Labs are on the front lines as molecular diagnostics assume a prominent role

By Gary Tufel

According to estimates compiled by the US Centers for Disease Control and Prevention (CDC), there are currently 1.2 million people in the United States living with human immunodeficiency virus (HIV).

Among those infected with the virus, members of certain subpopulations are disproportionately affected—notably including men who have sex with men, African-Americans, and Latinos. Briefing materials developed by CDC highlight the staggering impact of HIV among these groups, and also speak to the socioeconomic factors affecting HIV risk.1

In the United States, the healthcare community has more or less battled the growth rate of the HIV epidemic to a standstill. Since the 1990s, the number of new HIV infections occurring in the United States each year has remained relatively stable. On the downside, however, every year’s number remains quite large. In 2010—the most recent year for which such data are available—there were an estimated 47,500 new infections.2

Working with healthcare providers, CDC collects information about drug-resistant mutations of the virus as part of its HIV surveillance program. “At CDC, the data are unlinked from individuals to look at overall trends in drug resistance around the country,” says Michele Owen, PhD, acting senior advisor for laboratory diagnostics in CDC’s division of HIV/AIDS prevention. “Viral load data are also collected in the context of surveillance to determine the number of infected individuals who have viral suppression.”

CDC also tracks reported US cases of chlamydia, gonorrhea, and syphilis, and publishes the resulting data in its annual surveillance report on sexually transmitted diseases (STDs).3 These data provide insight into the scope and trends related to STD diagnoses across the country, and show that STDs continue to have a severe public health impact in the US. Key findings in the surveillance report for 2013 include:

  • Chlamydia: 1.5% decrease in the overall number of cases from 2012 to 2013.
  • Gonorrhea: Overall number of cases considered stable (0.6% decrease from 2012 to 2013).
  • Syphilis: 10% increase in primary and secondary syphilis cases from 2012 to 2013.

Although the overall incidence of STD infection remained stable in 2013, CDC notes that the drop in chlamydia cases marks the first year since national reporting began that chlamydia case rates have decreased. However, the agency also cautions that more information will be needed to understand whether this single-year drop represents the beginning of a long-term decline in new infections. And in the meantime, chlamydia still ranks as the most prevalent STD by far, with some 1.4 million cases reported in 2013.


The human and economic toll of STDs in the United States is substantial, with an estimated 20 million new STD infections each year (including new cases of HIV infection), and annual healthcare costs totaling an astonishing $16 billion.

Although the incidence of HIV infection has somewhat stabilized in the United States and some other countries, HIV continues to be a major public health issue throughout the world. The World Health Organization (WHO) reports that more than 2 million people worldwide were newly infected with HIV in 2013, the most recent year for which data are available. And in the United States, where it is estimated that more than 1.2 million people are living with HIV, CDC believes that almost 14% of them are not aware of their infection.

Globally, it is believed that approximately 35 million people are currently living with HIV or AIDS, with the vast majority living in low- and middle-income countries. According to WHO, the most affected region is sub-Saharan Africa, where 24.7 million people were thought to be living with HIV in 2014.

Syphilis is also a global public health problem, resulting in an estimated 12 million new infections per year in 2012. CDC estimates that 55,400 people in the United States get new syphilis infections annually. Reflecting a complex interplay that exists between syphilis and HIV, syphilis facilitates both HIV transmission and HIV acquisition. Among those who are living with HIV infection, the rate of coinfection with syphilis is approximately 20% to 70%.4,5

Louise Sigismondi, PhD, Chembio Diagnostic Systems.

Louise Sigismondi, PhD, Chembio Diagnostic Systems.

In the United States, HIV and syphilis are transmitted mainly through anal or vaginal sex. HIV can be spread through sharing drug-injection paraphernalia with an infected person, while syphilis can be spread through oral sex if an open sore is present. According to Louise Sigismondi, PhD, regulatory affairs director of R&D at Chembio Diagnostic Systems Inc, Medford, NY, some population groups have higher rates of HIV or syphilis, thus raising the risk of new infections with each sexual or drug-use encounter. In addition, pregnant women can transmit STDs to their unborn children, she says.

Gonorrhea ranks as the second-most prevalent STD in the United States. In 2013, the US incidence of gonorrhea infection was 106 cases per 100,000 people, for a total of 330,000 cases nationwide. This rate was a considerable increase over the historic low-point achieved in 2009, when the incidence was 98 cases per 100,000. Although the infection rate has decreased among women of all ages, it has increased among men aged 25 and over.

According to CDC, roughly half of the 20 million sexually transmitted infections that occur each year in the United States involve young people aged 15 to 24. The transmitted infections include chlamydia, gonorrhea, HIV, and syphilis—with the rate of syphilis infection especially on the rise.

“Young people overall are at greatest risk of STDs and face a number of unique prevention challenges,” says Donnica Smalls, a member of the news media team in CDC’s National Center for HIV/AIDS, Viral Hepatitis, Sexually Transmitted Diseases, and Tuberculosis Prevention. “Some may be unwilling to discuss risk behaviors with their doctors because they are embarrassed or have confidentiality concerns. Others can’t access healthcare because they lack insurance or transportation to healthcare facilities. Young women’s bodies are also more biologically susceptible to STDs—and the health consequences of STDs can be particularly severe for young women, who may face lifelong infertility.”


Significant efforts have been made to address this epidemic of sexually transmitted diseases, particularly in the past decade. Prevention programs have helped to reduce HIV prevalence rates in a small but growing number of countries, and the number of new HIV infections in the United States and globally has declined slightly.

Joe Medeiros, Alere.

Joe Medeiros, Alere.

Viral load monitoring is one strategy that has attracted significant attention both in the US and globally. For example, one of the three main goals of the US national HIV/AIDS strategy is to increase the proportion of HIV-diagnosed gay and bisexual men, blacks, and Latinos with undetectable viral load by 20%. Known as the “community viral load,” the prevalence of HIV-diagnosed individuals with undetectable viral load is a key indicator of the health of a population. According to Joe Medeiros, director of North American marketing for virology solutions at Alere Inc, Waltham, Mass, landmark studies have shown that a high incidence of undetectable viral load reduces the likelihood of transmission and improves health outcomes.

Resistance testing is another strategy that has proven its value to patients. US Department of Health and Human Services guidelines on the use of antiretroviral agents in HIV-1 infected adults and adolescents recommend resistance testing to help guide selection of an antiretroviral (ARV) regimen, says Medeiros.6 Resistance testing is normally done upon entry into care, upon initiation or modification of antiretroviral therapy (ART), or when treatment failure occurs.

The continued development of ART has enabled physicians to make significant progress in keeping HIV-infected mothers alive and preventing mother-to-child transmission of HIV. According to WHO, in 2013, 67% of pregnant women living with HIV in low- and middle-income countries (970,000 women) received ART to avoid transmitting HIV to their children. The percentage of women treated in 2013 represents a significant increase over the 47% who were treated in 2010. (For more information, see the companion article, “HIV: The Treatment Outlook.”)

At the end of 2013, according to WHO, 12.9 million people living with HIV were receiving ART. Of these, 11.7 million were in low- and middle-income countries, and about 740,000 were children. Compared to the number of infected persons who received ART in 2010, the 2013 figure represents an increase of 5.6 million people.

Although the increased number of patients receiving ART is a dramatic improvement in treatment worldwide, there are still almost 22 million other people living with HIV—or 3 out of every 5—who remain unable to gain access to ART.

Robert Bilkovski, MD, MBA, Abbott Molecular.

Robert Bilkovski, MD, MBA, Abbott Molecular.

Treatment of chlamydia is hindered by the fact that most infected individuals do not have symptoms. According to Robert Bilkovski, MD, MBA, senior associate medical director at Abbott Molecular Inc, Des Plaines, Ill, the most effective means of identifying and treating new chlamydia infections have come through screening programs targeting higher risk, sexually active populations.

“Public health authorities are also becoming increasingly concerned about antibiotic-resistant strains of gonorrhea, which make the disease more difficult to treat,” says Bilkovski. “Gonorrhea has progressively developed resistance to quinolone antibiotics and the oral cephalosporin cefixime. As a result, CDC now recommends that physicians treat gonorrhea with injectable ceftriaxone in combination with one of two oral antibiotics, either azithromycin or doxycycline.”


Today’s clinical laboratories rely on molecular testing methods for the detection of gonorrheal infections. “Compared to immunoassays and culture tests, molecular tests offer improved sensitivity and specificity,” says Bilkovski. “Combination molecular assays for gonorrhea and chlamydia are recommended for both screening and to aid in patient diagnosis.”

Molecular testing has also become the widely accepted standard for monitoring the efficacy of HIV treatment using antiretroviral therapy. “The tests quantify viral load levels to determine whether treatments are successfully suppressing viral replication,” says Bilkovski. “From a clinical perspective, the goal of antiviral drug treatment is to drive viral loads to undetectable levels—or at least decrease them to the accepted threshold of 200 copies per ml.

“In cases where patients do not respond to ART, physicians may consider switching to second-line therapies, based on HIV viral load level changes over time,” Bilkovski adds. “When the decision is made to switch to next-line ART, it becomes increasingly important to evaluate the patient’s HIV resistance level. Sequencing of viral DNA can also provide information that is clinically valuable for treating refractory cases of known HIV mutations.”

The Alere Determine HIV-1/2 Ag/Ab combo assay was the first fourth-generation HIV test in a rapid POC format to receive FDA approval and CLIA-waived status.

The Alere Determine HIV-1/2 Ag/Ab combo assay was the first fourth-generation HIV test in a rapid POC format to receive FDA approval and CLIA-waived status.

As a model for the successful use of viral load monitoring to guide antiviral therapy, Bilkovski cites the emergence of testing protocols and antiviral drugs for hepatitis C (HCV), which have transformed the disease from a life-threatening chronic condition to an infection that is curable in more than 90% of cases. Today, HCV viral load testing is used to gauge the initial level of viral replication, to aid in determining a patient’s suitability for antiviral therapy, and to assess the success of therapy.

“The very high cost of the new HCV medications prohibits treatment of every HCV-positive patient,” says Bilkovski. “Therefore, after using viral load assays or qualitative HCV tests to assess viral replication, physicians will assess liver function to determine whether treatment is necessary. If liver function tests are negative for HCV-induced liver damage, patients may be monitored to determine when treatment is warranted. In such cases, the risk of non-treatment is low.”

When seeking to adopt molecular diagnostic platforms, says Medeiros, “clinical labs should be aware of the technologies, methods, capabilities/limitations, and approved applications related to those platforms.” He offers the following summary of distinctive characteristics that can affect a platform’s suitability for use in a particular lab.

  • Technologies: transcription-mediated amplification, isothermal nucleic acid amplification, reverse transcription-polymerase chain reaction (RT-PCR), nucleic acid sequence-based amplification, branched chain DNA.
  • Methods: DNA/RNA qualitative and quantitative viral load assays, genotypic/phenotypic drug resistance testing, and molecular point-of care testing.
  • Capabilities/limitations: Analytical range, limit of detection, limit of quantification, time to results, sample requirements, samples per hour.
  • Applications: HIV treatment and monitoring, primary HIV diagnosis and enhanced surveillance, early infant HIV diagnosis, blood donor screening, resistance testing, subtyping, and polymorphism testing.

The testing environment, resources, and turnaround time should also be considered when selecting a molecular diagnostic platform, Medeiros adds. “There are certain scenarios where turnaround time is of critical importance, such as when a pregnant woman with unknown HIV status presents for delivery, and a rapid diagnostic test must be performed to determine if ARV therapy is warranted to prevent perinatal transmission. As new molecular point-of-care (POC) tests become available, their ability to rapidly differentiate HIV-1 from HIV-2, and identify subtypes, will enhance the information bases for clinical decision-making, which in turn can lead to improved health outcomes.”

In the United States, screening recommendations by the US Preventive Services Task Force combined with CDC’s strategy of high-impact prevention have helped to identify many more individuals infected with HIV.7,8

Clinical labs currently play a critical role in providing the results of HIV testing to healthcare professionals, but they remain challenged by the need to provide timely results and greater convenience to HIV patients. Currently, patients must undergo laboratory testing prior to an office visit with their physician. But doing so places an additional burden on patients, and may even strain available funding if extensive transportation is required. In some rural settings where HIV has become prevalent, such as the southeastern United States, many patients have difficulty accessing healthcare services.

As molecular POC diagnostic platforms become more widely available, it should become possible to carry out a patient’s laboratory tests while the patient is still in the clinic or physician’s office, says Medeiros. This will enable HIV-treating physicians to make and discuss clinical and therapeutic decisions before the patient leaves.


Chembio’s DPP HIV-1/2 assay makes use of the company’s dual path platform (DPP), a patented technology for rapid POC tests that increases sensitivity and specificity for the detection of infectious diseases by enabling separate paths for delivery of the sample and the conjugate. Compared to lateral-flow devices, DPP devices minimize “hook effect” and offer multiplexing—the ability to detect multiple analytes concurrently from a single sample and device. Chembio’s DPP HIV-1/2 assay has waived status under the requirements of the Clinical Laboratory Improvement Amendments of 1988 (CLIA). The company’s DPP HIV-syphilis assay is not yet approved for use in the United States, but is in use abroad.

 The DPP HIV-1/2 assay by Chembio makes use of the company’s dual path platform (DPP), a patented technology for rapid POC tests that uses separate paths for delivery of the sample and the conjugate.

The DPP HIV-1/2 assay by Chembio makes use of the company’s dual path platform (DPP), a patented technology for rapid POC tests that uses separate paths for delivery of the sample and the conjugate.

“For most public health and hospital laboratories, molecular testing is now the standard diagnostic tool for chlamydia and gonorrhea infections due to its excellent sensitivity and specificity,” says Bilkovski. “Previously used immunoassay and culture testing methods have longer reporting times, which often caused physicians to treat empirically, especially in the emergency department where patients are more likely to be lost to follow up. Molecular assays can provide results in 4 hours or less, and enable doctors to counsel patients appropriately on effective antibiotic treatments and prevention of future infections.”

In 2010, efforts to improve HIV testing in US laboratories received a boost when FDA approved the introduction of two fourth-generation HIV diagnostic tests, which detect both antibodies to the virus as well as the HIV p24 antigen. Fourth-generation tests are capable of identifying HIV infection earlier—within 1 to 2 weeks of a patient’s exposure—potentially facilitating quicker initiation of therapy. The agency’s first approvals went to the Abbott Architect HIV Ag/Ab combo assay and the Bio-Rad GS HIV Ag/Ab combo assay.

In 2013, FDA granted approval to the Alere Determine HIV-1/2 Ag/Ab combo assay, making it the first fourth-generation test in a rapid POC format to receive US market authorization. Offering results in 20 minutes, the assay can be performed using serum, plasma, or whole blood (fingerstick or venous). When performed using fingerstick whole blood, the test has CLIA-waived status.

In June 2015, FDA granted premarket approval to Siemens Healthcare Diagnostics, Tarrytown, NY, for the company’s HIV Ag/Ab combo assay for use on the Advia Centaur immunoassay analyzer. The test has been available in Europe since 2010. Its addition to the menu of the popular Advia Centaur analyzer will enable laboratories using the analyzer to offer fourth-generation HIV testing on their existing platform.

According to CDC’s Owen, FDA is currently reviewing another new fourth-generation test, the BioPlex 2200 HIV Ag/Ab test kit by Bio-Rad Laboratories, Hercules, Calif. The Bio-Rad kit simultaneously screens and differentiates antibodies to HIV-1 (groups M and O) and HIV-2, and the HIV-1 p24 antigen.

Molecular technologies have also played a vital role in HIV diagnosis and monitoring. Molecular assays are used to assess the status of a patient’s immune system, by providing either a count of the patient’s CD4 cells or a viral load measurement. In addition, says Medeiros, molecular tests are essential for identifying HIV mutations as new strains become more virulent. The newly discovered CRF19 strain in Cuba, for instance, has been shown to be an aggressive strain of HIV that can develop into AIDS within 3 years of infection.


In spite of the need for better and more convenient testing methods, FDA has not recently granted market authorization to any breakthrough technologies or products for HIV viral load or drug-resistance testing. But according to CDC’s Owen, that situation may change significantly in the not-too-distant future.

FDA recently approved a fourth-generation HIV Ag/Ab combo assay for performance on the Advia Centaur immunoassay analyzer by Siemens Healthcare Diagnostics.

FDA recently approved a fourth-generation HIV Ag/Ab combo assay for performance on the Advia Centaur immunoassay analyzer by Siemens Healthcare Diagnostics.

HIV investigators have already begun to explore research techniques using next-generation sequencing (NGS), and in the future these techniques may also be adapted for clinical applications. An advantage of such tests may be their ability to detect minority (low level) drug-resistance mutations that could be of clinical importance. However, commercially available versions of such NGS applications for clinical HIV management are likely a few years away.

Meanwhile, a number of companies are involved in developing less-complex molecular HIV tests that will likely be submitted for FDA evaluation in the next year or so. Among populations where HIV is most prevalent, the introduction of molecular POC technologies is expected to facilitate appropriate intervention strategies and reduce further transmission of the virus. According to Medeiros, such interventions will be necessary in order to achieve the future goal of an AIDS-free generation.

Molecular POC tests for HIV are either under development or being introduced to the market by Alere, Cepheid, Roche Molecular Diagnostics, Wave 80, and several other entities.

For markets in Africa and developing nations, Alere has recently introduced the Alere q nucleic acid testing platform and the Alere q HIV-1/2 Detect assay. The Alere q analyzer is designed to facilitate multiple assays and deliver lab-accurate results, even in demanding, resource-limited environments.

“These POC technologies utilize cartridge formats for the rapid qualitative or quantitative detection of HIV-1 and HIV-2 RNA,” says Medeiros. “They are able to make use of either plasma or whole blood (fingerstick, venous, or heelstick), giving them greater flexibility over instrument-based platforms, which can only make use of plasma samples. Results are typically available in 1 hour or less, depending on the technology used.”

Earlier this year, CDC issued a request for collaboration with industry, and it has now made arrangements to evaluate at least four new nucleic acid-based tests that are currently in various stages of development. According to Owen, the tests to be evaluated include both qualitative tests to aid in diagnosis and viral load tests for use in patient monitoring, and each of them is believed to offer some advantage of quicker turnaround or better near-patient access. Some of the tests are expected to enter clinical trials this year, in order to collect data for submission to FDA.

Gary Tufel is a contributing writer for CLP. For more information contact CLP chief editor Steve Halasey via [email protected].


  1. Today’s HIV/AIDS epidemic. [CDC Fact Sheet.] Atlanta: Centers for Disease Control and Prevention, 2015. Available at: Accessed June 29, 2015.
  1. New HIV/AIDS infections in the United States. [CDC Fact Sheet.] Atlanta: Centers for Disease Control and Prevention, 2012. Available at: Accessed June 29, 2015.
  1. Sexually transmitted disease surveillance 2013. Atlanta: Centers for Disease Control and Prevention, 2014. Available at: Accessed June 29, 2015.
  1. Syphilis and MSM (men who have sex with men). [CDC Fact Sheet.] Atlanta: Centers for Disease Control and Prevention, 2015. Available at: Accessed June 29, 2015.
  1. Incidence, prevalence, and cost of sexually transmitted infections in the United States. [CDC Fact Sheet.] Atlanta: Centers for Disease Control and Prevention, 2013. Available at: Accessed June 29, 2015.
  1. Panel on Antiretroviral Guidelines for Adults and Adolescents. Guidelines for the use of antiretroviral agents in HIV-1-infected adults and adolescents. Washington, DC: US Department of Health and Human Services, 2015. Available at: Accessed June 29, 2015.
  1. Moyer VA [on behalf of the US Preventive Services Task Force]. Screening for HIV: Preventive Services Task Force recommendation statement. Ann Intern Med. 2013;159(1):51–60, W-15–W-16.
  1. High-impact prevention. [CDC briefing online.] Atlanta: Centers for Disease Control and Prevention, 2014. Available at: Accessed June 29, 2015.ST