This is the report activity of my exchange program in Kobe University Jan 9 th - Feb 2th 2018

DATE ACTIVITY
Jan 9th That was my first day in Kobe University. I entered Infectious Disease Therapy department, the
activity in this department started at 07.30, In the morning we saw and learned about patient
medical record and directly do clinical round. In the afternoon we always have conference with the
doctors and professor to discussed about patient condition and made best decision for patient.
This department divided into 2 group that lead by a doctors to handle the patient. I followed one
of the group that must check patient condition when clinical round every day.
This is the data of the patient :
Gender Assesment Treatment
Man (Noda) Pneumonia Sulbactam
Ceftazidime
Woman Infection post kidney
(ikeda/HCU transplantation
patient)
Man Prosthetic joint Vancomycin
(Nakagawa) infection (MRSA)
Woman Secondary peritonitis Meropenem
(sadakata) after colonostomy Azobactam
caused by Ca colon
70 y.o woman Rectal cancer after Ampicillin/Sulbactam
hysterectomy totalize
55 y.o woman Sub acute meningitis 12/18-19 Ampicillin
12/20-21 Ampicillin+
Multiple lung lession
Cefotaxime+
Liver function test Vancomycin

abnormality 12/21 Liposomal

Amphotericin B
12/20 INH + RFP + EB +
PZA (TB antibiotic)
12/19-21 ACV (Acyclovir)

Man Otitis externa (MRSA)

This day I choosed one of patient that I want to observed. I choosed 55 years old woman that
diagnosed with subacute meningitis with unknown etiology. She was presented to hospital in
December 20 2017 with complain of headache and prolong fever. At the first time she was
diagnosed with influenza. But her condition getting bad and she diagnosed with common fever and
 she get CT scan and MRI. From there we knew there was abnormality in her brain MR imaging
and also her lung, then subacute meningitis diagnosed. Now, she treated with Liposomal
Amphotericin B and TB antibiotic (INH + RFP + EB + PZA).
Jan 10th That day I done the activity like first day.

And this is my patient condition today :

Patient (55 y.o woman diagnosed with sub acute meningitis) condition getting better, no more
fever, but the CT Scan show ground glass opacity in her lung that means she had abnormality in
her lung.

Patient can go home if we change into oral antibiotic.

She also had History of ovarian cyst when 25 years old.

Discussion :
- The laboratory test show CSF mononuclear cell +, VCM stopped because the culture result is
negative, ADA (Adenosine deaminase) of patient isn’t high (not refer TB infection)
- The patient must continue the medication until we knew the etiology or patient condition
completely back to normal.
- Still waiting the liver culture result in about 4 weeks and PCR of the CSF (the result is faster)
- In case the patient condition improving can we let the patient go home and continue the
medication in home?
The patient can continue the medication in home if we can change the main antibiotic,
Liposomal Amphotericin B that give intravena with the other oral antibiotic. This antibiotic
function is to treat fungal infection, this patient treated as fungal infection because she had
subacute meningitis but the culture test always negative, sometimes fungal infection can
spread to another part of body included menings with no fungal in that part so the culture test
become negative. And the patient condition improved after treated with L-AMB so the doctor
continue to give it.
Jan 11th That day I done the activity like first day.

And this is my patient condition today :

Clinical appearance of patient is good, no fever
She had lumbar punction to check beta-D-glucan (to check however she had fungal infection).
The result of Cryptococcus neoforman culture: -
 Discussion :
- In case if will check the therapeutic response for cryptoccosis just check the opening pressure
from lumbal punction. Normal value is 3-4 cm if meningitis it can reach until 10cm.
- The clinical laboratory result show the abnormality of her red blood cell that always decrease,
so we suspicious that she had autoimune disease not infection, but before that we must check
her CT, MR and also the level of beta D-glucan (is attractive antigen for broad range of
fungal). If the all components is normal we can stop the antibiotic.
- She must continue her medication until the result done.
Jan 12th That day I done the activity like first day.

And this is my patient condition today :

- Clinical appearance of patient is good, no fever
- She had Brain MR Examination, the result have same consolidation with the MR result in
20/12/2017.
- She had Aorta CT, the result is she had some mass near her sacral vertebrae

Lab
CRP : [11/1/18] 0,48 (n= 0,00-0,14)
AST : [11/1/18] 30 (n= 13-30)
ALT : [11/1/18] 45 (n= 7-23)
Gamma GTP : [11/1/18] 116 (n= 9-32)
ALP : [11/1/18] 376 (n= 106-322)
eGFRcreat : [11/1/18] 72.7 (< 60)
Beta D glucan : [12/1/18] 64 (N= 0-11)

Discussion
- The consolidation show the abnormality of her brain but the condition still same as her first
brain MR, that means the treatment just improve her clinical condition not the lesion.
- The Beta D Glucan value is more than normal, but with that value we can’t diagnosed as fungal
infection, we need more >60. And in fact, Beta D Glucan very specific to detect PCP.
- She must continue her medication
Jan 15th That day I done the activity like first day.

And this is my patient condition today :

 - Clinical appearance of patient is good, no fever
- In 14/01/18 her headache is improving
- In 15/01/18 Her eGFRcreat decrease again, may be caused by L-AMB or TB antibiotic (mainly
rifampicin)
- She still continue the medication

Discussion
- If the renal function is getting low the first we must stop is L-AMB and then if still decrease
we must change rifampicin with other drug.
- Why she gave TB antibiotic even though she has no history of TB infection?
Because in japan the main causes of subacute meningitis are :
1. TB infection
2. Bacterial infection
3. Outoimune
4. Others
And for extra pulmonal TB the duration of therapy is 8-12 month
Jan 16th That day I done the activity like first day.

And this is my patient condition today :

- Clinical appearance of patient is good, no fever
- She had high blood pressure before today, and it started improving yesterday. This is the data
:
[20/12/17] T = 37,8 | BP = 164/71
[21/12/17] T = 38,2 | BP = 188/74
[22/12/17] T = 38,7 | BP= 153/74
[23/12/17] T= 37,8 | BP = 184/87
[14/01/18] T= 36,4| BP = 140/76
[15/01/18] T= 36,6| BP = 120/70
[16/01/18] T= 36,4| BP = 120/62

Discussion
Not stable blood pressure in old people is normal condition (?)
Jan 17th That day I done the activity like first day.

And this is my patient condition today :

 - Clinical appearance of patient is good, no fever
- Suspected collagen disease
Main doctors have differential diagnose :
 Ovaryan teratoma
 Presacral teratoma
 Meningocele
 Curranino syndrome

Discussion

High beta D glucan is false positive can be caused by L-AMB

The diagnosis of Neuro-bachet lupus

Jan 18th That day I done the activity like first day.

And this is my patient condition today :

- Clinical appearance of patient is good, no fever

She will have MR test again for her brain and teratoma to make sure the curanino syndrome
diagnosis or other diagnosis

Vital sign
BP = 105/69
T = 36,1
HR = 98
RR= 18

CSF
Cell count = 17
Monosite = 98
PMN = 2
Protein = 76
Glucose = 64
 Beta -2- mikroglobulin = 2210
ADA = on process
Cryptococcus = on process

Blood glucose =94

MR result show hyperdensity mass near sacral vertebra the size is 71,72 x 80,29 x 86,90 mm
Jan 19th That day I done the activity like first day.

And this is my patient condition today :

- Clinical appearance of patient is good, no fever

Around 6 o'clock

The result of blood test to check collagen disease

IgG = 1303 (n= 861-1747 mg/dl)
IgA = 246 (n= 93-393 mg/dl)
IgM= 126 (n= 50-269 mg/dl)

Cylothorax -
Hemolisis -

Lupus anticoagulant on process (n=0-1.299)

M-protein on process
IgG4 on process
Clinical Virology Department
th
Jan 22
Jan 23th Protein purification (23-1-2018)
Sample :
- vero old  from monkey
- vero new  from monkey
- 293  from human

 Put @2ml sample to small tube

 Centrifuge it in 4oC, 5000 rpm, 1 minutes
 Take the supernatant
 Prepare reagent Na2HPO4 and Nickle-NTA agarose beads
 Add @100ul Na2HPO4 to each 2ml tube, 500 ul to 293 in 10ml tube (big), and 550 ul to vero
new in 11ml tube (big)
  Add @40ul Ni-beads to each 2ml tube and 200 ul to 293 in 10ml tube (big) and vero new in
11ml tube (big)
 Mix
 Rotate overnight

Jan 24th Continuing purification

 Prepare the ice bowl to put sample

 Centrifuge the sample (Vero new, vero old, 293) in 4oC, 5000 rpm, 1 minutes
 Take the supernatant and place it in the different tube and take the nickel beads in the
different tube
SDS PAGE
 Making separation gel
 Making stacking gel
 Prepare the sample for SDS PAGE
The sample for SDS PAGE must be in small volume and add with (yg biru2).
 SDS PAGE process
Install the power supply at 120V, Amax, wait until 20 minutes and after that we can
increase the voltage to make the process faster. The proses finished when the sample reach
1/3 part of the gel bottom.
 Staining
We do Coomassie Brilliant Blue (CBB) and Western Blot (WB) staining in this
experiment. But, the western blot do tomorrow. The differences between the 2 process of
staining are the western blot more specific because in WB we do blocking process and give
it antibody to block another protein, so the protein that we want can show clearly (but,
sometimes the contamination can occur) and in WB process we just need shorten time than
if we do CBB that need overnight incubation if we want the clear result.
th
Jan 25 We do western blot staining
 Prepare the gel, transfer membrane, filtration paper and the device
 Transfer process
Run the transfer unit power supply at 15V in 1 hour (don’t take a long time, it can makes
the device burn)
 Blocking step
 After the transfer process, Incubate with shake and cover the transfer membrane in the
blocking buffer that made from skim milk and PBST in 1 hours. And add it with primary
antibody to........ and shake it for 1 hour.
After that the process continue with give it secondary antibody (but before that remove and
wash the solution of primary antibody)
 Prepare the Application for see the result
Before we read it, add the filtration membrane with substrate to increase the signal.
Jan 26th We do CBB and western blot with the result from HPLC peak (peak 1, peak 2, peak 3), the same
sample as yesterday, vero and 293 sample that made by another researcher. The CBB result show
that :
1. Peak 1 : didn’t show protein
2. Peak 2 : we can see the protein that we want, there are : gH, gQ1, gL,gQ2 (tetramer)
3. Peak 3 : in normal condition the protein shouldn’t be appear, maybe it’s contamination.
4. Control: saw the protein appear
5. 12/24 293: we can’t see tetramer
1/24 vero before protease : tetramer appear with the background from some protein
6. 1/25 vero after protease : tetramer appear with the background from some protein
7. 1/25 vero after protease : tetramer appear with the background from some protein

The western blot result :

1. Peak 1 : didn’t show protein
2. Peak 2 : we can see the protein that we want, there are : gH, gQ1, gL,gQ2 (tetramer)
3. Peak 3 : in normal condition the protein shouldn’t be appear, maybe it’s contamination.
4. Control: saw the protein appear
5. 293 elution :
6. Vero old elution :
7. Vero new elution :
8. 12/24 293: we can’t see tetramer
9. 1/24 vero before protease : tetramer appear with the background from some protein
10. 1/25 vero after protease : tetramer appear with the background from some protein
11. 1/25 vero after protease : tetramer appear with the background from some protein

Jan 29th We do transfection with CaCl2 method to make the plasmid that contain CD134 (HHV6B receptor)
entered the eukaryote cell.
 We also change the medium of another sample. If the sample adhere we must be careful when suck
it up, just suck it from the wall don’t touch the base of plate that contain many cell. But if the
sample isn’t adhere we can suck it freely, it doesn’t matter because we want to throw away the old
cell and propagate the new cell in the new medium from the remaining cells.
The other things that we do is collect Ni-NTA agarose Beads. Ni-NTA agarose Beads is a nickel-
charged affinity resin that can be used to purify recombinant proteins containing a polyhistidine
(6xHis) sequence. Proteins bound to the resin may be eluted with either low pH buffer or by
competition with imidazole or histidine. The first step to collect it is washing process that divided
into fast and slow process. In the fast process we used vacum device. After washing process we
must do ellution process to separate the Ni-NTA beads from the protein.
Jan 30th We continue to change the medium, but now we put the old medium that contain the cell and drop
it into 5L (293-). After that we add Ni-NTA beads to bind the protein and stir it overnight.
We also continue the transfection process. After we mix luria bertani medium with CBPC we must
check the density to know about bacteria growth. The normal value 0,6-0,7. If the density is more
than that normal value we can't use it for the experiment.
And we continue the process after collect Ni-NTA beads, now we do SDS PAGE to check the
protein.
Jan 31th We continue transfection process, now we collect the pellet and freeze it in -80C
We also make luria bertani medium that consist of DMEM, L-glutamin, gentamicin, FBS 8% for
nutrition, and NaHCO3
Today we also learn about HPLC (High-performance liquid chromatography), its a technique in
analytical chemistry that used to separate, identify, and quantify each component in a mixture. It
relies on pumps to pass a pressurized liquid solvent containing the sample mixture through a
column filled with a solid adsorbent material. Each component in the sample interacts slightly
differently with the adsorbent material, causing different flow rates for the different components
and leading to the separation of the components as they flow out the column.
Feb 1th We collect the highest peak fraction from HPLC result and do SDS PAGE to check the protein on
that peak fraction.
We also continue the transfection process (now we do purification step), the first that we must do
is making lysis buffer from tris HCL pH 7,4 , NaCl, EDTA, and Tryton X(to lysis the eucaryote
cell wall) that mix together.
Feb 2th We collect the supernatan from the transfection plate (The medium or supernatan collected in the
day 3 and 4) and to check the success of the transfection process we do IFA (Immunofluorecent
Assay).