Improving diagnostic testing for infectious diseases

Most visits to a doctor or hospital occur when a patient feels under the weather, but is unsure of the cause of the symptoms or the best way to treat them. For example, sniffles and a sore throat could be caused by seasonal allergies or the flu, each of which must be treated with different medications. In such cases, physicians rely on diagnostic tests to help them decide on the appropriate course of treatment.

The improvement of existing diagnostic tests and the development of better ones is an active area of scientific research, especially with respect to infectious disease. Existing tests may be inadequate for many reasons, including cost, complexity, and the time it takes to get results. Researchers and public health officials are attempting to overcome these issues by simplifying assays for at-home testing, making tests more available in resource limited settings, and improving their reliability, speed, and affordability. Previously faced with these challenges, the diagnostic tests for human immunodeficiency virus (HIV), malaria, and tuberculosis (TB) have recently improved.

An at-home test for HIV

In the United States, one in five persons infected with HIV does not know he or she is infected and improved access to HIV testing could close this gap (1). Most HIV testing has involved the detection of human antibodies in the blood that recognize the virus. An antibody is a protein made by the immune system to protect the person from a foreign agent. Antibodies bind to pathogens like bacteria, parasites, or viruses to prevent and stop an infection. If infected with HIV, a person will produce antibodies that specifically bind to proteins on the virus to block the virus from infecting a cell, or that bind to an already infected cell to help immune cells kill it. In HIV testing, HIV proteins are attached to the surface of a plate and incubated with patient blood. If the patient has antibodies to HIV, the antibodies will bind the HIV proteins and give a positive reading. This positive reading is shown through a parallel chemical reaction that causes a color change in the test.

This month, the FDA approved the sale of a rapid HIV diagnostic test over-the-counter at select pharmacies. Developed by Orasure, OraQuick Advance ® Rapid HIV-1/2 Antibody Test is a simple at home test that can detect an HIV infection using saliva (2). Oraquick contains an applicator and a small cassette in which HIV proteins are bound to a membrane. The applicator is used to swab the outer gums and is then placed in a vial of solution to help the collected antibodies to flow to the membrane. If there are HIV-specific antibodies, they are captured by the bound proteins and a colored band appears. OraQuick is an advance in HIV testing since this method takes under 30 minutes to complete and can be easily performed and read by a patient in the privacy of his or her own home, though it does have a higher false negative rate than laboratory tests.

Figure 1. The OraQuick Advance ® Rapid HIV-1/2 Antibody Test looks similar to an at-home pregnancy test. The applicator is used to swab the gums and placed in a solution that helps HIV specific antibodies travel up to cartridge to give a colored band indicating a  positive result. Stanvanloon\ 2 March 2007

The hope is that over-the-counter approval of OraQuick will ease access to HIV testing and improve HIV awareness. At-home testing should reduce anxiety about seeking testing and may reduce the time between infection and appropriate treatment of the person and their partner. Priced at $60 per test, cost may be a barrier, but improvements in insurance coverage and an increase in market competition may help.

Simplifying malaria testing for resource limited settings

Malaria is a disease endemic to many developing nations and is of great global health concern, as it is treatable but still caused an estimated 216 million infections and 655,000 deaths in 2010 alone  (3) (see the SITN Flash from May 2012 for more on malaria). Deaths were primarily in African children living in resource-limited settings. Because malaria is so prevalent in some regions and fever is a common symptom, any child in these areas with a fever is usually treated with antimalarial drugs even if they have no malaria infection. Not only is this inappropriate treatment for a patient without malaria, this overuse of antimalarial drugs has led to the emergence of parasites resistant to them (4). Therefore, access to quick diagnosis of malaria that can be performed in resource limited settings is necessary for correct treatment.

The most commonly used malaria test in these settings is microscopic examination of patient blood samples. Plasmodium, the parasite that causes malaria, infects and replicates in red blood cells. The parasites are visible under the microscope in dry blood smears stained with dye. While this test sounds simple, it still requires a trained staff and lab equipment to conduct. Even if parasites are detected, a doctor has no information about which of the four Plasmodium species is causing the infection, which may be important in determining the correct course of treatment.

Figure 2. Malaria parasites infect red blood cells. Blood from an infected patient, seen here, is dyed to make parasites dark purple and easier for doctors to observe and confirm an infection. CDC/ Neva Gleason/1968

To address these issues and provide a method that does not require a lab setting, rapid diagnostic tests like OptiMAL detect infection and differentiate between species in a format similar to the OraQuick HIV test. OptiMAL detects proteins produced by the parasite circulating in patient blood instead of the patient-derived antibodies that OraQuck detects. One disadvantage of detecting particular malaria proteins is that they may remain present in blood days after an infection is clear, which could produce a false positive resulting a patient who no longer has malaria.

Advances in diagnostic testing for malaria are currently limited by funding and access but more widespread use of rapid diagnostic tests will allow better treatment and care for patients as well as a reduced risk of antimalarial resistance.

Improving the speed of tuberculosis testing

One third of the world’s population is asymptomatically infected with Mycobacterium tuberculosis (Mtb). In 2010, nine million people had active, symptomatic, tuberculosis (TB) and 1.4 million died of the disease (5). With such a large number of infections, good diagnostic testing is absolutely critical.

There are multiple diagnostic tests used by health care professionals to detect active tuberculosis infections. The commonly accepted test is a sputum smear, in which sputum coughed up by the patient is smeared and stained using a method called acid-fast staining to better observe the Mtb from the lung.  All of the cells in the sputum are dyed except for Mtb and other mycobacteria, which have a characteristically thick and waxy exterior that prevents stain from entering the cell. This test may give false negatives since most patients have very low levels of mycobacteria in sputum. Conversely, it might also give false positives since non-dyed cells may or may not be Mtb. Therefore, this test must be done twice and paired with a different test before results are confirmed. Growing Mtb from sputum can confirm a TB infection but it takes 2 months to grow enough Mtb to observe under the microscope. The long lag time between sample collection and confirmed results is problematic for quickly treating the patient and reducing the time that the patient spends unknowingly spreading the infection to others.

Figure 3. Doctors collect and stain patient sputum smears using acid-fast staining in which all cells except mycobacteria is stained. Here the white patches indicate the presence of Mtb.

Researchers have stepped in to provide a simplified gene-based test to identify patients with Mtb in their sputum samples by finding and increasing the number of copies of Mtb-specific genes in sputum samples to levels high enough to detect. The most popular commercial form is called GeneXpert MTB/RIF, which surpasses older tests in its ability to detect small amounts of Mtb in less than 2 hours (6). This test can be performed by an unskilled worker and does not need the additional safety measures required for growing infectious tuberculosis cultures. However, the test still requires a reliable power source and costs $17,000 per instrument and $14 per test. Charitable organizations like the Foundation for Innovative New Diagnostics and the Gates Foundation are currently subsidizing the cost for resource-limited areas (7).

The common goals of diagnostic test development for infectious disease are simplicity, speed, accuracy, and ease of access. For HIV testing, FDA approval of the over-the-counter sale of OraQuick allows patients to easily acquire a basic HIV test with rapid and dependable results. In the same way, use of rapid tests for malaria like OptiMAL should make healthcare professionals aware of confounding symptoms and help them decide on the correct treatment. Advances in TB genetic testing will shorten the time to appropriate treatment and will provide the foundation for the development of similar but better tests for other neglected diseases.

Nicole Espy is a graduate student at the Harvard School of Public Health.

References

1. Centers for Disease Control and Prevention. “Monitoring selected national HIV prevention and care objectives by using HIV surveillance data—United States and 6 U.S. dependent areas – 2010: HIV Surveillance Supplemental Report 2012;17 (No.3, part A)”: http://www.cdc.gov/hiv/topics/surveillance/resources/reports/.

2. Orasure Technologies, Inc, “OraQuick Advance Rapid HIV-1/2 Antibody Test Customer Letter,”: http://www.orasure.com/docs/pdfs/products/oraquick_advance/OraQuick-Advance-Package-Insert-English.pdf.

3. World Health Organization, “World Malaria Report 2011”: http://www.who.int/malaria/world_malaria_report_2011/9789241564403_eng.pdf.

4. World Health Organization, “Universal Access to Diagnostic Testing: An Operational Manual,”: http://whqlibdoc.who.int/publications/2011/9789241502092_eng.pdf.

5. World Health Organization, “Media Centre: Tuberculosis Fact Sheet,” http://www.who.int/mediacentre/factsheets/fs104/en/index.html.

6. BoehmeCC, et al. Rapid molecular detection of tuberculosis and rifampin resistance. 2010 Sep 9;363(11):1005-15. Epub 2010 Sep 1.

7. Foundation for Innovative New Diagnostics, “FIND-negotiated prices for Xpert® MTB/RIF and country list,”: http://www.finddiagnostics.org/about/what_we_do/successes/find–negotiated–prices/xpert_mtb_rif.html. December 2010.