Diagnosis of Infectious Disease

Infectious diseases are caused by microorganisms, such as bacteria, viruses, fungi, and parasites .

Doctors suspect an infection based on the person's symptoms, physical examination results, and risk factors. First, doctors confirm that the person has an infection rather than another type of illness. For example, a person with a cough and difficulty breathing may have pneumonia (a lung infection). However, the person may have asthma or heart failure. In such a person, a chest x-ray can help doctors distinguish pneumonia from the other possible disorders.

Once doctors confirm that the person has an infection, they usually need to know which specific microorganism is causing the infection. Many different microorganisms can cause a given infection. For example, pneumonia can be caused by viruses, bacteria, or, rarely, fungi. The treatment is different for each microorganism.

Many different types of laboratory tests can identify microorganisms. Laboratory tests use a sample of blood, urine, sputum, or other fluid or tissue from the body. This sample may be

• Stained and examined under a microscope
• Cultured (placed in conditions that encourage the growth of microorganisms)
• Tested for antibodies, produced by the person's immune system in response to the microorganism
• Tested for a microorganism's antigens (molecules from the microorganism that can trigger an immune response in the body)
• Tested for genetic material (such as DNA or RNA) from the microorganism

No single test can identify every microorganism, and tests that work well for one microorganism often do not work well for another. Doctors must choose the test based on which microorganisms they think are most likely to cause a disorder.

Sometimes several different tests are done, typically in a specific order, based on the results of the previous test. Each test further narrows the possibilities. If the right test is not done, doctors may not identify the cause of infection.

Some samples sent for testing, such as sputum, stool, and swabs from the nose or throat, normally contain many types of bacteria that do not cause disease. Doctors distinguish between these bacteria and those that could cause the person's illness. Other samples come from areas that normally do not contain any microorganisms (that are sterile), such as urine, blood, or cerebrospinal fluid (the fluid that surrounds the brain and spinal cord). Finding any bacteria in such samples is abnormal.

When a microorganism is identified, doctors can then do tests to determine which drugs are most effective against it (susceptibility tests), and effective treatment can be started sooner.

STAINING AND EXAMINATION USING A MICROSCOPE

Doctors sometimes can identify a microorganism simply by looking at it under a microscope.

Most samples are treated with stains. Stains are special dyes that color the microorganisms, causing them to stand out from the background. Some microorganisms have a distinctive size, shape, and stained color that enable doctors to recognize them.

However, many microorganisms look alike and cannot be distinguished using a microscope. Also, there must be enough of them, and they must be large enough to be seen with a microscope. For example, viruses cannot be identified using a microscope because they are too small.

For bacteria, doctors often first use Gram stain (a violet-colored stain). Bacteria are classified as follows:

• Gram-positive (they look blue because they retain the violet Gram stain)
• Gram-negative (they look red because they do not retain the stain)

Doctors can make some treatment decisions based on whether bacteria are gram-positive or gram-negative.

In addition to Gram stain, other stains can be used depending on the microorganisms thought to be present.

CULTURE OF MICROORGANISMS

Usually, a sample contains too few microorganisms to see using a microscope or to be identified using other tests. Thus, doctors usually try to grow (culture) the microorganism in a laboratory until there are enough to identify. A sample is taken from an area of the person's body likely to contain the microorganism. Samples may include

• Blood
• Sputum
• Urine
• Stool
• Tissue
• Cerebrospinal fluid
• Mucus from the nose, throat, or genital area

The sample is placed on a dish or in a test tube that contains specific nutrients to encourage growth of microorganisms. Different nutrients are used depending on which microorganisms doctors suspect. Often, doctors add substances to the dish or test tube to stop the growth of microorganisms that do not cause the disease doctors suspect.

Many microorganisms, such as the bacteria that cause urinary tract infections or strep throat, can easily be grown in a culture. Some bacteria, such as the bacteria that cause syphilis, cannot be cultured at all. Other bacteria, such as those that cause tuberculosis, can be cultured but take weeks to grow. Some viruses can be cultured, but many cannot.

TESTING OF A MICROORGANISM'S SUSCEPTIBILITY TO ANTIMICROBIAL DRUGS

Although doctors know in general which antimicrobial drugs are effective against different microorganisms, microorganisms are constantly developing resistance to drugs that were previously effective. Thus, susceptibility testing is done to determine how effective various antimicrobial drugs are against the specific microorganism infecting the person. This testing helps doctors determine which drug to use for a particular person's infection (see Antibiotics : Selecting an Antibiotic).

Cultures are often used for susceptibility testing. Once a microorganism has been grown in a culture, doctors add different antimicrobial drugs to see which ones kill the microorganism. They also test how sensitive the microorganism is to a drug—that is, whether a small or a large amount of a drug is needed to kill the microorganism. If a large amount is needed to kill the microorganism in the laboratory, doctors usually do not use that drug.

Sometimes genetic testing can be used to detect genes in the microorganism that cause resistance to certain antimicrobial drugs. For example, methicillin-resistant Staphylococcus aureus (MRSA) bacteria can be identified by testing for the mecA gene.

Because susceptibility testing occurs in the laboratory, the result does not always match what happens in the person's body when a drug is given. Factors related to the person receiving the drug can influence how effective a drug is . They include the following:

• How well the person's immune system is working
• How old the person is
• Whether the person has other disorders
• How the person's body absorbs and processes the drug

ANTIBODY TESTS

Antibody tests are usually done on a sample of the infected person’s blood. They also can be done on samples of cerebrospinal fluid or other body fluids.

Antibodies are substances produced by a person's immune system to help defend against infection . They are produced by certain types of white blood cell when these white blood cells encounter a foreign substance or cell. It typically takes several days to produce the antibody.

An antibody recognizes and targets the specific foreign substance (antigen) that triggered its production, so each antibody is unique, made for a specific type (species) of microorganism. If a person has antibodies to a particular microorganism, it means that the person has been exposed to that microorganism and has mounted an immune response. However, because many antibodies remain in the bloodstream long after an infection has resolved, finding antibodies to a microorganism does not necessarily mean the person is still infected. The antibodies may remain from a previous infection.

Notes; Finding antibodies to a microorganism in a person's blood does not necessarily mean that the person is still infected because the antibodies may remain from a previous infection.

ANTIGEN TESTS

Antigens are substances that can trigger an immune response in the body. Microorganisms have antigens on their surface and inside them. Antigen tests detect the presence of a microorganism directly, so that doctors can diagnose an infection quickly, without waiting for a person to produce antibodies in response to the microorganism. Also, these tests can be used in people whose immune system cannot produce many antibodies, such as people who have recently had bone marrow transplantation or who have AIDS.

To do antigen tests, doctors take a sample from a person and mix it with a test antibody to the suspected microorganism. If there are antigens from that microorganism in the person's sample, they attach to the test antibody. Different methods can be used to detect the antigen-antibody combination. But whatever method is used, the presence of the antigen means that the microorganism is present and probably is the cause of the infection.

Tests used to identify Gram Positive Bacteria

• Catalase Test

This test is used to identify organisms that produce the enzyme, catalase. This enzyme detoxifies hydrogen peroxide by breaking it down into water and oxygen gas.

2H2O2 -Catalase-> 2H2O + O2

 The bubbles resulting from production of oxygen gas clearly indicate a catalase positive result. The sample on the right below is catalase positive. The Staphylococcus spp. and the Micrococcus spp. are catalase positive. The Streptococcus and Enterococcus spp. are catalase negative.

• Mannitol Salt Agar (MSA)

This type of medium is both selective and differential. The MSA will select for organisms such as Staphylococcusspecies which can live in areas of high salt concentration (plate on the left in the picture below). This is in contrast to Streptococcus species, whose growth is selected against by this high salt agar (plate on the right in the picture below).

The differential ingredient in MSA is the sugar mannitol. Organisms capable of using mannitol as a food source will produce acidic byproducts of fermentation that will lower the pH of the media. The acidity of the media will cause the pH indicator, phenol red, to turn yellow. Staphylococcus aureus is capable of fermenting mannitol (left side of left plate) while Staphylococcus epidermidis is not (right side of left plate).

• Blood Agar Plates (BAP)

This is a differential medium. It is a rich, complex medium that contains 5% sheep red blood cells. BAP tests the ability of an organism to produce hemolysins, enzymes that damage/lyse red blood cells (erythrocytes). The degree of hemolysis by these hemolysins is helpful in differentiating members of the genera Staphylococcus, Streptococcus and Enterococcus.

• Beta-hemolysis is complete hemolysis. It is characterized by a clear (transparent) zone surrounding the colonies. Staphylococcus aureus, Streptococcus pyogenes and Streptococcus agalactiae are b-hemolytic (the picture on the left below shows the beta-hemolysis of S. pyogenes).
• Partial hemolysis is termed alpha-hemolysis. Colonies typically are surrounded by a green, opaque zone. Streptococcus pneumoniae and Streptococcus mitis are a-hemolytic (the picture on the right below shows the a-hemolysis of S. mitis).
• If no hemolysis occurs, this is termed gamma-hemolysis. There are no notable zones around the colonies. Staphylococcus epidermidis is gamma-hemolytic.

• Streak-stab technique

• Taxos P (optochin sensitivity testing)
• Taxos A (bacitracin sensitivity testing)
• CAMP Test
• Bile Esculin Agar
• Nitrate Broth
• Spirit Blue agar
• Starch hydrolysis test
• Motility Agar
• Coagulase Test

Tests used to identify Gram Negative Bacteria

• Oxidase Test

This test is used to identify microorganisms containing the enzyme cytochrome oxidase (important in the electron transport chain). It is commonly used to distinguish between oxidase negative Enterobacteriaceae and oxidase positive Pseudomadaceae.

Cytochrome oxidase transfers electrons from the electron transport chain to oxygen (the final electron acceptor) and reduces it to water. In the oxidase test, artificial electron donors and acceptors are provided. When the electron donor is oxidized by cytochrome oxidase it turns a dark purple. This is considered a positive result. In the picture below the organism on the right (Pseudomonas aeruginosa) is oxidase positive.

• Sugar (eg glucose) broth with Durham tubes

This is a differential medium. It tests an organism's ability to ferment the sugar glucose as well as its ability to convert the end product of glycolysis, pyruvic acid into gaseous byproducts. This is a test commonly used when trying to identify Gram-negative enteric bacteria, all of which are glucose fermenters but only some of which produce gas.

Like MSA, this medium also contains the pH indicator, phenol red. If an organism is capable of fermenting the sugar glucose, then acidic byproducts are formed and the pH indicator turns yellow. Escherichia coli is capable of fermenting glucose as are Proteus mirabilis (far right) and Shigella dysenteriae (far left).  Pseudomonas aeruginosa (center) is a nonfermenter.

The end product of glycolysis is pyruvate. Organisms that are capable of converting pyruvate to formic acid and formic acid to H2 (g) and CO2 (g), via the action of the enzyme formic hydrogen lyase, emit gas. This gas is trapped in the Durham tube and appears as a bubble at the top of the tube. Escherichia coli and Proteus mirabilis (far right) are both gas producers. Notice that Shigella dysenteriae (far left) ferments glucose but does not produce gas.

*Note - broth tubes can be made containing sugars other than glucose (e.g. lactose and mannitol).  Because the same pH indicator (phenol red) is also used in these fermentation tubes, the same results are considered positive  (e.g. a lactose broth tube that turns yellow after incubation has been inoculated with an organism that can ferment lactose).

• Methyl Red / Voges-Proskauer (MR/VP)
• Kliger’s Iron Agar (KIA)
• Nitrate Broth
• Motility Agar
• MacConkey agar
• Simmon’s Citrate Agar
• Urease test
• Sulfur Indole Motility Media (SIM)