Cover Story

 CONTINUING EDUCATION

To earn CEUs, see current test at www.mlo-online.com  under the CE Tests tab. The CE test covers all material in the Cover Story section.

LEARNING OBJECTIVES

Upon completion of this article, the reader will be able to:

1. Describe the HIV infection to include the pathogenesis, HAART treatment, and HIV-associated nephropathy.
2. Discuss the various tests used to diagnose HIV-1 infection.
3. Explain TB infection to include historical and current morbidity and mortality, TB incidence among U.S.-born individuals versus immigrant populations, and BCG vaccination for TB.
4. Differentiate among TB identification techniques such as TST or IGRA to include the advantage and limitations of IGRA.

Infectious Diseases

HIV infection and nephropathy

By Shu-Ling L. Fan, PhD, D(ABCC), F(ACB)

In 1985, the first description of acute human immunodeficiency virus (HIV) infection, a "mononucleosis-like" illness, was published based upon the clinical records of 12 men with documented seroconversion to HIV during the preceding six months; 11 of these individuals experienced a remarkably similar illness.¹ Since that time, larger studies have described the clinical and laboratory features of primary or acute HIV infection.

HIV infection and the advanced disease, acquired immunodeficiency syndrome (AIDS), remain leading causes of illness and death in the United States. As of 2008, 33 million people were estimated to be living with HIV/AIDS, and more than 37 million had died since the beginning of the epidemic. In terms of the recent growth of the epidemic, an estimated 2.7 million people became newly infected with HIV in 2008.²

Pathogenesis and states of infection

Viral transmission. HIV-1 most often enters the host through the genital mucosa during sexual intercourse. The viral envelope protein, glycoprotein 120 (gp120), binds to the CD4+ molecule on dendritic cells in cervicovaginal epithelium as well as tonsillar and adenoidal tissue, which may serve as initial target cells in infection transmitted via genital-oral sex.³

Newly acquired HIV infection can result from transmission of macrophage tropic and T-cell tropic viruses mediated by different co-receptors.⁴ HIV-infected cells fuse with CD4+ T-cells, leading to the spread of the virus. HIV is detectable in regional lymph nodes within two days of mucosal exposure and in plasma within another three days.³ Once virus enters the blood, there is widespread dissemination to organs such as the brain, spleen, and lymph nodes.

The intestinal mucosa is also a primary target during initial infection.⁵ Massive CD4+ T-cell depletion during acute infection has been demonstrated with simian immunodeficiency virus (SIV) in rhesus macaques.⁶ This can lead to an early and disproportionate loss of CD4+ T-cells in the gastrointestinal compartment.⁷ It has also been proposed that microbial translocation, due to changes in the gut mucosal barrier, may be the etiology of chronic immune activation in HIV infection.⁸

Viremia. Viremia was documented between five to 30 days after experimental intravaginal HIV exposure. Patients with acute HIV have a markedly elevated viral load, easily detectable with regular (as opposed to ultrasensitive) viral-load tests. In one study, for example, all patients with acute HIV had values >100,000 copies/mL.⁹ HIV RNA levels rapidly increase from the earliest quantifiable measure to a peak level that usually coincides with seroconversion.¹⁰

A variety of symptoms and signs may be seen in association with acute HIV infection. Published series consistently report that the most common findings are fever, lymphadenopathy, sore throat, rash, myalgia/arthralgia, and headache.¹¹ None of these findings is specific, but several features, especially prolonged duration of symptoms and the presence of mucocutaneous ulcers, are suggestive of the diagnosis.

Seroconversion. Most patients seroconvert to positive HIV serology within four to 10 weeks after exposure using newer diagnostic tests, and ≥95% seroconvert within six months.¹²

Clinical latent period. The period of early HIV disease extends from seroconversion to six months following HIV transmission. During the period of asymptomatic infection, patients generally have no findings on physical examination except for possible lymphadenopathy.

The lymphoid tissue serves as the major reservoir for HIV. The follicular dendritic cells in lymphoid tissue filter and trap free virus and infected CD4+ T-cells. The viral burden in peripheral blood mononuclear cells is relatively low at this time. The lymph-node architecture is disrupted, and more HIV is released peripherally into the bloodstream as the disease progresses.

AIDS is primarily a consequence of continuous, high-level replication of HIV-1, leading to virus and immune-mediated killing of CD4+ lymphocytes.¹³ Once the diagnosis of HIV infection has been established, the severity of disease and rate of progression can be estimated by measurement of the CD4+ count and the HIV viral load.

Diagnosis

Establishing the diagnosis of primary HIV infection is clearly important from the public-health perspective. Patients are typically highly infectious during acute HIV due to an enormous viral burden in blood and genital secretions.¹⁴

Such patients may be unaware that they are infected and continue to engage in high-risk behaviors such as unprotected sex or needle sharing, putting others at risk. Pregnant women can transmit HIV perinatally unless a timely diagnosis is made and antiretroviral therapy is initiated.¹⁵

There are several types of tests used to diagnose acute HIV infection:

Viral Load. Nucleic-acid amplification testing (NAT) is a sensitive method to detect acute HIV viremia in patients who are antibody-negative. The preferred test is the reverse transcription polymerase chain reaction (RT-PCR) version with a lower-limit cutoff of 400 copies/mL. A false-positive test should be suspected if the viral load is low (<10,000 copies/mL) in the setting of suspected acute HIV infection.¹⁶ A repeat sample should be drawn in this setting since a rising viral load suggests a true-positive result. NAT, however, is an expensive test to utilize as a screening tool for the detection of acute HIV infection in large populations.

Serologic tests. The serologic tests for HIV infection are based upon detection of IgG antibody against HIV-1 antigens in serum. These HIV antigens include p24 (a nucleocapsid protein) and gp120 and gp41 (envelope proteins). Antibodies to p24 antigen are the first detectable serologic markers following HIV infection.¹⁷ IgG antibodies appear six to 12 weeks following HIV infection in the majority of seroconverted patients and by six months in 95% of patients.¹⁸ IgG antibodies to HIV generally persist for life. Positive tests should be confirmed with repeat tests or corroborating laboratory data (e.g., Western blot assays). Assays for IgM antibodies are not used because they are relatively insensitive.

Rapid HIV tests. Because of the time that elapses before results of conventional HIV tests are available, providing patients with their test results can be resource intensive and challenging for screening programs, especially in episodic care settings (e.g., emergency departments, urgent-care clinics, and STD clinics) in which continuing relationships with patients typically do not exist. The use of rapid HIV tests can substantially decrease the number of persons who fail to learn their test results and reduce the resources expended to locate persons identified as HIV infected. Positive rapid HIV test results are preliminary and must be confirmed by a Western blot assay before the diagnosis of HIV infection is established.

When acute retroviral syndrome is a possibility, a plasma RNA test should be used in conjunction with an HIV antibody test to diagnose acute HIV infection.¹⁹

HIV-associated nephropathy

The introduction of the first protease inhibitor in 1995, combination antiretroviral therapy (ART), or highly active antiretroviral therapy (HAART), had a dramatic impact on the natural history of HIV, with significant reductions in opportunistic infections and mortality. An unanticipated consequence of prolonged survival has been the increasing prevalence of serious non-AIDS complications, including kidney, liver, and cardiovascular disease, which have emerged as leading contributors to morbidity and mortality in patients with HIV infection.²⁰

As patients infected with HIV live longer while receiving antiretroviral therapy, kidney diseases have emerged as significant causes of morbidity and mortality due to the nephrotoxicity of antiretrovirals. Being a member of the black race, being of an older age, and/or suffering from hypertension, diabetes, low CD4+ cell count, and high viral load remain important risk factors for kidney disease in this population.²¹

HIV-associated nephropathy (HIVAN) is a renal syndrome in HIV-1 seropositive patients, characterized by heavy proteinuria, renal dysfunction, and rapid progression to renal failure, first described in 1984 by Rao, et al.²² HIVAN is now the third leading cause of end-stage renal disease (ESRD) in African-Americans between the ages of 20 and 64 and the most common cause of ESRD in HIV-1 seropositive patients.²³

As patients infected with HIV live longer while receiving antiretroviral therapy, kidney diseases have emerged as significant causes of morbidity and mortality due to the nephrotoxicity of antiretrovirals.

Studies using animal models of HIVAN suggest that the renal pathogenesis is due to viral infection of the renal cells rather than immune dysregulation in the setting of systemic HIV-1 infection.²⁴ In humans, the presence of HIV-1 in renal epithelial cells has been shown. One study examined human kidney biopsy samples from HIV-1 seropositive patients. Using in situ hybridization, HIV-1 RNA was detected in renal epithelial cells. The presence of HIV-1 was further confirmed using DNA in situ PCR. Of note, HIV-1 RNA and DNA were also detected in renal epithelia from several patients with undetectable viral loads, suggesting that renal cells may act as a reservoir for HIV-1.²⁵

Light microscopy of HIVAN biopsies is characterized by frequently collapsing focal glomerulosclerosis. The term "collapse" refers to an implosive retraction of the glomerular basement membrane. There is marked hypertrophy and hyperplasia of the overlying visceral epithelial cells. These cells may display mitotic figures and intracytoplasmic protein resorption droplets.²⁶ Prominent lymphocytic infiltration of the interstitium is frequently present. On immunofluorescence, there may be staining for IgM, C3, and, less frequently, C1.²⁷

Kidney disease tends to be asymptomatic and is usually not the primary focus of a visit to an HIV clinic. HIVAN is the most common cause of chronic renal failure in HIV-1 seropositive patients and is especially prevalent among patients of African descent. Though HAART has dramatically reduced mortality of patients with HIV/AIDS, the incidence of ESRD due to HIVAN has not shown signs of decrease and is likely to increase. The presence of kidney disease should be anticipated, and screening and proper interpretation of the relationship between serum creatinine level and glomerular filtration rate, or GFR, are recommended. Just as optimal control of HIV replication is achievable for most patients, so is the control of hypertension and diabetes. The future holds enormous opportunities for research in new markers for early detection of kidney disease, prevention strategies, novel therapeutics, and a better understanding of the interaction between black race and kidney disease.

Shu-Ling L. Fan, PhD, D(ABCC), F(ACB), is an instructor in Pathology at Harvard Medical School and assistant director of Clinical Chemistry in the Department of Pathology at Beth Israel Deaconess Medical Center in Boston, MA.

References

1. Cooper DA, Gold J, Maclean P, et al. Acute AIDS retrovirus infection. Definition of a clinical illness associated with seroconversion. Lancet. 1985;325(8428)1:537-540.
2. UNAIDS Annual Report 2008. Towards Universal Access. http://data.unaids.org/pub/Report/2009/jc1736_2008_annual_report_en.pdf . Accessed January 18, 2010.
3. Kahn JO, Walker BD. Acute human immunodeficiency virus type 1 infection. N Engl J Med. 1998;339(1):33-39.
4. Zhu T, Wang N, Carr A, et al. Genetic characterization of human immunodeficiency virus type 1 in blood and genital secretions: Evidence for viral compartmentalization and selection during sexual transmission. J Virol. 1996;70(5):3098-3107.
5. Nilsson J, Kinloch-de-Loes S, Granath A, et al. Early immune activation in gut-associated and peripheral lymphoid tissue during acute HIV infection. AIDS. 2007;21(5):565-574.
6. Li Q, Duan L, Estes JD, et al. Peak SIV replication in resting memory CD4+ T cells depletes gut lamina propria CD4+ T cells. Nature. 2005;434(7037):1148-1152.
7. Kotler DP. HIV infection and the gastrointestinal tract. AIDS. 2005;19(2):107-117.
8. Haynes, BF. Gut microbes out of control in HIV infection. Nat Med. 2006;12(12):1351-1352.
9. Daar ES, Little S, Pitt J, et al. Diagnosis of primary HIV-1 infection. Los Angeles County Primary HIV Infection Recruitment Network. Ann Intern Med. 2001;134(1):25-29.
10. Fiebig E, Wright D, Rawal B, et al. Dynamics of HIV viremia and antibody seroconversion in plasma donors: implications for diagnosis and staging of primary HIV infection. AIDS. 2003;17(13):1871-1879.
11. Niu MT, Stein DS, Schnittman SM. Primary human immunodeficiency virus type 1 infection: Review of pathogenesis and early treatment intervention in humans and animal retrovirus infections. J Infect Dis. 1993;168(6):1490-1501.
12. Sheppard HW, Busch MP, Louie PH, et al. HIV-1 PCR and isolation in seroconverting and seronegative homosexual men: Absence of long-term immunosilent infection. J Acquir Immune Defic Syndr. 1993;6(12):1339-1346.
13. Ho DD, Neumann AU, Perelson AS, et al. Rapid turnover of plasma virions and CD4 lymphocytes in HIV-1 infection. Nature. 1995;373(6510):123-126.
14. Daar ES, Moudgil,TM, Meyer RD, et. al. Transient high levels of viremia in patients with primary human immunodeficiency virus type 1 infection. N Engl J Med. 1991; 324(14):961-964.
15. Patterson K, Leone P, Fiscus S. Frequent detection of acute HIV infection in pregnant women. AIDS. 2007;21:2303-2308.
16. Rich JD, Merriman NA, Mylonakis E, et al. Misdiagnosis of HIV infection by HIV-1 plasma viral load testing: a case series. Ann Intern Med. 1999;130(1):37-39.
17. Jacquez JA, Koopman JS, Simon C et al. Role of the primary infection in epidemics of HIV infection in gay cohorts. J Acquir Immune Defic Syndr. 1994;7(11):1169-1184.
18. Pilcher CD, Tien HC, Eron JJ Jr, et al. Brief but efficient: acute HIV infection and the sexual transmission of HIV. J Infect Dis. 2004;189:1785-1792.
19. U.S. Department of Health and Human Services, Panel on Clinical Practices for Treatment of HIV Infection. Guidelines for the use of antiretroviral agents in HIV-1–infected adults and adolescents. Washington, DC: U.S. Department of Health and Human Services; 2006.
20. Palella FJ, Delaney KM, Moorman AC, Loveless MO, et al, for The HIV Outpatient Study Investigators. Declining morbidity and mortality among patients with advanced human immunodeficiency virus infection. N Engl J Med.1998;338(13):853-860.
21. Winston J, Deray G, Hawkins T, et al. Kidney disease in patients with HIV infection and AIDS. Clin Infect Dis. 2008;47(11):1449-1457.
22. Rao TK, Filippone EJ, Nicastri AD, et al. Associated focal and segmental glomerulosclerosis in the acquired immunodeficiency syndrome. N Engl J Med. 1984;310(11):669-673.
23. U.S. Renal Data System. USRDS 2003 Annual Data Report: Atlas of End-Stage Renal Disease in the United States. Bethesda, MD: National Institutes of Health, National Institute of Diabetes and Digestive and Kidney Diseases; 2003.
24. Bruggeman LA, Dikman S, Meng C, et al. Nephropathy in human immunodeficiency virus-1 transgenic mice is due to renal transgene expression. J Clin Invest. 1997;100:84-92.
25. Bruggeman LA, Ross MD, Tanji N, et al. Renal epithelium is a previously unrecognized site of HIV-1 infection. J Am Soc Nephrol. 2000;11(11):2079-2087.
26. D’Agati V, Suh JI, Carbone L, et al. Pathology of HIV-associated nephropathy: a detailed morphologic and comparative study. Kidney Int. 1989;35:1358-1370.
27. D’Agati V, Appel GB. HIV infection and the kidney. J Am Soc Nephrol. 1997;8(1):138-152.

The melting pot: new immigration guidelines affect TB testing

By Cara Weibrod, PhD

A century of scientific advancement has ensured that tuberculois (TB) is no longer a Top 10 killer (although the World Health Organization [WHO] estimates that 1.7 million people die from TB each year). TB’s resurgence (1985 to 1992) and current morbidity and mortality estimates show that TB lurks incessantly, mostly unrecognized.¹

Surveillance and control of TB in foreign-born U.S. residents is still a major issue, as the United States remains the prime destination for immigrants worldwide. By 2007, the TB rate among immigrants was 9.7 times higher than in U.S.-born citizens. In fact, the proportion of cases in foreign-born vs. U.S.-born individuals has increased each year since 1993.² Immigrants from Mexico, the Philippines, India, and Vietnam account for more than half of foreign-born TB cases. ² Increasing rates of multidrug resistant (MDR) TB cases globally are a major concern; and almost all MDR-TB cases in Chinese-Americans are from those who are foreign-born.

Guidelines for TB testing

Prior to 2007, the U.S. TB testing strategy focused on identifying active disease in immigrants and refugees. Federal guidelines mandated extensive TB screening by a registered "panel physician" (or "civil surgeon" if the immigrant was already residing in the U.S.) through a combination of medical history, physical exam, and a chest X-ray. If any result indicated TB, the applicant would undergo three consecutive sputum smears for confirmation. Without conclusive results and proper documentation, immigrant status was denied.

A significant change occurred in 2007 when the Centers for Disease Control and Prevention (CDC) issued new technical instructions to identify latent TB infection (LTBI) in immigrants.³ These re-introduced the 100-year-old tuberculin skin test (TST) for applicants aged 2 to 14 years from countries with an incidence of >20 cases per 100,000 population. If TST positive, the immigrant has further testing to rule out active TB; if active TB is not confirmed, he is considered to have LTBI and requires follow-up and treatment after entering the U.S.

Implementing the TST even in this targeted group had repercussions. Many locally run TB-control programs in the U.S. were burdened with an increase in the number of immigrants categorized as having LTBI, Class 2B, based on a positive TST. These people required further expensive evaluation and potential treatment. Much of this effort, however, may have been wasted. Many TST-positive immigrants may be falsely recognized as having LTBI because of prior bacille Calmette-Guérin (BCG) vaccination and the cross-reactivity between TST’s in vivo purified protein derivative and the Mycobacterium bovis strains used in the BCG vaccine. Billions of people have been BCG-vaccinated; the most common countries of origin for U.S. immigrants have high vaccination coverage (India, 99%; Vietnam, 93%; Mexico, >80%; China, 99%; and the Philippines, 93%).^4,5

The poor specificity of the TST in immigrants may be the reason that the CDC has recently recommended new TB diagnostics in its 2009 update for panel physicians.⁶ The most important change is that panel physicians may now choose an interferon-gamma release assay (IGRA) instead of the TST.

What are IGRAs?

Interferon-gamma release assays (IGRAs) are blood tests that determine whether a person has TB infection. Infected individuals have T-cells in their blood responding to the antigens of the disease being tested. When a disease antigen is added to blood collected from such individuals, a rapid re-stimulation of antigen-specific T-cells occurs, with the release of a cytokine called interferon-gamma (IFN-γ) which is then detected in the assay. The antigens used in IGRAs are highly specific for TB and absent from the BCG vaccine.

Two types of IGRA tests are currently available: the enzyme-linked immunosorbent assay (ELISA) and an enzyme-linked immunospot. Despite their recent introduction to TB control, IGRAs have a large body of supporting clinical evidence. In excess of 400 peer-reviewed, published studies for the ELISA-based test and 80 for the enzyme-linked immunospot are the basis for the broad clinical indications for IGRAs.

What are advantages of IGRAs?

IGRAs have distinct advantages over TST applicable both inside and outside the immigration setting. The greatest benefit for immigrants is that IGRAs do not cross-react with BCG vaccinations. By using IGRA instead of TST, users can avoid false-positive results in BCG-vaccinated individuals.

IGRAs are cost-effective. Many health officials across the U.S. report that switching from TST to the ELISA-based strategy may harness major cost-savings through reductions in the number of chest X-rays, sputum smears, and the public-health resources required to evaluate and treat immigrants who are TST-positive but unlikely to have true TB infection. Oxlade, et al, found when BCG vaccination among immigrants was widespread, the ELISA test was significantly cheaper than TST.⁷

IGRAs are significantly more sensitive in detecting people with active TB than the TST. A recent meta-analysis of published literature found the ELISA-based test had a sensitivity of 84.5% in studies of developed countries vs. 71.5% for the TST. The sensitivity of the enzyme-linked immunospot was reported at 88.5%, determined from studies that used a cut-off lower than that recently approved for use in the U.S.⁸

High test specificity, the percentage of people without infection who test negative, is essential to avoid false-positive results and their consequences. The Diel, et al, meta-analysis found specificity of >99.2% for the ELISA-based test, and — again at different cutoffs than were recently approved for use in the U.S. — 86.3% for the enzyme-linked immunospot. A previous meta-analysis found that the specificity of the TST in BCG-vaccinated populations is 59%.⁹

Confidence in the diagnostic capabilities of IGRAs is high. Data indicates acceptance of latent TB prophylaxis is higher in healthcare workers tested with one of the IGRA tests compared to TST.¹⁰ In addition, the 2008 American College Health Association TB testing guidelines now recommend the ELISA-based IGRA technology for international visitors studying in the United States.¹¹

Limitations of IGRAs

Compared to the TST, new IGRA technology has few limitations in performance. As they are not administered at the point-of-care, however, IGRAs require access to laboratory testing. Cost of any assay is a consideration, but when compared to the cost of further evaluation following the TST, IGRAs can lead to better outcomes for immigrants and all other populations tested.

Tuberculosis remains a contemporary disease with extensive global consequences. Affecting more than one million immigrants entering the U.S. every year, the CDC’s new immigrant TB Technical Instructions are a major improvement that can raise efficiency and lower costs of global migration to the United States. The inclusion of IGRA technology for immigrant TB testing is significant not only for the U.S., but also for individual immigrants worldwide.

Minnesota native, Cara Weisbrod, PhD, is a medical writer for Cellestis, a medical technology company based in Melbourne, Australia. The IGRA tests referred to are (ELISA)-based QuantiFERON-TB Gold In-Tube assay (QFT; Cellestis, Melbourne, Australia) and enzyme-linked immunospot (ELISPOT)-based
T-SPOT.TB test (Oxford Immunotec, Abingdon, U.K.).

References

1. Centers for Disease Control and Prevention. Achievements in Public Health, 1900-1999: Control of Infectious Diseases. MMWR 1999;48(29):621-629.
2. Centers for Disease Control and Prevention. Trends in tuberculosis - United States, 2007. MMWR 2008;57:281-285.
3. Centers for Disease Control and Prevention. 2007 Technical Instructions for Tuberculosis Screening and Treatment for Panel Physicians. http://www.cdc.gov/ncidod/dq/panel_2007.htm . Accessed January 25, 2010.
4. WHO Vaccine Preventable Disease Monitoring System 2009 Global Summary. http://www.who.int/immunization_monitoring/en/globalsummary/scheduleselect.cfm . Accessed January 25, 2010..
5. Zwerling A, Behr M, Verma A, et al. World Atlas of BCG Policies & Practices. http://www.bcgatlas.org . Accessed January 25, 2010.
6. CDC Immigration Requirements: Technical Instructions for Tuberculosis Screening and Treatment: Using Cultures and Directly Observed Therapy, CDC, October 1, 2009. http://www.cdc.gov/ncidod/dq/pdf/tuberculosis-ti-2009.pdf .
   Accessed January 25, 2010.
7. Oxlade O, et al. Interferon-gamma release assays and tuberculosis screening in high-income countries: a cost-effectiveness analysis. IJTLD. 2007;11(1):16-26.
8. Diel R, et al. Comparative Performance of Tuberculin Skin Test, QuantiFERON-TB-Gold In Tube Assay and T-Spot.TB Test in Contact Investigations for Tuberculosis. Chest. 2009. http://chestjournal.chestpubs.org/content/135/4/1010.abstract?sic=effa3710-2811-469d-8b . Accessed January 25, 2010..
9. Pai M, et al. Systematic Review: T-Cell-Based Assays for the Diagnosis of Latent Tuberculosis Infection: An Update. Ann Intern Med. 2008. http://www.annals.org/content/149/3/177.abstract?sid=7dc7a5fb-a58b-43d6-b4a2-c92178a . Accessed January 25, 2010.
10. Fox BD, et al. The QuantiFERON-TB-GOLD Assay for Tuberculosis Screening for Healthcare Workers: A Cost-Comparison Analysis. Lung. 2009;187(6):413-419.
11. American College Health Association. Tuberculosis Screening and Targeted Testing of College and University Students. July 2008. http://www.acha.org/Publications/Guidelines_WhitePapers.cfm . Accessed January 25, 2010.