Infection and Immunity

I- INTRODUCTION:

The patient in the case is a 21-year old Asian female that is diagnosed to

have acute myeloid leukaemia. The female was transferred to a haematological unit

to be treated from the acute leukaemia. She started a full intensity conditioning

treatment so that she can be capable of having an allogeneic stem cells transplant

that has both chemotherapy and total body irradiation. The female also conditioned

broad-spectrum antibiotics so she will have a reduced infection in the respiratory

tack, and as a result she’ll have a lower fever that makes her capable of doing the

stem cells transplant. The patient then showed a neutropenic fever for four days that

didn’t respond to treatment. The infection caused other symptoms such as having

shortness of breath, coughing blood and chest pain. The patient’s recovery progress

after the stem cells transplant, was monitored by Full Blood Counts (FBC) and in

specific the Absolute Neutrophil Count (ANC) which recorded 0.3 x109/L which is quite

lower than the normal range that is in the range of 1.5x109/L to 8x109/L. In the lab, many

tests were performed such as Isolation and identification of microorganisms,

Identification of suspicious colony on blood agar, Identification of suspicious colony

on SDA, Chest CT scan, Nucleic acid detection, and ELISA for detection of

galactomannan antigen. In this lab report, the sequence of events will be discussed

from the specimen reception, diagnosis, treatment and infection control. It will also

include analysing the results and discussing them to reach clear conclusions.

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 II- Methods:

The tests were performed according to the schedule provided, and the tests

included are isolation and identification of microorganism’s test, identification of

suspicious colony on blood agar, optochin sensitivity test, Gram staining,

identification on SDA, wet preparation of colonies, chest CT scan, polymerase chain

reactions (PCR) test, Agarose gel electrophoresis test, and ELISA for detection of

galactomannan antigen test.

III- Results:

 In the isolation and identification of microorganism’s test, a sputum sample

was taken from the patient, and as a result a suspicious colony was detected

on SDA whereas no suspicious colonies were detected on chocolate agar.

 In the identification of suspicious colony on blood agar, the suspicious colony on

blood agar was subculture onto a new blood plate that was then incubated at 37 °C.

As a result, small rounded structures that have a brown colour showed and other small

white structures that have a circular form too. Beta hemolysis showed around the

colony as shown in figure 1.1 below.

Figure1.1: Macroscopic appearance on blood agar.

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 According to the optochin sensitivity test that was carried, the microorganism

showed to be   to optochin with no zone of inhibition as shown in figure1.2

below.

Figure 1.2: Results of optochin (P) sensitivity test.

 In the Gram staining, the cells are tiny and rounded, and shown to be aligned

and having a dark blue as shown in figure 1.3 below.

Figure 1.3: Results of Gram staining.

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 The suspicious colony on SDA was subculture onto a new SDA plate for

purification and identification. As a result, a large circular structure showed on

the plate, having a grey colour in the middle and white from the exterior, as

shown in figure 2.0 below.

Figure 2.0: Macroscopic appearance on SDA.

 The wet preparation of colonies grown on SDA that was carried out resulted in

showing fungus. Shown in figure 3 below:

Figure 3.0: Structures seen by wet preparation from SDA (Magnification x400)

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 In the pulmonary computed tomography scan that was performed, the results

show that the lung cavity that is pointed at with an arrow in figure 4.0 below is

infected.

Figure 4.0: Results of chest CT scan.

 Total DNA was extracted from the whole blood of the patient, and the

polymerase chain reactions (PCR) test was done to fine if the nucleic acid of

Aspergillus fumigatus is present. According to table 1.0 shown below, the

results show that Aspergillus fumigatus is present since it is positive.

Table 1.0: Details of PCR reactions

PCR reaction # PCR reaction

1 Day 1 blood sample

2 Day 2 blood sample

3 Day 3 blood sample
4 Day 4 blood sample
5 Negative control

6 Aspergillus fumigatus positive control

 Agarose gel electrophoresis was performed on PCR products, a strand is

shown for the samples on days 1,2, 4 and 6 which indicates that it is positive

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 and Aspergillus is present, as shown in figure 5.0 below .

L 1 2 3 4 5
6

100bp

Figure 5.0: Gel electrophoresis results from the PCR reactions.

 In the ELISA for detection of galactomannan antigen test, Serum samples

from the patient were obtained on two consecutive days to screen for

detection of galactomannan antigen. As a result the test showed yellow

markers at A1-A6, B1-B6 , A7-A12,B7-B12, and +CTR for both days, as

shown in figure 6.0 below.

Patient
A1-A6 Patient
B1-B6 A7-A12
B7-B12

+ CTR + CTR
 F1-F6

- CTR - CTR
G1-G6 G7-G12
H1-H6 H7-H12

Figure 6.0: Results of the ELISA for galactomannan

IV- Discussion:
The disease diagnosed in this case is Aspergillus fumigatus. In order to diagnose

this disease, many tests should be taken into consideration such as taking a sputum

sample and culturing it to detect if the disease is growing, then perform a chest x-ray

in order to detect if there are any signs of being infected by this disease such as

aspergillomas, having a blood test to detect any presence of antibodies

to Aspergillus in the blood flow, PCR test, skin or other blood tests to make sure that

there is an allergy to Aspergillus spores, and tests that are necessary to detect

fungal cell wall of fungal species.

According to the given results, the identification of suspicious colony on blood

agar showed the presence of the disease, and further tests such as subculturing the

suspicious colony on a new SDA plate that showed the growing of the disease, and

wet preparation of colonies grown on SDA showed clearly the fungus. The PCR test

showed the patient being tested positive for the disease, and the GEL

electrophoresis test also showed clearly the strands on each test, and this indicates

that the patient has the disease. All these tests performed helps to identify and

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diagnose accurately the disease.

Patients having this disease can predict having it since it shows many

obvious symptoms. As long as the disease is limited inside the lungs, the patient will

experience high fever, hard cough, coughing up blood, difficulty in breathing, chest

pain that gets worse while taking deep breaths. Nevertheless, when the infection

spreads outside of the lungs symptoms vary depending the part of the body that is

infected. Generally, common symptoms include headaches, swollen eyes,

nosebleed, joint pain, lesions on the skin, difficulties with speech, confusion and

seizures

For treatment, oral corticosteroids can sometimes be used. Patients are

recommended to take corticosteroids in combination with antifungal medications

such as itraconazole. Some patients that don’t show any symptoms do not require

any treatments even though they have the infection. Nevertheless, it is always

preferred to check up and get a treatment so the infection doesn’t spread up and the

symptoms starts showing. There are many examples of medications that are

considered effective such as voriconazole, itraconazole, and amphotericin B.

To prevent such infections, some steps can be followed especially if a person

is at a group that could transfer the infection. The first step is to prevent doing any

type of activities that is in contact with Aspergillus species. The second step if being

infected is take prophylactic antifungal medication, and to test directly if having any

symptoms of the infection.

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