Seminario 11

PERSPE C T I V E

The New Age of Molecular Diagnostics for Microbial Agents

FOCUS ON RESE ARCH

The New Age of Molecular Diagnostics for Microbial Agents Richard Whitley, M.D. Related article, p. 991

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ow, in this era of molecular diagnostic tests, can we best determine whether there is a causal relationship between the presence of a genetic signature of an infectious agent and disease? In recent years, molecular techniques have been applied successfully in the identification of infectious agents such as Borna virus, Kaposi’s sarcoma–associated herpesvirus (human herpes­ virus 8), West Nile virus, and the severe acute respiratory syndrome (SARS) coronavirus.1 Currently, the majority of surveillance and discovery efforts use methods based on sequences of known agents — namely, competitive polymerase chain reaction (PCR) and microarrays. Such efforts fail, however, when the agents in question are truly novel or sufficiently distant in sequence from related agents that they confound hybridization. A recent case in point was that of a febrile illness that, after similar clinical courses, led to the deaths of three patients who had recently received visceral-organ transplants from a single donor. All three patients had identical viral sequences, and in one of them, seroconversion to this agent occurred, indicating recent exposure. As Palacios and colleagues report in this issue of the Journal (pages 991–998), new diagnostic techniques were required in order to identify what turned out to be a new arenavirus that had been transmitted through organ transplantation. In brief, RNA from two recipi-

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ents’ transplanted organs — a liver and a kidney — was used as starting material to create a library of more than 100,000 sequences. Bioinformatics tools were then used to subtract human sequences and identify the residual sequences that might represent microbial pathogens. In the short term, the findings of Palacios et al. clarify some aspects of the natural history of arena­ virus infection in immunocompromised patients at high risk for infection. More significantly, this application of new diagnostic techniques may ultimately transform microbiologic testing. The investigators describe a method of adapting high-throughput DNA pyrosequencing to pathogen discovery and discuss the innovations in specimen preparation and bioinformatics that permitted them to identify this new arenavirus in a cluster of cases of fatal encephalitis. Pyro­ sequencing entails the enzymatic incorporation of complementary nucleotides into an elongating DNA chain; this process results in pyrophosphate release. A camera registers this emission as light. This method has been adapted to a slide format, wherein hundreds of thousands of wells containing individual microbeads coated with DNA are sequenced in parallel (see diagram). High-throughput pyrosequencing has been used primarily to sequence large genomes; to my knowledge, it has not previously been applied to pathogen discov-

ery. However, when Palacios et al. found that the classic application of typical diagnostic assays, including viral culture, electron microscopy, PCR, and panmicrobial microarrays, failed to identify an infectious agent, they turned to pyrosequencing. Although derived sequences at the nucleic acid level were not informative, bioinformatic analyses at the protein level revealed footprints of a potential causative agent. Using these sequences as a foundation for developing specific molecular reagents, the investigators cloned the remainder of the viral genome by means of PCR, propagated it in cell culture, defined its morphologic characteristics by means of electron microscopy, and created molecular and serologic assays for detection of infection. Such metagenomic pyro­ sequencing permits the rapid identification of all nucleic acid sequences in a sample. It has been the primary method used for all sequencing of both human and bacterial genomes. Other applications have included studies of microbial diversity — for example, the sequencing of HIV and of bacterial microflora detected in either healthy people or those with a given disease.2,3 Unlike competitive PCR or array methods, in which investigators are limited by sequence information for known organisms and must make choices regarding the range of pathogens to be considered, meta­ genomic sequencing is unbiased and provides the opportunity to

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PERSPECTIVE

The New Age of Molecular Diagnostics for Microbial Agents

DNA-covered microbeads

Clinical specimen sequenced by chemiluminescence

The potential for the application of this technique is significant. Infections of the central nervous system often pose difficult diagnostic dilemmas. In spite of the Unexplained Encephalitis Project sponsored by the Centers for Disease Control and Prevention, as many as 40% of patients with altered mentation, fever, and inflammation of the central nervous system never receive a diagnosis that establishes the cause of their illness. Acute respiratory tract infections are a leading cause of childhood illness and death, accounting for nearly 2 million deaths annually. Even in state-ofthe-art laboratories, an agent is identified in only 30 to 60% of cases. Similarly, in the United States alone, enteric infection Draft 2 in the 2/01/08 results hospitalization of Author Whitley nearly 500,000 adults and 160,000 1 Fig # Title children annually. Although the vast majority of these cases reME Hogan Malina DE solve without permanent sequelArtist KMK/SW ae, as many asNOTE: 5000 deaths each AUTHOR PLEASE Figure has been redrawn and type has been reset year are to foodborne Pleaseattributed check carefully Issue date 3/06/08 infections of unknown cause.4 This new application of meta­ genomic pyrosequencing may well aid in the identification of unknown microbial agents that cause human disease. Palacios et al. also expand our knowledge of arenavirus infection that occurs after organ transplantation. Previously, two clusters of arenavirus transmission through solid-organ transplantation had been reported. The prototypic arenavirus is lymphocytic choriomeningitis virus (LCMV). Typically, disease attributed to LCMV in an otherwise healthy host is asymptomatic or associated with a mild febrile illness; however, it has been implicated in aseptic COLOR FIGURE

Nucleic acid sequences obtained

Viral genome cloned

Virus propagated in tissue culture

Infection confirmed through serologic and molecular assays Use of High-Throughput DNA Pyrosequencing for Pathogen Discovery.

consider a broader spectrum of organisms. Thus, it is ideal for applications in which unknown bacteria, fungi, parasites, or viruses may be involved.

meningitis in rare instances. According to the current report, all three patients died 4 to 6 weeks after transplantation following an encephalopathic illness; this indicates that this arenavirus may be more virulent than others. Because of the protracted incubation period, health care providers might easily have sought the cause of the illness somewhere other than the transplanted organs and might have failed to pursue and identify the new infectious agent. As contemporary molecular diagnostic approaches are applied to medical conditions with potentially infectious causes, meta­ genomic pyrosequencing should permit us to expand our list of offending pathogens. The identification of new agents, in turn, may well lead to the development of new prophylactic and therapeutic interventions. No potential conflict of interest relevant to this article was reported. This article (10.1056/NEJMp0708085) was published at www.nejm.org on February 6, 2008. Dr. Whitley is a professor of pediatrics, microbiology, medicine, and neurosurgery at the University of Alabama at Birmingham, Birmingham. 1. Lipkin WI, Briese T. Emerging tools for microbial diagnosis, surveillance and discovery. In: Lemon SM, Hamburg MA, Sparling PF, Choffnes ER, Mack A, eds. Global infectious disease surveillance and detection: assessing the challenges — finding solutions: workshop summary. Washington, DC: Institute of Medicine, 2007:177. 2. Ley RE, Knight R, Gordon JL. The human microbiome: eliminating the biomedical/ environmental dichotomy in microbial ecology. Environ Microbiol 2007;9:3-4. 3. Turnbaugh PJ, Ley RE, Hamady M, FraserLiggett CM, Knight R, Gordon JL. The human microbiome project. Nature 2007;449:804-10. 4. Frenzen PD. Deaths due to unknown foodborne agents. Emerg Infect Dis 2004; 10:1536-43. Copyright © 2008 Massachusetts Medical Society.

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