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Diagnosis of Infectious Disease

By Kevin C. Hazen, PhD, Duke University Health System

Infectious diseases are caused by microorganisms, such as bacteria, viruses, fungi, and parasites (see Table: Types of Infectious Organisms).

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

  • Tested for antibodies to the microorganism

  • 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 (are sterile), such as the 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 gram-positive (they look blue because they retain the violet Gram stain) or 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 and Susceptibility Testing


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 most cannot.

Susceptibility testing

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 (see Diagnosis of Infectious Disease: Tests That Detect Genetic Material in Microorganisms) 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 mecAgene.

Because susceptibility testing occurs in the laboratory (rather than the person's body), it is not entirely accurate. Factors related to the person receiving the drug can influence how effective a drug is. They include how well the person's immune system is working, how old the person is, whether the person has other disorders, and how the person's body absorbs and processes the drug.

Tests That Detect Antibodies to Microorganisms

Some microorganisms, such as the bacteria that cause syphilis, cannot be cultured. To diagnose such infections, doctors may use tests that detect antibodies to the microorganisms. Antibody tests are usually done on a sample of the infected person’s blood. They also can be done on cerebrospinal fluid or other body fluids.

Antibodies are substances produced by the body's immune system to help defend against infection (see Acquired Immunity : Antibodies). They are produced by certain types of white blood cell when the white 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 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 (antibodies are part of the immune system—see Acquired Immunity : Antibodies). 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 be from a previous infection.

Doctors may test for several antibodies, depending on which infections they think are likely. Sometimes doctors just test whether an antibody is present or not. But usually they try to determine how much antibody is present. They determine the amount of antibody by repeatedly diluting the sample in half until it no longer tests positive for the antibody. The more dilutions it takes until the test is negative, the more antibody is present.

Because it takes several days to weeks for the immune system to produce enough antibody to be detected, diagnosis of an infection may be delayed. Antibody tests done right after people become ill are often negative. Thus, doctors may take one sample immediately and then take another one several weeks later to see whether antibody levels have increased. If levels of an antibody are low on the first test after people become ill, finding an increase in the antibody levels several weeks later suggests an active, current (rather than a previous) infection.

Tests That Detect Genetic Material in Microorganisms

If a microorganism is difficult to culture or identify by other methods, doctors can do tests to identify pieces of the microorganism’s genetic material. This genetic material consists of nucleic acids: deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). Thus, these tests are called nucleic acid–based tests. The polymerase chain reaction (PCR) is an example of this type of test.

Each genetic test is specific to only one specific microorganism. That is, a genetic test for hepatitis C virus detects only that virus and not any other. Thus, these tests are done only when a doctor already suspects a particular disease.

Most nucleic acid–based tests are designed to identify the presence of a microorganism (called qualitative testing). However, a few of these tests can measure the amount of genetic material present (called quantitative testing) in certain microorganisms, such as HIV and hepatitis C, and thus determine how severe the infection is. Quantitative tests can also be used to monitor how well treatment is working.

Nucleic acid–based tests can sometimes be used to check the microorganisms for genes or gene mutations that make the microorganism resistant to a drug. However, these tests are not completely accurate because not all resistance mutations are known. Thus, tests cannot check for all the genes for resistance that may be present. Also, these tests are expensive, not widely available, and available for only a few microorganisms.

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