Detecting Emerging Infectious Diseases: An Overview of the Laboratory Response Network for Biological Threats

Abstract

The Laboratory Response Network (LRN) was established in 1999 to ensure an effective laboratory response to high-priority public health threats. The LRN for biological threats (LRN-B) provides a laboratory infrastructure to respond to emerging infectious diseases. Since 2012, the LRN-B has been involved in 3 emerging infectious disease outbreak responses. We evaluated the LRN-B role in these responses and identified areas for improvement. LRN-B laboratories tested 1097 specimens during the 2014 Middle East Respiratory Syndrome Coronavirus outbreak, 180 specimens during the 2014-2015 Ebola outbreak, and 92 686 specimens during the 2016-2017 Zika virus outbreak. During the 2014-2015 Ebola outbreak, the LRN-B uncovered important gaps in biosafety and biosecurity practices. During the 2016-2017 Zika outbreak, the LRN-B identified the data entry bottleneck as a hindrance to timely reporting of results. Addressing areas for improvement may help LRN-B reference laboratories improve the response to future public health emergencies.

The Laboratory Response Network (LRN), established in 1999, is a partnership of the Centers for Disease Control and Prevention (CDC), the Federal Bureau of Investigation, and the Association of Public Health Laboratories. The objective of the LRN is to ensure an effective response to chemical and biological terrorism by improving the nation’s public health laboratory infrastructure. LRN laboratories provide timely, accurate laboratory test results for various chemical threats (eg, cyanide, toxic metals, nerve agents) and biological threats (eg, anthrax, plague, tularemia) to inform public health decision-making.

The LRN for biological threats (LRN-B) is a component of the LRN that comprises sentinel-, reference-, and national-level laboratories.¹ Sentinel laboratories include thousands of private and commercial laboratories that perform routine diagnostic testing services and have the microbiology subspecialty capabilities to perform standardized protocol-driven steps² in identifying infectious disease agents. When sentinel laboratories have determined that the causative agent in a specimen cannot be identified and additional testing must occur (rule-out and refer), specimens may be sent to LRN-B reference laboratories.

There are 123 domestic LRN-B reference laboratories, including federal, state, and local public health, veterinary, military, and food testing facilities. These laboratories can detect and confirm the presence of biological threat agents. National laboratories (CDC, US Army Medical Research Institute of Infectious Diseases, and Navy Medical Research Center) have pathogen-specific subject matter expertise and can provide complete characterization of a pathogen using tools such as whole-genome sequencing. When LRN-B reference laboratories detect an unusual or unique bacteria or virus, they refer the agent to a national laboratory.

The LRN-B structure enables rapid distribution of diagnostic tests and reliable communication of testing results back to CDC. CDC’s LRN-B program office provides LRN-B reference laboratories with standardized reagents, protocols, and training on safety controls, including the use of Biosafety Level 3 laboratories and personal protective equipment. Biosafety levels are associated with the risks of working with biological organisms and have specific controls for use and containment of each organism. LRN-B reference laboratories are required to demonstrate proficiency in performing LRN-B assays. They are also required to report results back to CDC by using an electronic data exchange mechanism.

Expansion of LRN-B Capability: Emerging Infectious Diseases

Since its founding, the LRN has expanded its role beyond testing for a bioterrorism response to including testing for emerging infectious diseases such as Middle East Respiratory Syndrome Coronavirus (MERS-CoV), Ebola virus, and Zika virus.

Middle East Respiratory Syndrome Coronavirus

MERS-CoV is a novel coronavirus that was discovered in 2012 in the Kingdom of Saudi Arabia.³ MERS-CoV infection causes severe acute respiratory illness with symptoms of fever, cough, and shortness of breath. As of the end of April 2019, epidemiologic data showed that 838 of the 2428 patients infected with MERS-CoV had died (case fatality rate: 34.5%).⁴ To date, all cases have been associated with living in or traveling to the Arabian Peninsula.⁵ Much like other coronaviruses, human-to-human transmission is believed to occur through respiratory droplets spread by coughing. This zoonotic coronavirus has been primarily associated with camels but may have additional animal reservoirs.

Ebola Virus

Ebola virus was first discovered in 1976 near the Ebola River in what is now the Democratic Republic of the Congo.⁶ Since then, 4 other viruses in the same genus have been discovered. These viruses, collectively called ebolaviruses, have appeared sporadically, primarily in Africa. It is believed that the virus is zoonotic and that bats are the most likely reservoir. Ebola virus can cause severe, often deadly, disease in humans and nonhuman primates (monkeys, gorillas, and chimpanzees).⁷ Medical or response personnel can become infected through exposure to patient blood or other bodily fluids.

Zika Virus

The Zika virus, a flavivirus primarily transmitted by mosquitos, was identified in a non-human primate in Uganda in 1947.⁸ Zika virus can also be transmitted sexually and from a pregnant woman to her fetus.⁸ Disease in humans has been characterized as mild with fever, conjunctivitis, skin rash, malaise, and muscle and joint pain. Many persons infected with Zika virus are asymptomatic, making case identification difficult. Before 2015, outbreaks of Zika virus disease had been documented in Africa, the Americas, Asia, and the Pacific. In May 2015, Brazil identified an outbreak of Zika virus disease and later that year reported an association between infection and congenital Zika syndrome, including microcephaly, and Guillain-Barré syndrome.⁹ The outbreak spread throughout the Americas in 2015-2016. The first case identified in the United States was in Puerto Rico in December 2015.¹⁰

The LRN-B reference laboratories have taken a leading role in diagnostic testing for emerging infectious diseases, including MERS-CoV, Zika virus, and Ebola virus. Generally, when a novel threat such as MERS-CoV, Ebola, or Zika emerges, no commercial products are available to detect the causative agent of these diseases. CDC is often the first organization to develop and validate a detection assay that can be distributed to LRN-B national and reference laboratories as soon as a US Food and Drug Administration (FDA) authorization is granted. Results generated by LRN-B reference laboratories can then assist public health officials in making decisions and taking actions that mitigate or prevent disease.

This case study provides an overview of 3 challenging emerging infectious disease outbreak responses in which LRN-B reference laboratories responded to domestic biological threats: MERS-CoV, Ebola virus, and Zika virus. We also identify lessons learned and areas for improvement. To our knowledge, no studies have documented the activities of the LRN-B under the conditions of an active infectious disease outbreak. Such analysis would be invaluable for improving preparedness for future outbreaks.

LRN-B Response to Emerging Infectious Diseases

In all 3 responses to emerging infectious disease outbreaks described in this case study, the Secretary of the US Department of Health and Human Services, under section 319 of the Public Health Service Act,¹¹ determined that the emerging infectious disease presented a public health emergency in the United States, or a material threat to the US population sufficient to affect national security, prompting the initiation of numerous response activities.¹² Because no FDA-cleared or -approved medical countermeasures existed that could be deployed immediately to address the emerging threat, the FDA was able to use its emergency use authorization (EUA) authority to facilitate the availability and use of the medical countermeasures needed in these circumstances. In the United States, an EUA is a legal means for the FDA to approve new medical countermeasures or new indications for previously approved medical countermeasures during a declared emergency. One medical countermeasure often needed in a public health emergency is a clinical diagnostic assay for patients meeting criteria for testing.

The FDA granted EUAs to CDC (for MERS-CoV, Ebola virus, and Zika virus) and the US Department of Defense (DoD, for Ebola virus) to manufacture and deploy diagnostic tests to detect these viruses in human clinical specimens. The agencies manufactured, quality control–tested, and deployed diagnostic tests to LRN-B reference laboratories. The LRN-B program office at CDC provided verification panels, protocols, and technical support for these EUA assays in collaboration with CDC subject matter experts. CDC provided diagnostic testing algorithms and procedures to identify cases, collect and ship the correct specimens, perform testing, and report results.

LRN-B reference laboratories submit testing results to CDC via dedicated electronic laboratory reporting systems. Transmission of these data uses data element standards and security measures to address laboratory data management and exchange. All results from the EUA assays for the 3 viruses were reported to CDC by using these electronic laboratory reporting methods.

Middle East Respiratory Syndrome Coronavirus

Because MERS-CoV was a novel coronavirus, no known medical countermeasures existed when it was discovered in 2012. The United States needed a diagnostic assay to test suspected infections in travelers from the Arabian Peninsula. CDC subject matter experts, public health experts, and virologists collaborated to develop the CDC Novel Coronavirus 2012 real-time reverse transcriptase polymerase chain reaction (rRT-PCR) assay to detect MERS-CoV in clinical specimens from suspected cases.¹³ The FDA granted an EUA to develop the assay on June 5, 2013.¹⁴ LRN-B reference laboratories completed CDC requirements, including testing, interpreting, and reporting results on a panel of contrived specimens, to perform this test on clinical specimens. Health care providers, clinical laboratories, and epidemiologists used new case definitions created by subject matter experts to identify suspected cases of MERS-CoV. In accordance with LRN-B algorithms described previously, respiratory specimens from these suspected cases were referred by sentinel laboratories to LRN-B reference laboratories for testing.

In 2013, LRN-B reference laboratories tested and reported results on 141 specimens for MERS-CoV with no presumptive positive cases detected. In May 2014, CDC worked closely with LRN-B reference laboratories to confirm 2 travel-associated cases of MERS-CoV in the United States: 1 in Indiana and 1 in Florida. The cases were not linked, although both infections were in health care providers who had lived and worked in the Kingdom of Saudi Arabia, where they are believed to have been infected. As part of the public health investigation for both cases, specimens were collected from household members and the health care workers with whom the 2 infected US persons had close contact. Because the initial EUA did not contain an approval to test case contacts, an amendment to the EUA was submitted to FDA for authorization to test this population. With FDA authorization, LRN-member laboratories tested specimens using the CDC Novel Coronavirus 2012 rRT-PCR assay. All household members and the health care workers who cared for the patients tested negative for infection with MERS-CoV.¹⁵ In 2014, 49 LRN-B reference laboratories and the DoD reported 1097 MERS-CoV test results to CDC (unpublished data).

Although no cases of MERS-CoV have occurred in the United States since 2014, 66 LRN-B reference laboratories continue to test clinical specimens from patients suspected of infection with MERS-CoV. More than 2500 test results for MERS-CoV have been reported to CDC by LRN-B laboratories since the assay was deployed in 2013 (unpublished data). Infected persons continue to be identified in the Arabian Peninsula, emphasizing the need to maintain diagnostic testing capability in LRN-B reference laboratories for specimens from symptomatic travelers.

Ebola Virus

In 2014, an unprecedented outbreak of Ebola virus disease occurred in 3 countries in West Africa, with many local cases and 7 countries reporting imported cases.^16,17 As with MERS-CoV, no FDA-cleared or -approved medical countermeasures were available in the United States for Ebola virus disease. On August 5, 2014, DoD received an EUA for the DoD EZ1 rRT-PCR assay for the presumptive detection of Ebola Zaire virus.¹⁴ CDC collaborated with DoD to acquire and distribute this test to LRN-B reference laboratories that were prepared to conduct Ebola virus testing. Blood specimens that were presumptive positive by the DoD assay had to be referred to the CDC Viral Special Pathogens Branch for confirmation using molecular test methods. LRN-B reference laboratories used the DoD assay through spring 2015, when the FDA granted 2 EUAs for the CDC Ebola Virus NP rRT-PCR assay and the CDC Ebola Virus VP40 rRT-PCR assay¹⁴ developed by the CDC Viral Special Pathogens Branch. LRN-B reference laboratories subsequently transitioned to the CDC assays, which are still in use today. As of this writing, 68 LRN-B reference laboratories had completed CDC requirements for performing the 2 CDC assays for emergency use.

The diagnostic tests for Ebola virus disease presented biosafety and biosecurity challenges that LRN-B reference laboratories typically do not encounter. The laboratories that performed testing for Ebola virus needed to establish and practice heightened safety precautions and procedures in high-containment facilities, based on CDC guidance for specimen collection, inactivation, reporting results, and waste management.¹⁸ Furthermore, LRN-B reference laboratories, as an extension of their routine outreach to public health partners, coordinated and communicated best practices for infection control and biosafety with sentinel laboratories, hospitals, and public health officials. This outreach was needed because local public health was not prepared to respond to such a deadly, contagious disease.

During this international outbreak, dozens of suspected cases were identified in several US states. As part of the testing algorithm, Ebola testing was performed only for patients who met criteria for persons under investigation, in consultation with CDC.¹⁸ These criteria were having clinical signs and symptoms consistent with Ebola virus disease and an epidemiologic risk factor (eg, contact with a person with confirmed Ebola virus disease) within 21 days before the onset of symptoms. CDC and LRN-B reference laboratories worked closely with clinicians and sentinel laboratory staff members to identify suspected cases. Since 2014, LRN-B reference laboratories have tested and reported to CDC the results for 180 specimens of Ebola virus disease, including the 4 confirmed US cases.¹⁹

Zika Virus

State and local public health laboratory preparation was essential for testing travelers arriving in the United States from Zika-affected areas. CDC collaborated with the FDA to obtain EUAs for a Zika immunoglobulin M antibody capture enzyme-linked immunosorbent assay (Zika MAC-ELISA) and an rRT-PCR assay (Trioplex rRT-PCR) in February 2016 and March 2016, respectively.¹⁴ CDC deployed the Zika MAC-ELISA to detect anti-Zika antibodies and the Trioplex rRT-PCR assay to detect Zika, dengue, and chikungunya virus RNA. rRT-PCR can usually detect the presence of RNA when patients are symptomatic soon after infection. The Zika MAC-ELISA detects antibodies to Zika up to 12 weeks postexposure. By December 2016, 61 LRN-B reference laboratories had implemented the Zika MAC-ELISA, and 89 laboratories had implemented the Trioplex rRT-PCR assay.

From March 2016 through April 2017, LRN-B reference laboratories reported 92 686 test results to CDC, of which 50 492 (54.5%) were from the Zika MAC-ELISA and 42 194 (45.5%) were from Trioplex. LRN-B reference laboratories tested specimens by one or both tests based on the CDC testing algorithm.²⁰ Of the 50 492 Zika MAC-ELISA results, 2790 (5.5%) specimens tested were presumptively positive for Zika virus antibodies (Table 1). CDC or CDC-designated laboratories confirmed presumptive positive Zika MAC-ELISA results by using the plaque reduction neutralization test. Of the 42 194 Trioplex results, 1636 (3.9%) specimens tested positive for Zika virus RNA (Table 2). LRN-B reference laboratories also detected dengue and chikungunya RNA in 195 and 22 specimens, respectively (data not shown).

Table 1.

Zika MAC-ELISA results reported by Laboratory Response Network for Biological Threats Laboratories to CDC, March 2016 through April 2017

Specimen Type	No. of Tests	Zika-Positivea Tests, No. (%)
Serum	50 242	2790 (5.6)
Cerebrospinal fluid	250	8 (3.2)
Total	50 492	2798 (5.5)

Abbreviations: CDC, Centers for Disease Control and Prevention; MAC-ELISA, immunoglobulin M antibody capture enzyme-linked immunosorbent assay.

^aPositive results are presumptive until confirmed by plaque reduction neutralization test per CDC’s Zika MAC-ELISA instructions for use.

Table 2.

Zika virus RNA test results from Trioplex real-time reverse-transcriptase polymerase chain reaction assay, reported by Laboratory Response Network for Biological Threats Laboratories to CDC, March 2016 through April 2017

Specimen Type	No. of Tests	Zika-Positive Tests, No. (%)
Serum	23 725	756 (3.2)
Urine	17 510	862 (4.9)
Amniotic fluid	55	1 (1.8)
Whole blood	861	17 (2.0)
Cerebrospinal fluid	43	0
Total	42 194	1636 (3.9)

Abbreviation: CDC, Centers for Disease Control and Prevention.

Lessons Learned

Each public health emergency described here was unique and required its own solution. For example, support from LRN-B reference laboratories enabled rapid testing of specimens from health care workers and household contacts associated with the 2 cases of MERS-CoV infection.¹⁵ Because MERS-CoV was novel, data on human-to-human transmission were unknown. A similar virus, the severe acute respiratory syndrome coronavirus, was first found in China in November 2002 and led to severe respiratory illness detected in more than 8000 persons in 24 countries and 774 deaths.²¹ The LRN-B reference laboratories provided diagnostic evidence that person-to-person transmission of MERS-CoV did not occur in the United States in 2014.¹⁵

Laboratory biosafety and biosecurity must be addressed in every laboratory response, but there were heightened challenges with handling and testing specimens from patients with suspected Ebola virus disease. The infectious dose of Ebola virus via aerosolization has been estimated at only a few virus particles²² and the virus is categorized as an agent to be handled in Biosafety Level 4 laboratories. LRN-B reference laboratories consulted with CDC subject matter experts for biosafety guidance; the subject matter experts, in turn, communicated identified best practices for infection control to the sentinel laboratories. As a result, CDC and the Association of Public Health Laboratories collaborated to form the CDC/Association of Public Health Laboratories Biosafety and Biosecurity Program.²³ This initiative, funded through the Epidemiology and Laboratory Capacity for Infectious Diseases cooperative agreement, aims to enhance biosafety practices in all laboratories by providing additional training, supporting salaries for biosafety officers in public health laboratories, and identifying site-specific risks and gaps.

During the Zika virus outbreak, the volume of testing was substantially greater for Zika virus than for MERS-CoV or Ebola. LRN-B reference laboratories tested 1097 Zika virus specimens during the MERS-CoV outbreak, 180 Zika virus specimens during the Ebola outbreak, and 92 686 Zika virus specimens during the Zika outbreak. In high-volume testing LRN-B reference laboratories, hundreds of specimens arrived daily for Zika virus testing. Because of the potential pregnancy-related consequences of infection with Zika virus, physicians prioritized specimens tested from pregnant women. Public health laboratories worked closely with CDC and other public health partners to implement complex testing algorithms^20,24-27 to support patient care. LRN-B reference laboratories had to establish communication with health care providers who did not normally submit specimens to public health laboratories. An additional challenge for reference laboratories was complying with the requirement to report all test results to the LRN-B program office at CDC while accessioning, testing, and reporting to submitters large numbers of specimens. In 2017, one solution implemented to address specimen management in an emergency was to cross-train other public health staff members to assist with accessioning and reporting. In response to the Zika virus outbreak and in collaboration with the Association of Public Health Laboratories, the LRN-B program office has identified and is implementing reporting processes that will require less laboratory staff member time to submit data to CDC.

Conclusion

When the LRN was first established in 1999, its capability to respond to biological threats was limited to only a few public health laboratories. Currently, the LRN is a robust network of laboratories that have demonstrated that they can adopt new diagnostic tests within days or weeks, create new biosafety procedures to manage high-risk specimens, report test results in a timely manner to influence public health action, and collaborate with public health authorities to detect emerging pathogens. LRN-B reference laboratories will continue to prepare to test for biological threat agents such as anthrax and smallpox. However, detection of emerging infectious diseases is increasingly important in public health. LRN engagement in emerging infectious disease response may depend on the size, scale, and consequences of the public health threat. But, because the LRN is integrated with subject matter experts at CDC and other agencies, preparedness efforts for other threatening emerging infectious diseases will continue.