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Human Immunodeficiency Virus (HIV) is estimated to infect 1,00,000 people in the United States. The most recent statistics from the World Health Organization indicate that worldwide AIDS cases number 7.7 million, with just 7% of cases in the U.S. and >75% of cases in Africa. Serologic test methods for the diagnosis of HIV infection have been commercially available since 1985. Screening assays are currently available for use with serum, urine, oral fluid, and dried blood spots.
The serologic response to HIV infection is characterized by the appearance of antigen and antibodies to several viral proteins. Core, or p24, antigen is the first serologic marker to be detected, as early as 2 weeks after infection. Antigen levels fall as p24 antibody appears, at 6-10 weeks post infection. Late in the disease course, p24 antibody drops, and p24 antigen may reappear. Antibodies to envelope glycoprotein (gp41, gp120, and gp160) begin to rise at 6 weeks after infection and are characteristically present throughout the disease course. Although seroconversion typically occurs within 2-3 months and as early as 3 weeks after HIV exposure, a delay of 5-6 months is not unusual. There is a case report of late seroconversion after 34 months.
The most commonly used initial laboratory test for the diagnosis of HIV infection is an enzyme immunoassay (EIA) for viral antibody, performed on a serum sample obtained by venipuncture. Antibodies to core protein and envelope glycoprotein are detected. Within the laboratory, if the initial test is negative, the result is reported and no further testing is necessary on that sample. If the initial test is positive, the EIA must be repeated in duplicate on the same sample, and if either repeat test is positive, the sample is considered repeat reactive. Additional testing by a method more specific for HIV antibodies (Western blot or Immunofluorescent assay) is then performed. The sensitivity and specificity of serum EIA are 99-100% and 98-99%, respectively. Possible causes of false positive and false negative HIV antibody reactions are listed in the following tables.
Causes of False Positive HIV Antibody Reactions
Causes of False Negative HIV Antibody Reactions
EIA testing methods are also available for urine specimens. However, there is currently no confirmatory assay for urine so that a positive result must be followed by serum testing. Oral fluid, collected by a specialized swab-type device, can be tested by EIA with follow-up testing by Western blot.
Western blot (WB) is a method of separating proteins that has been used for supplemental testing for HIV antibodies. Antibodies to the viral proteins p17, p24, p31, gp41, p51, p55, p66, and gp120/160, may be identified by WB and appear as bands. The Association of State and Territorial Public Health Laboratory Directors (ASTPHLD) criteria for interpretation of Western blots are most commonly used. By these criteria, a reactive WB must contain two of three major bands of diagnostic significance (anti-gp120/160, anti-gp41, and anti-p24), a nonreactive WB is defined as one without any HIV-1 specific bands, and an indeterminate WB contains one or more viral specific bands but insufficient bands to meet the definition of reactive. Overall, false positive WBs are estimated to occur no more frequently than 1 in 20,000 tests. False negative WBs may occur very early after HIV-1 infection or late after the development of AIDS. Indeterminate WBs may occur in 13-48% of low risk populations and are usually caused by cross-reacting antibodies to p24, p55 or p66 bands. In low-risk populations, an indeterminate WB is rarely associated with true HIV infection. In high-risk patients, an indeterminate pattern is variably followed by seroconversion. Repeat EIA and Western blot testing is generally recommended 2-3 months following an indeterminate result. Western blot testing should not be requested prior to screening by EIA.
With the advent of more effective early treatment regimens for HIV infection, additional testing may be desired sooner than 2-3 months after an indeterminate result. Qualitative polymerase chain reaction (PCR) tests for viral DNA may be of use in resolving indeterminate WBs. Sensitivity and specificity of the qualitative PCR for HIV-1 DNA are both 98-100%, and detection of as few as six viral DNA copies per mL of whole blood is possible. Again qualitative PCR is not applicable for use prior to EIA screening.
Another application of PCR in the treatment of HIV infection is the quantitative, or viral load assay. There are currently two commonly used quantitative methods, both measuring HIV viral RNA. Reverse transcriptase PCR is a target amplification method that is sensitive to a level of 400 copies per mL of whole blood. An alternative quantitative test is bDNA which utilizes a branched DNA signal to detect HIV RNA. Ultrasensitive assays that may detect as few as 50 viral copies are in development for both tests. Quantitative methods are most appropriately used for monitoring known HIV disease, in conjunction with CD4 counts, and adjustment of anti-retroviral therapy. Because of lower sensitivity for small amounts of viral RNA, quantitative tests are not appropriate for the confirmation of HIV infection.
Although earlier detection of seroconversion has come about with the development of more sensitive HIV antibody EIAs, assays for the p24 antigen may still be of use prior to seroconversion. During acute infection, p24 antigenemia precedes seroconversion, usually resolves within 2-3 weeks as antibodies to p24 appear, but may persist for 6-14 months before seroconversion.
The Kansas City community recently lost a prominent physician and business leader. Pierre W. Keitges, M.D., pathologist and President of Physicians Reference Laboratory (PRL), died on August 5. Dr. Keitges’ vision was to maintain a reference laboratory system that provided unparalleled laboratory service without compromising the highest standards of quality for issues of reimbursement, cost and convenience, while treating all patients in a manner that always recognizes the dignity of the human individual.
PRL was founded in 1976 by Dr. Keitges who was then director of the laboratory at the University of Kansas Medical Center (KUMC). Dr. Keitges had just developed a concept for a reference laboratory that was unique and, since the opportunity to develop the concept at KUMC was not available, Dr. Keitges, along with Daniel Duffin, then the controller of KUMC, opened PRL.
With a vision, philosophy and core management group, PRL began in an office in the basement of the old Saint Joseph Hospital with card tables and folding chairs. Since then, PRL has developed into a major regional reference laboratory with a reputation for quality and excellence second to none in the health care, pharmaceutical, and drug testing industries. Not only has PRL’s business grown over 20% yearly for the past 17 years, but the staff has grown to 321 employees including 13 board certified pathologists.
PRL continues today with a core management group led by Dr. Keitges’ appointed successor, Spencer Kerley, M.D., who assumes the office of President of PRL. Dr. Kerley has been with PRL a little over six years. He was recently appointed Chairman of the Department of Pathology at Saint Joseph Health Center. The management group of 8 vice presidents, including Executive Vice President of Finance and Business and Business Affairs, Daniel Duffin, and Greg Keitges, Vice President of Information Systems and Human Resources, comprises a combined total of over 130 years of PRL management experience. PRL continues with exactly the same vision and philosophy as defined by Dr. Keitges some 21 years ago.