Chapter 4 AEROBIC GRAM NEGATIVE BACTERIA

Chapter 4            AEROBIC GRAM NEGATIVE BACTERIA

Bordetella pertussis

Morphology

Small, ovoid coccobacillus

Characteristics

·         Gram negative bacteria

·         Aerobic bacteria

·         Non-motile

·         Non-sporing

·         Capsulated

Biochemical reactions

Oxidase and catalase positive

Toxins

·         Pertussis toxin (PTx)

·         Adenylate cyclase (CyaA)

Disease caused

Pertussis or whooping cough

Mode of transmission

Airborne droplets

Incubation period

7–14 days

Common symptom

The patient might make a “whooping” sound when breathing in or vomit

Pathogenesis

1.    Infects its host by colonizing lung epithelial cells

2.    The bacterium contains a surface protein, filamentous hemagglutinin,

         which binds to sulfatides that are found on cilia of epithelial cells.

3.    Once anchored, the bacterium produces tracheal cytotoxin, which stops the cilia from beating.

4.    This prevents the cilia from clearing debris from the lungs,

         so the body responds by sending the host into a coughing fit.

5.    These coughs expel some bacteria into the air, which are free to infect other hosts.

Hemophilus influenza

Morphology

Gram-negative coccobacillus

 

Characteristics

·         small (1 µm X 0.3 µm)

·         pleomorphic

·         Nonmotile

·         non–spore-forming

·         fastidious

·         facultative anaerobe

Biochemical reactions

Nitrates are reduced to nq8uj

Pathogenicity

Normally inhabits the nasopharynx and tonsillar region in many normal persons.

Mode of transmission

Direct contact or by inhalation of respiratory tract droplets

Incubation period

Unknown

Laboratory diagnosis

Sample : CSF, sputum, nasopharyngeal swab

Laboratory test :

Direct

-       Coagulation slide test

-       Latex agglutination slide test

-       Quelling reaction

Direct fluorescent antibody test

Centrifuge

-       Inoculate the deposits

Blood

Inoculate on thioglycollate broth

Tryptone soya broth

Add 3ml blood to 15 to 30 ml molten nutrient agar and pour into petridish.

Incubate under 36^oC. Under moist CO₂ atmosphere

Other samples

Inoculate on horse blood agar (with commercially avaibale V and X factor impregnated paper disc

Chocolate agar (with commercially avaibale X factor  impregnated paper disc

Chapter 17 : Serology Test

ASOT test

Definition

Antistreptolysin O (ASO) titer is a blood test to measure antibodies against streptolysin O, a substance produced by group A Streptococcus bacteria.

Alternative Names

ASO titer; ASLO

Introduction

The ASO test is primarily ordered by itself or along with an anti-DNase B to help determine whether a person has had a recent streptococcal infection. In most cases, strep infections are identified and treated with antibiotics and the infections resolve. In cases where they do not cause identifiable symptoms and/or go untreated.

This test is important in the investigation of poststreptococcal disease. Most complications develop at a stage when it is not possible to isolate streptococci (Group A) in culture. In 80% – 85% of the patients with rheumatic fever there is a rise in ASO antibody, which is highest soon after the onset of the disease. Infection with group ‘C’ and group ‘G’ streptococci can also produce a rise in ASO.

Streptolysin-O is a type of exotoxins produced by β-haemolytic group A Streptococcus. People infected with β-haemolytic group A Streptococcus will produce specific antibodies against the streptococcal exotoxins. Antistreptolysin-O Test (ASOT) is a rapid latex particle agglutination test that uses latex reagent, a suspension of uniform sized polystyrene latex particles coated with streptolysin-O, which allows visual observation of the antigen-antibody reaction. Presence of antistreptolysin-O in the serum will result in agglutination of the latex particles.

ASOT test kit

Why the test is performed?

This test is used to detect prior infection by group A Streptococcus, the bacteria responsible for diseases such as:

• Bacterial endocarditis
• Glomerulonephritis
• Rheumatic fever
• Scarlet fever
• Strep throat

The ASO antibody may be found in the blood weeks or months after the strep infection has gone away.

Normal Results

Normal value ranges may vary slightly among different laboratories. Talk to your doctor about the meaning of your specific test results.

What abnormal results mean?

Abnormal results may be due to:

• Active streptococcal infection
• Bacterial endocarditis
• Post-streptococcal glomerulonephritis
• Rheumatic fever
• Scarlet fever

Principle

The qualitative slide agglutination test. The reagent contains an aqueous suspension of polystyrene latex particles which are sensitized with streptolysin O. These particles agglutinate in the presence of ASO present in patient serum.

Requirement

·         Latex ASO reagent kit

·         Positive and negative serum controls

·         Normal saline

·         Test plate or dark slide

·         Patient’s serum

Procedure

1.    Bring reagents and samples to room temperature.

2.    Dilute patient serum 1:5 with normal saline.

3.    Place one drop each of diluted patient serum and positive and negative control sera on the respective zones of a test plate.

4.    Add one drop of latex ASO reagent to each of these drops.

5.    Mix well with stirring rods and look for agglutination within 2 minutes.

Interpretation

Marked agglutination indicates the presence of ASO. The results are reported in Todd units/ml or international units(IU). ASO titres higher than 200 Todd units/ml are indicative of prior streptococcal infection. For quantitative test prepare further dilutions of serum (1:10,1:20, 1:30, 1: 40) which indicates ASO concentration 400, 800, 1200 and 1600 Todd units/ml respectively.

     

Interpretation

Uses

1.    Simple to perform

2.    Rising titre indicates active and progressive streptococcal infection

3.    Declining titre indicates recovery of the patient.

ANA test

Definition

Anti-nuclear antibodies (ANAs, also known as anti-nuclear factor or ANF) are auto-antibodies directed against contents of the cell nucleus.

Alternative names

Fluorescent Antinuclear Antibody; FANA

Introduction

The ANA test was designed by Dr. George Friou in 1957. The ANA test is performed using a blood sample. The antibodies in the serum of the blood are exposed in the laboratory to cells. It is then determined whether or not antibodies are present that react to various parts of the nucleus of cells. Thus, the term anti-"nuclear" antibody. Fluorescence techniques are frequently used to actually detect the antibodies in the cells, thus ANA testing is sometimes referred to as fluorescent antinuclear antibody test (FANA). The ANA test is a sensitive screening test used to detect autoimmune diseases.

ANA test kit

The ANA test is ordered when someone shows signs and symptoms that are associated with SLE or another autoimmune disorder. It may also be ordered when a person has been diagnosed with an autoimmune disorder and the doctor suspects that she may have developed an additional autoimmune disorder. Those with autoimmune disorders can have a wide variety of symptoms such as low-grade fever, joint pain, fatigue, and/or unexplained rashes that may change over time.

Taking these drugs may affect test results

• Acetazolamide, Aminosalicylic acid (PAS).
• Chlorothiazide,Chlorpromazine, Chlorprothixene, Clofibrate.
• Ethosuximide.
• Gold salts, Griseofulvin.
• Hydralazine.
• Isoniazid.
• Mephenytoin, Methyldopa, Methysergide.
• Oral contraceptives.
• Para-aminosalicylic acid, Penicillin, Phenylbutazone, Phenytoin, Primidone, Procainamide, Propylthiouracil.
• Quinidine.
• Reserpine.
• Streptomycin, Sulfonamides.
• Tetracyclines, Thiouracil, Trimethadione.

Purpose of test

·         Screens for systemic lupus erythematosus. (SLE)

·         Monitors effectiveness of treatment with drugs for systemic lupus erythematosus.

Interpretation

ANA tests are performed using different assays (indirect immunofluorescence microscopy or by enzyme-linked immunoabsorbant assay, ELISA) and results are reported as a titer, often with a particular type of immunofluroscence pattern (when positive). Low-level titers are considered negative, while increased titers, such as 1:320, are positive, indicating an elevated concentration of antinuclear antibodies.

ANA shows up on indirect immunofluorescence as fluorescent patterns in cells that are fixed to a slide that is evaluated under a microscope. Different patterns have been associated with a variety of autoimmune disorders, although overlap may occur. Some of the more common patterns include:

·            Homogenous (diffuse) - associated with SLE and mixed connective tissue disease

·            Speckled - associated with SLE, Sjogren syndrome, scleroderma, polymyositis, rheumatoid arthritis, and mixed connective tissue disease

·            Nucleolar - associated with scleroderma and polymyositis

·            Centromere pattern (peripheral) - associated with scleroderma and CREST (Calcinosis, Raynaud's syndrome, Esophogeal dysmotility, Sclerodactyly, Telangiectasia)

Interpretation

How is the ANA titer determined?

A titer is determined by repeating the positive test with serial dilutions until the test yields a negative result. The last dilution which yields a positive result (fluorescence) is the titer which gets reported. For example, if a titer performed for a positive ANA test is:

1:10 positive
1:20 positive
1:40 positive
1:80 positive
1:160 positive
1:320 negative

Uses

Sample handling : urine

(a)        Method of collection

(b)        Method of transport

(c)        Types of preservatives used

(d)        Common type of pathogen found in urine

Sample handling : sputum

Collection

1.    Give the patient a sterile wide-necked, leak-proof container

2.    Patient to be advised that he or she should cough deeply and collect sputum sample (not saliva) in the morning and before using mouthwash or tooth-brush

3.    If sputum sample is very little or patient cannot cough out the sputum, it may be possible to collect a satisfactory sample by placing the patient comfortably so that his head and shoulder are lower than his or her chest. If he or she remains thus for about ten minutes sputum may drain into trachea and then the patient will be able to cough it out.

4.    Sputum should be collected upon rising in the morning as overnight incubation and growth of fungi in the lungs will increase the likelihood of isolating pathogenic fungi.

5.    Patients should not eat before specimen collection.

6.    Twenty-four hour samples are unacceptable because they become overgrown with bacteria and fungal contaminants.

Transport

1.    Specimens must be sent to the laboratory and processed as soon as possible, a delay of longer than two hours at room temperature may impede the detection of some fungi.

2.    Store at 4C if short delays in processing are anticipated.

3.    If pneumonia or bronchopneumoniae or M. tuberculosis is suspected, collect purulent part of sputum on a sterile cotton swab and insert in Amie’s transport medium.

4.    If pneumonic plague or mycobacterium is suspected, sample should be marked as HIGH RISK and transported in Cary-Blair medium.