Penicillin G 10U (49) 6 12 12 12 12 12 R 20
Cefoxine 30µg (50) 6 18 18 18 18 18 R 22
Azothromycin 15µg
(54)
6 6 6 6 6 6 R 18
Erothomycin 15µg (13) 6 7 7 7 7 7 R 23
Clindamycin 2µg (55) 6 14 14 14 14 14 R 21
Doxycyclin 30µg (53) 6 15 15 15 15 15 R 16
0
2
4
6
8
10
12
14
16
18
20
4 6 8 10 20 24
Cefoxine 30µg (50)

5. Interpretations
All the gram-positive isolates and gram-negative isolates except
Pseudomonas ssp. had readable zones during the early observation periods
i.e. 6h. Pseudomonas ssp. lawns could be read after 8h of incubation.
By using a modified protocol, we were able to find that after 8 hours the
inhibition zone size become fixed in the range of sensitivity or resistant as
compare to 22 hours.
Protocol Day Hours
Old Second 22
Modified Same 08
Result Day Hours
Time saved Half 14

5. Interpretations
 The projected cost saving for patients who were changed to less expensive
therapy was 2,500 rupees per patient.
 If we presume that cost-effective changes and the same compliance rate would
occur in the group randomized to routine processing, then additional patients
would have benefited, with an estimated overall cost savings of 50,000 rupees.
 These cost savings in antimicrobial susceptibility, coupled with changes in
therapy made for efficacy reasons, support the usefulness of earlier reporting
of the antibiotic susceptibility patterns of blood culture isolates.
Method Cost (rupees)
Automated 3,000
Manual 500
Result Cost (rupees)
Money saved (test) 2,500

Thank You

Dessertation - Early treatment of Bloodstream Infections

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