key: cord-0796447-1h87wldg
authors: Dong, J.; Ismail, N.; Walker, D.H.
title: Molecular Testing in Emerging Infectious Diseases
date: 2016-10-14
journal: Diagnostic Molecular Pathology
DOI: 10.1016/b978-0-12-800886-7.00015-7
sha: ea10c63c06c75f54b579ed1840db4f5ac4a667e7
doc_id: 796447
cord_uid: 1h87wldg

It was widely believed in the late 1960s that infectious diseases had been conquered by vaccines and antibiotics and humans were no longer under threat by microbial pathogens. Yet, since that time more than 60 pathogens have been discovered that can cause serious emerging infectious diseases. Molecular methods have played critical roles in the discovery, monitoring, and clinical diagnostics of emerging pathogens. In this chapter, we present well-recognized emerging pathogens. We provide examples of the utility of molecular assays in research and clinical care of emerging infectious diseases. We also discuss some theoretical and practical limitations of molecular tests and the future prospects of expanding molecular diagnostics for emerging pathogens based on new advances of knowledge and technologies.

By the late 1960s there was a widespread opinion that the era of infectious diseases was finished and that vaccines and antibiotics had controlled microbial pathogens. Indeed, it was commonly believed that we had discovered the important agents of infections and that there was little left to do in this scientific field. ". . .The war on infectious diseases is over and we have won. . ." was an often repeated conclusion. Yet in the quarter of a century between 1967 and 1992 more than 30 previously unrecognized pathogens were discovered as the etiologic agents of human infectious diseases (Table 15 .1). Some of the diseases were well characterized, but the causes had been unknown. Other novel syndromes were recognized and the etiologic agents identified including acquired immunodeficiency syndrome (AIDS) and human immunodeficiency virus (HIV). Nevertheless, the general belief was that infectious diseases were less important than cardiovascular diseases and cancer, and they were not favored for research support and public health attention.

In 1992, the concept of emerging infectious diseases was defined and brought to the attention of physicians and scientists by a very widely distributed and read publication from the Institute of Medicine of the National Academies of Sciences, Emerging Infections: Microbial Threats to Health in the United States. The emergence of at least 16 novel infectious agents over the following 12 years (Table 15. 2) emphasized that this phenomenon would be a continued series of events. The causes of awareness of the presence of an unknown pathogen are the abrupt onset of a cluster of severe illness (eg, Legionella pneumonia at a convention of the American Legion), recognition of distinct gross or microscopic pathologic lesions (eg, pseudomembranous colitis caused by Clostridium difficile), and clinical laboratory microscopy (eg, intramonocytic inclusions of Ehrlichia chaffeensis in patients with human monocytotropic ehrlichiosis). In numerous other instances application of an advanced technologic method identified the etiology of a well-defined syndrome (eg, noroviruses in Norwalk diarrheal illness; an outbreak had occurred and samples retained from years earlier).

Many methods have been employed for the initial detection and identification of novel emerging pathogens including microscopy, bacterial culture, cell culture, animal inoculation, electron microscopy, archaic serologic tests, cross-reactive serologic tests, serendipitous serologic testing, and immunohistochemistry. However, currently molecular methods including probe hybridization, polymerase chain reaction (PCR) that amplifies the target or the signal, and nucleic acid sequencing are the most prominent methods for detection and characterization of newly emerging pathogens, both for discovering the agent and for determining that it is truly novel [1À11] .

An example of the application of molecular methods to the identification of previously unidentified 

agents of human infection is that of hepatitis C virus (HCV). After the discoveries of hepatitis A and B viruses, it was clear that the majority of cases of posttransfusion hepatitis were due to a condition designated non-A non-B hepatitis. The disease was transmissible to chimpanzees. In 1989, plasma from an infected chimpanzee was pelleted by ultracentrifugation and nucleic acids extracted from the pellet. cDNA was synthesized from both RNA and DNA with random primers and reverse transcriptase. Screening identified an RNA-encoded clone that expressed an antigen that reacted with antibodies of infected subjects. Eventually the complete genomes of all of the genotypes of HCV were determined, and a novel species most closely related to flaviviruses was established [12, 13] .

Another dramatic emergence of a viral disease occurred in 1993 in the Four Corners region of the southwestern United States. A mysterious highly lethal respiratory illness was investigated by a team from the Centers for Disease Control and Prevention (CDC). Extensive serologic screening of numerous antigens revealed unexpected reactivity with antigen of hantaviruses from other parts of the world that caused renal disease and hemorrhagic fever, and immunohistochemistry detected hantaviral antigen in pulmonary endothelium. Regions within the M segment of the RNA hantaviral genomes encoding G2 protein that are highly conserved were targeted by primers for nested PCR after reverse transcriptase generation of cDNA. Tissues from infected patients were analyzed, and the PCR products sequenced revealing a novel hantavirus subsequently named Sin Nombre virus. Viral sequences were identified in other patients and in Peromyscus maniculatus rodents, the reservoir. The story of hantaviral pulmonary syndrome unfolded to reveal related agents in many locations in North, Central, and South America [14, 15] .

A novel coronavirus in association with cases of severe acute respiratory syndrome (SARS-CoV) emerged in southern China in late 2002 and spread to 37 countries in five continents with 8273 confirmed cases and 775 deaths. No further cases have been reported since July 2003 [16] . RT-PCR, cloning, and sequencing contributed to identification of the SARS-CoV within weeks of the first cases reported in 2003 [17À20] and enabled rapid development of effective molecular diagnostic assays for routine clinical use [21, 22] . SARS-CoV is associated with high mortality. Thus, timely and accurate diagnosis is needed to prevent the spread of this contagious disease. SARS-CoV spreads by respiratory secretions and airborne transmission. Early in the illness, SARS cannot be distinguished from common respiratory infections based on clinical symptoms [16] . During the SARS epidemic, PCR-based molecular testing was helpful because of its ability to rapidly screen for many viruses. After the identification of SARS-CoV, specific RT-PCR and serological assays were developed, and RT-PCR detected infection before the appearance of antibodies when the risk of transmission is greatest [16À22] .

The bacterial rrs gene encoding 16S rRNA was recognized as a valuable phylogenetic tool for discrimination and identification of bacterial species. David Relman crafted this tool into an approach to identify an unknown etiologic agent by PCR of the rrs gene with primers that corresponded to genomic regions that were conserved among eubacteria. Using this approach, he amplified and determined bacterial DNA sequences from bacillary angiomatosis lesions of patients with AIDS. Comparison with a bacterial gene database revealed that the DNA sequences matched bacteria that are currently named Bartonella henselae and B. quintana. Serendipitous testing of a patient who also had been diagnosed with cat scratch disease led to the recognition that B. henselae was also the long sought-after etiology of this well-characterized disease [23, 24] .

The same approach to discovery using rrs gene amplification and DNA sequencing led to the identification of what is currently classified as Anaplasma phagocytophilum as the etiologic agent of tick-transmitted human granulocytotropic anaplasmosis [25, 26] . Subsequently Ehrlichia ewingii was recognized as another human tick-borne pathogen among patients evaluated in a molecular diagnostics laboratory who tested negative for E. chaffeensis infection [27À29]. More recently, Bobbi Pritt at Mayo Clinic noted that the melting curve of the DNA amplicons in a real-time PCR assay for Anaplasmataceae differed from the expected curves of known pathogens for a group of patients in Wisconsin and Minnesota. Sequence analysis identified another novel tick-borne pathogen tentatively designated Ehrlichia muris-like agent [8, 9] .

The discovery of a novel bunyavirus that has caused thousands of human infections with a case fatality rate of 12% in 15 provinces in China relied upon a molecular approach to identify the viral agent. Xue-Jie Yu investigated an outbreak in China that was thought to be due to severe infection with A. phagocytophilum. He noted that some of the clinical manifestations differed from those of anaplasmosis. He observed cytopathic effect in DH-82 cells inoculated with clinical samples rather than the typical morulae formed by Anaplasma species in infected cells. Ultrastructural analysis suggested that the pathogen causing the outbreak was a virus that belongs to the family of bunyaviruses. Based on the known sequence of bunyaviruses, PCR primers were designed, which yielded no amplicons. Subsequently, he began sequencing the RNA of heavily infected cells and discarded the sequences of the culture host species, Canis familiaris. This approach enabled him to determine that he had recovered a novel Phlebovirus of the family Bunyaviridae. He accomplished this feat without the use of nextgeneration sequencing (NGS) [10] . The application of NGS now allows us to obtain an abundance of viral gene sequences from infected host cells and the discovery of further novel viral and bacterial agents.

Molecular technologies have been critical in the initial discovery of agents of emerging infectious diseases. These methods have also been routinely used for further characterization of pathogen strains and sequence variations. Molecular data are now widely used in molecular epidemiological studies and phylogenetic analyses, and sequence comparisons have been performed to facilitate the specific detection of genetically diverse strains/sequences and investigate the origin, transmission, distribution, biology, and diversity of these pathogens [12,13,21,30À33] , which are fundamentally important in the prevention and tracking of disease outbreaks. Knowledge of sequence variations is used in the development of accurate diagnostic assays and for the design of effective treatment strategies of diseases caused by these agents. Molecular epidemiological studies are critical for public health surveillance [14,15,34À65] . We provide here examples of how molecular tests contributed to public health surveillance and patient care.

Continuing challenges in influenza include the sporadic human cases of highly pathogenic avian H5N1 influenza, emergence of pandemic H1N1 influenza in 2009 [62, 69] , and human infections with avian H7N9 influenza in 2013 [11] . Influenza A virus undergoes continuous antigenic drift and sporadic antigenic shifts in the viral surface glycoproteins, hemagglutinin (H) and neuraminidase (N). Influenza A has 15 H and 9 N subtypes. Antigenic H and N subtypes to which humans lack immunity are introduced by reassortment of virus genes and cause pandemics, whereas H and N antigenic variants determined by point mutations cause seasonal influenza epidemics [66, 67] . Molecular assays are the preferred method for identification and surveillance of new strains of influenza A infections [11, 62, 67] . Influenza A has no pathognomonic symptoms, and diagnosis based on clinical signs is correct in only two-thirds of patients [68, 70] . Therefore, sensitive and rapid laboratory tests are required to diagnose and guide antiviral treatment. Recently, multiplex molecular assays for respiratory viruses including influenza viruses have been developed, and several have received approval/clearance by the US Food and Drug Administration (FDA) for routine clinical use (Table 15 .2, listed under pandemic H1N1 influenza virus). These assays provide rapid and sensitive tests for respiratory viral infections.

Human immunodeficiency virus 1 (HIV-1) was discovered in 1983 (Table 15 .1). It is a single-stranded, positive-sense, enveloped RNA retrovirus (http:// www.hiv.lanl.gov/). HIV-1 can cause AIDS, a chronic disease leading to immunodeficiency and susceptibility to opportunistic infections (http://www.who.int/ hiv/en/). Three groups of HIV-1 have been identified based on sequence similarity, including M (main), O (outlier), and N (non-M/non-O) (http://www.hiv.lanl. gov/). Of the three groups of HIV-1, group M dominates the global epidemic and is further classified into subtypes A, B, C, D, F, G, H, J, and K. In addition, circulating recombinant forms (CRFs), mosaic viruses formed between subtypes during co-or superinfection, have also been recognized (http://www.hiv. lanl.gov/). Although subtype B is predominant in North America and Europe, non-B variants represent more than 90% of HIV-1 circulating globally [71] . In recent years, the prevalence of non-B subtypes and CRFs in the United States is steadily increasing due to increased international travel and immigration [72À74] . Sequencing data of HIV-1 genomes have been used for tracking HIV epidemics and for the design of accurate viral detection, viral load, and HIV-1 drugresistance genotype assays to guide clinical use of antiretroviral treatment [38, 75] . The recent availability of the NGS approach has greatly facilitated generation of HIV-1 sequences and detection of quasispecies, which can improve understanding of HV-1 infection, pathogenesis, and epidemics [38, 76, 77] .

It is believed that 150 million people worldwide are infected with HCV (http://www.who.int/mediacentre/factsheets/fs164/en/). Between 70% and 80% of people infected with HCV will develop chronic infection. Chronic hepatitis C is closely associated with the development of cirrhosis and hepatocellular carcinoma and is the most common cause of adult liver transplantation in the United States and the world (http://www.cdc.gov/hepatitis/hcv/). A comparison of HCV genomic sequences from around the world revealed substantial heterogeneity of nucleotide sequences. Phylogenetic analyses have shown that HCV strains can be classified into six genotypes (numbered 1À6) and a large number of subtypes within each genotype [78] . HCV genotypes 1, 2, and 3 appear to have a worldwide distribution, but their relative prevalence varies from one geographic area to another. HCV genotype 1 is reported to be the most common in the United States [79À81]. HCV virus genome sequencing has been used to study HCV genotypes, subtypes, quasispecies, and mutations. The information is important for epidemiological studies, to trace the source of infection, for development of direct acting antiviral (DAA) therapy, and for understanding of susceptibility and resistance to antiviral treatment [82À85].

Many methods have been employed for the clinical diagnostics of emerging pathogens including microscopy, bacterial culture, cell culture, and serologic tests. However, each of these methods has its own limitations that must be considered by the clinical laboratory. For example, even though cell culture could be considered as the gold standard in diagnosis of infection with emerging obligate intracellular bacteria such as Rickettsia or Ehrlichia, the requirement for biological safety laboratory level 3 (BSL-3) (for Rickettsia) or BSL-2 (for Ehrlichia) makes this test difficult to implement in many conventional clinical microbiology laboratories. Further, the prolonged turnaround time (TAT) (eg, detection by culture at 7À10 days after sample processing) makes this approach impractical. Results from such a test are not clinically useful due to failure to guide therapy during the early stages of infection when appropriate antibiotic treatment is highly effective. Similar to culture, serologic tests such as indirect immunofluorescence assays, which rely on detection of antigen-specific antibodies, have several limitations such as low sensitivity during the early stages of infection when there is a low level of specific antibodies and false-positive results due to cross-reaction of antibodies to antigens from closely-related bacterial species. In addition, diagnosis of acute infection by IgG serology using single or paired (acute and convalescent) serum samples has the limitation of lack of a standardized cutoff titer among laboratories if a single sample is obtained, or the frequent inability to obtain convalescent serum when paired samples are required. In the latter case, while IgG serology could be useful for epidemiologic surveillance, paired sera are not optimal for timely diagnosis and treatment of acute infection. Thus, the emergence of molecular methods including probe hybridization, target or signal amplification, and sequencing provides better diagnostic advantages compared to microscopy, culture, and serology such as rapid TAT, higher sensitivity and higher specificity in different patient populations, and using different specimen types (eg, blood, plasma, cerebrospinal fluid, tissues, fluids). These molecular tests have become the gold standards due to their high negative and positive predictive values and their ability to detect and characterize newly emerging pathogens for clinical purposes [1À11].

Molecular assays are routinely used in clinics for the diagnosis, prognosis, and treatment decisions of various emerging infectious diseases [12,13,38,75, 86À91] (Tables 15.1 Tables 15.1 and 15.2, there are US FDA-approved/cleared tests for some of these pathogens, and CDC has tests for all these agents. There are also laboratory-developed tests brought to clinical use after significant research and development and validation studies by individual laboratories [12,13,38,75,86À91] . As in other infectious diseases, clinical molecular tests for emerging infectious diseases include (1) nucleic acid detection assays with defined limit of detection cutoffs, (2) quantitative methods with broad dynamic ranges, lower and higher limit of quantification values, (3) genotyping and subtyping assays, and drug resistance mutation assays at even single base-pair resolution are used for disease prognosis and guiding treatment strategies [71, 81, 92] . General quality management protocols that cover preanalytic, analytic, and postanalytic phases also apply to molecular tests of emerging pathogens.

Following the discovery of HIV-1 in 1983 and HCV in 1989, molecular tests were developed and implemented for routine clinical use to detect viral infection, monitor viral load, and examine specific HIV-1 drugresistant mutations and HCV genotypes to guide patient management. Several practice guidelines have incorporated HIV-1 and HCV molecular tests (eg, http://www.who.int/hiv/pub/guidelines/en/; http://www.hcvguidelines.org/full-report-view). For example, because detection of HCV RNA, not IgG antibody, is diagnostic of current HCV infection, and HCV genotype 1 is more difficult to treat than genotype 2 or 3, testing for HCV genotype is recommended to guide selection of the most appropriate treatment regimen. HCV RNA detection and genotyping assays are routinely performed in clinical diagnostics laboratories (http://www.hcvguidelines.org/full-report-view).

Over the years, with advances in molecular technology, HIV-1 and HCV clinical molecular tests have improved significantly with respect to performance characteristics including sensitivity, specificity, and dynamic range. Currently, there are several FDAapproved/cleared molecular tests for HIV-1 and HCV (Table 15 .1), and new methods are continuously developed and evaluated for better care of patients with HIV-1 and HCV infection [38, 75, 89, 93] .

A high portion of emerging infectious diseases are vector-borne zoonoses that have emerged from natural cycles. The underlying causes of their emergence are a combination of environmental changes, such as increased populations and geographic distribution of their reservoir hosts and vectors, and development of new scientific tools that contribute to their detection and identification. For example, PCR-based molecular methods have enabled the discovery of a large number of bacterial and viral organisms in ticks, which preceded the identification of these organisms as etiologic agents of emerging infectious diseases.

Among these emerging infectious diseases are two contrasting tick-borne infections, Lyme borreliosis [37, 47] , and human monocytotropic ehrlichiosis (HME) [27À29] . Lyme disease is well known, feared, at times inappropriately diagnosed, and very rarely fatal. HME is largely unknown, frequently misdiagnosed as another tick-borne disease such as Rocky Mountain spotted fever or a viral infection, and is often lifethreatening. Lyme borreliosis occurs particularly in suburban populations in the northeastern United States and has been investigated extensively in prominent academic medical institutions in this region. HME occurs particularly in the rural southeastern United States and has not been the focus of in-depth clinical studies in academic medical centers in this region. Both Lyme borreliosis and HME have high incidence although that of HME is not well recognized.

The effects of these conditions on the development and application of diagnostic tests including molecular diagnostics are far from satisfying. Diagnosis of Lyme borreliosis depends heavily on serological assays. Patients with Lyme disease frequently have developed antibodies to Borrelia burgdorferi by the time in their course of illness when they present for medical attention. These patients and those with a classic bulls-eye appearing rash are diagnosed, treated effectively with appropriate antibiotics and recover. As with other infections antibodies take time to be stimulated and produced. Thus, some patients' diagnoses may be delayed. Molecular methods seldom provide a diagnosis owing to the paucity of organisms in the blood and other readily obtained clinical samples [94] .

A tremendous problem is the large number of persons with atypical symptoms of a wide range that includes those similar to chronic fatigue syndrome or fibromyalgia who are convinced that they are suffering from chronic Lyme disease but whose results of validated tests do not support the diagnosis. Many of them are convinced that the tests are inadequate and that better tests are needed [94] . In contrast, patients with HME often have not developed antibodies to the etiologic agent, E. chaffeensis, at the time when they present for medical attention. The bacteria can infect mononuclear phagocytes and are present in circulating monocytes providing an often effective target for molecular diagnostics at a time when appropriate antibiotic treatment results in rapid recovery from an otherwise life-threatening infection [29] . Yet HME, which likely has an incidence similar to Lyme disease, lacks a readily available point-of-care diagnostic test. Effective molecular target genes have been identified, and in-house assays provide proof-of-concept that molecular diagnostics offer an effective approach [28, 95] . Moreover, low-cost instrument-free devices for nucleic acid amplification and specific identification have been developed that would be appropriate for point-of-care diagnosis.

Why have no more effective efforts been made to devise, develop, and commercialize molecular approaches to these two important emerging infections? For Lyme borreliosis, molecular diagnostics may not possess the solution when too few or no Borrelia are present. For HME, the issues lie in the realms of clinical practice, public health, and business. Physicians who are unaware of HME and note that febrile illnesses during the tick season often respond to doxycycline therapy are not inclined to order send out tests that would cost the patient. Serology that is based on comparing IgG antibody levels in paired sera often fails to provide a diagnosis of acute infection as it relies on the seldom-obtained convalescent serum. Public health agencies are powerless to address effectively a disease that is not diagnosed, and if diagnosed, is not reported. The epidemiologic reports depend on the data obtained by passive surveillance. In fact, active, prospective, population-based surveillance in endemic regions such as Missouri suggested that HME is a highly prevalent disease [96] . The combination of nonspecific clinical manifestations of HME, test underutilization, lack of a gold standard test that is effective when therapeutic decisions are made, and problems in interpretation of diagnostic tests such as serology, and misleading epidemiologic data have accounted for reported low incidence of HME. This situation has failed to stimulate interest in commercial development and marketing of a useful point-of-care assay, although there could be an adequate pull from the potential users of the test.

The advances of sequencing technology, nanotechnology, and bioinformatics have driven molecular tests including assays for emerging pathogens to be more comprehensive and precise. For example, the availability of various sequence databases permits quick identification of sequence identity and variations. For example, the HIV database http://www.hiv.lanl.gov/ contains data on HIV genetic sequences and drug resistance associated mutations. It is valuable for HIV epidemiological studies, research, development, and clinical validation studies of HIV clinical assays [38, 71] . It is well known that there are significant variations of clinical phenotypes in the presence of emerging infections ranging from asymptomatic carrier to lethal infection. Recently, assays to examine multiple pathogen panels have been developed [97À102], which should increase the diagnostic yield for many pathogens. A critical need for emerging pathogen analysis is quicker, easier, cost-effect assays that can be used in a point-of-care setting. New assays that are performed on platforms with a small footprint and detect pathogens quickly (in minutes instead of hours or days) have entered clinical use. For example, the FilmArray (BioFire Diagnostics, Inc.) and Simplexa (Focus Diagnostics, Inc.) molecular assays can generate results in approximately 60 min. The user-friendly Alere i (Alere Inc.) and Cobas Liat (Roche Molecular Systems) platforms are compact and portable, generate rapid molecular results in 15À20 min, can use electricity or rechargeable battery, and therefore are completely mobile and suited for point-of-care testing. It is obvious that the current rapid development of new technologies will further enhance the utility of molecular diagnostics in various emerging infectious diseases.

The advancement of molecular methods for emerging infections comes hand-in-hand with other areas including general infectious diseases, genetics and genomics, and oncology. There are needs to develop unified sequence databases for the input and search of emerging pathogens and other sequences, to understand pathogen/genotype/sequence correlation with phenotypes (eg, lethality or carrier with an emerging infection), to develop panels to more effectively diagnose patients based on shared clinical signs and symptoms, and to develop point-of-care molecular platforms and assays for emerging infectious diseases.

Over the last two decades, sequencing technology has evolved from labor-intensive and time-consuming methodologies to automatic and real-time sequence detection. Recent development and use of NGS has revolutionized the landscape of microbiology and infectious disease. The availability of sequencing data has speeded up pathogen discovery, and also helped improve diagnosis, typing of pathogens, detection of virulence and drug resistance, and development of new vaccines and targeted treatment [103À106] .

With the ever-extending use of NGS on a variety of clinical samples, rapid progress on determining the composition of the human microbiome and its impact upon human health are to be expected in the coming years. This deluge of sequencing data requires a consolidated and curated database to input and search sequences, sequence variations, associated symptoms and diseases, available tests, and treatment options. A unified reporting guideline for molecular epidemiology has been proposed recently [107] . Adoption of this guideline by the research and clinical communities should help to integrate the effort for the comprehension of genomics and metagenomics relevant to the field of medical microbiology, and to improve management of infectious diseases.

Traditional pathogen detection methods in infectious diseases rely upon the identification of agents associated with a particular clinical syndrome. The availability of a significant amount of sequence information and the emerging field of metagenomics using NGS have the potential to revolutionize pathogen detection by allowing the simultaneous detection of all microorganisms in a clinical sample, without a priori knowledge of their identities. This can identify new sequences and organisms that may be initially considered nonpathogenic and may cause infections in different human populations and health conditions. They may cause diseases not previously thought to have a microbial component, and the methods may determine previously unknown etiology of infections. For example, infection with certain emerging pathogens may only cause disease symptoms in patients with AIDS or immune suppression after organ transplantation, or in travelers not previously exposed to the agents. Further biological and clinical studies are necessary to categorize sequence information and interpret clinical relevance when a pathogen sequence is detected, which is critical for diagnosis, treatment, and public health surveillance of emerging infectious diseases.

Assays to examine multiple pathogen panels have been developed [97À102]. These assays are designed either to detect many infections that can cause similar symptoms (eg, FDA-approved/cleared respiratory viral panels as listed in Table 15 .2, multiple viruses that can trigger gastrointestinal symptoms) [100, 101] , pathogens that share homologous sequences, for example, 16S rRNA sequencing [98, 99, 102, 108] or are expected to occur under the circumstances of biothreat [97] . The availability of more pathogen sequences and further understanding of their correlation with clinical symptoms are necessary for the rational design of panels that can fit various needs.

New technological developments including microfluidics, nanotechnology, and lab-on-a-chip technologies have enabled development of user-friendly, easy, and quick point-of-care molecular tests including Alere i (Alere Inc.) and Cobas Liat (Roche Molecular Systems). In the setting of emerging infectious diseases, rapid and accurate identification of the causative agent is critical to facilitate effective patient management and enable prompt initiation of infection controls. Point-of-care assays are especially needed in resource-limited settings and in situations with lack of access to centralized medical facilities. Further development of point-of-care molecular tests for emerging pathogens is critical to timely diagnosis, treatment, and subsequent control of emerging infectious disease.

Development of a panel of recombinase polymerase amplification assays for detection of biothreat agents

Molecular diagnostics for Lassa fever at Irrua specialist teaching hospital, Nigeria: lessons learnt from two years of laboratory operation

Completion of the Lassa fever virus sequence and identification of a RING finger open reading frame at the L RNA 5 0 end

Hospital-based surveillance for Lassa fever in Edo State

Deep sequencing of norovirus genomes defines evolutionary patterns in an urban tropical setting

DNA microarray for detection of gastrointestinal viruses

Metagenomic analysis of viruses in feces from unsolved outbreaks of gastroenteritis in humans

Comparison of test specificity in commercial antigen and in-house PCR methods for rotavirus detection in stool specimens

Keeping pace with parvovirus B19 genetic variability: a multiplex genotype-specific quantitative PCR assay

Identification of past and recent parvovirus B19 infection in immunocompetent individuals by quantitative PCR and enzyme immunoassays: a dual-laboratory study

Low-level DNAemia of parvovirus B19 (genotypes 1-3) in adult transplant recipients is not associated with anaemia

An improved method for quantification of Vibrio vulnificus in oysters

Multiplex PCR assays for the detection of Vibrio alginolyticus, Vibrio parahaemolyticus, Vibrio vulnificus, and Vibrio cholerae with an internal amplification control

Comparative genomics of cryptosporidium

A new set of primers directed to 18S rRNA gene for molecular identification of Cryptosporidium spp. and their performance in the detection and differentiation of oocysts shed by synanthropic rodents

A novel nested multiplex PCR for the simultaneous detection and differentiation of Cryptosporidium spp., Enterocytozoon bieneusi and Encephalitozoon intestinalis

Evolutionary history of the Clostridium difficile pathogenicity locus

Diverse sources of C. difficile infection identified on whole-genome sequencing

Similar proportions of stool specimens from hospitalized children with and without diarrhea test positive for Clostridium difficile

Comparative analyses of Legionella species identifies genetic features of strains causing Legionnaires inverted question mark disease

Recombination drives genome evolution in outbreak-related Legionella pneumophila isolates

Clinical and molecular epidemiological features of hemorrhagic fever with renal syndrome in Korea over a 10-year period

Genetic susceptibility to haemorrhagic fever with renal syndrome caused by Hantaan virus in Chinese Han population

A one step real time PCR method for the quantification of hepatitis delta virus RNA using an external armored RNA standard and intrinsic internal control

Eighth major clade for hepatitis delta virus

Current methods for molecular typing of Campylobacter species

Detection, identification and quantification of Campylobacter jejuni, coli and lari in food matrices all at once using multiplex qPCR

Intragenomic sequence variation of the ITS-1 region within a single flow-cytometry-counted Cyclospora cayetanensis oocysts

Prevalence and molecular characterization of Cyclospora cayetanensis

Digital droplet PCR (ddPCR) for the precise quantification of human T-lymphotropic virus 1 proviral loads in peripheral blood and cerebrospinal fluid of HAM/TSP patients and identification of viral mutations

Development and validation of a new high-throughput method to investigate the clonality of HTLV-1-infected cells based on provirus integration sites

Molecular characterization of human T-cell lymphotropic virus type 1 full and partial genomes by Illumina massively parallel sequencing technology

Nucleotide sequence analysis of isolates of human T-lymphotropic virus type 1 of diverse geographical origins

Prevalence of enterotoxins and toxin gene profiles of Staphylococcus aureus isolates recovered from a bakery involved in a second staphylococcal food poisoning occurrence

Screening, detection, and serotyping methods for toxin genes and enterotoxins in Staphylococcus strains

Bacterial whole-genome sequencing revisited: portable, scalable, and standardized analysis for typing and detection of virulence and antibiotic resistance genes

Geographical and genospecies distribution of Borrelia burgdorferi sensu lato DNA detected in humans in the USA

Comparative population genomics of the Borrelia burgdorferi species complex reveals high degree of genetic isolation among species and underscores benefits and constraints to studying intra-specific epidemiological processes

DNA extraction protocol for rapid PCR detection of pathogenic bacteria

Genomic diversity and virulence characterization of historical Escherichia coli O157 strains isolated from clinical and environmental sources

A real time PCR platform for the simultaneous quantification of total and extrachromosomal HIV DNA forms in blood of HIV-1 infected patients

Next-generation sequencing of HIV-1 RNA genomes: determination of error rates and minimizing artificial recombination

Heteroresistance of Helicobacter pylori from the same patient prior to antibiotic treatment

Diagnosis of Helicobacter pylori: What should be the gold standard?

Genetic identification of cryptic genospecies of Haemophilus causing urogenital and neonatal infections by PCR using specific primers targeting genes coding for 16S rRNA

Lineage-specific virulence determinants of Haemophilus influenzae biogroup aegyptius

Evaluation of DNA extraction and PCR methods for detection of Enterocytozoon bienuesi in stool specimens

Comparison of real-time PCR and a microimmunofluorescence serological assay for detection of Chlamydophila pneumoniae infection in an outbreak investigation

In silico synteny based comparative genomics approach for identification and characterization of novel therapeutic targets in Chlamydophila pneumoniae

Detection of Herpesviridae in whole blood by multiplex PCR DNA-based microarray analysis after hematopoietic stem cell transplantation

Identification of chromosomally integrated human herpesvirus 6 by droplet digital PCR

Diagnostic assay for Rickettsia japonica

Complete genomic DNA sequence of the East Asian spotted fever disease agent Rickettsia japonica

HCV RNA quantification with different assays: implications for protease-inhibitor-based response-guided therapy

High-resolution hepatitis C virus (HCV) subtyping, using NS5B deep sequencing and phylogeny, an alternative to current methods

Comparison of real-time PCR and antigen assays for detection of hepatitis E virus in blood donors

Rapamycin and everolimus facilitate hepatitis E virus replication: revealing a basal defense mechanism of PI3K-PKB-mTOR pathway

Diagnosis of infections caused by pathogenic free-living amoebae

Genetic analysis among environmental strains of Balamuthia mandrillaris recovered from an artificial lagoon and from soil in

Genome sequence of human herpesvirus 7 strain UCL-1

Human endogenous retrovirus-K18 superantigen expression and human herpesvirus-6 and human herpesvirus-7 viral loads in chronic fatigue patients

Genetic diversity between and within the arenavirus species indigenous to western Venezuela

Establishment of conventional and fluorescence resonance energy transfer-based real-time PCR assays for detection of pathogenic New World arenaviruses

Species-specific detection of three human-pathogenic microsporidial species from the genus Encephalitozoon via fluorogenic 5 0 nuclease PCR assays

Gain and loss of multiple functionally related, horizontally transferred genes in the reduced genomes of two microsporidian parasites

Intravascular persistence of Anaplasma platys, Ehrlichia chaffeensis, and Ehrlichia ewingii DNA in the blood of a dog and two family members

Detection of medically important Ehrlichia by quantitative multicolor TaqMan real-time polymerase chain reaction of the dsb gene

Emerging and re-emerging tick-transmitted rickettsial and ehrlichial infections

Nucleotide sequence of the Barmah Forest virus genome

Genetic stability among temporally and geographically diverse isolates of Barmah Forest virus

Whole genome PCR scanning reveals the syntenic genome structure of toxigenic Vibrio cholerae strains in the O1/O139 population

Rapid oligonucleotide suspension array-based multiplex detection of bacterial pathogens

Detection of Bartonella species in the blood of veterinarians and veterinary technicians: a newly recognized occupational hazard? Vector Borne Zoonotic Dis

Bartonella henselae infections in solid organ transplant recipients: report of 5 cases and review of the literature

Widespread use of real-time PCR for rickettsial diagnosis

Phylogeny of Rickettsia spp. inferred by comparing sequences of 'gene D', which encodes an intracytoplasmic protein

Emerging and re-emerging tick-transmitted rickettsial and ehrlichial infections

Genetic characterization and phylogeny of Sabia virus, an emergent pathogen in Brazil

Establishment of conventional and fluorescence resonance energy transfer-based real-time PCR assays for detection of pathogenic New World arenaviruses

Variability in minimal genomes: analysis of tandem repeats in the microsporidia Encephalitozoon intestinalis

A novel nested multiplex PCR for the simultaneous detection and differentiation of Cryptosporidium spp., Enterocytozoon bieneusi and Encephalitozoon intestinalis

Temporal and geographic evidence for evolution of Sin Nombre virus using molecular analyses of viral RNA from Colorado, New Mexico and Montana

Naturally occurring Sin Nombre virus genetic reassortants

Measurements of human herpesvirus 8 viral load in blood before and after leukoreduction filtration

Detection of human herpesvirus 8 by quantitative polymerase chain reaction: development and standardisation of methods

Sensitive multiplex PCR assay to differentiate Lyme spirochetes and emerging pathogens Anaplasma phagocytophilum and Babesia microti

Anaplasma phagocytophilum-a widespread multi-host pathogen with highly adaptive strategies

Diagnosis of henipavirus infection: current capabilities and future directions

Sequence analysis of the Hendra virus nucleoprotein gene: comparison with other members of the subfamily Paramyxovirinae

Comparison of RT-PCR assay and virus isolation in cell culture for the detection of Alkhumra hemorrhagic fever virus

Complete genome sequencing and genetic characterization of Alkhumra hemorrhagic fever virus isolated from Najran, Saudi Arabia

Molecular characterization of Nipah virus, a newly emergent paramyxovirus

Development of a strand-specific real-time qRT-PCR for the accurate detection and quantitation of West Nile virus RNA

Detection of West Nile virus RNA (lineages 1 and 2) in an external quality assessment programme for laboratories screening blood and blood components for West Nile virus by nucleic acid amplification testing

Intravascular persistence of Anaplasma platys, Ehrlichia chaffeensis, and Ehrlichia ewingii DNA in the blood of a dog and two family members

Detection of medically important Ehrlichia by quantitative multicolor TaqMan real-time polymerase chain reaction of the dsb gene

Real-time reverse transcriptase PCR assay for improved detection of human metapneumovirus

Human metapneumovirus viral load is an important risk factor for disease severity in young children

Application of the Ibis-T5000 pan-Orthopoxvirus assay to quantitatively detect monkeypox viral loads in clinical specimens from macaques experimentally infected with aerosolized monkeypox virus

Detection of monkeypox virus with real-time PCR assays

Comprehensive detection and identification of human coronaviruses, including the SARS-associated coronavirus, with a single RT-PCR assay

Rapid and sensitive detection of multiple genes from the SARS-coronavirus using quantitative RT-PCR with dual systems

Identification of a novel coronavirus in patients with severe acute respiratory syndrome

A novel coronavirus associated with severe acute respiratory syndrome

Coronavirus as a possible cause of severe acute respiratory syndrome

Characterization of a novel coronavirus associated with severe acute respiratory syndrome

HTLV-3/STLV-3 and HTLV-4 viruses: discovery, epidemiology, serology and molecular aspects

Development and validation of a multiplex real-time PCR assay for simultaneous genotyping and human T-lymphotropic virus type 1, 2, and 3 proviral load determination

Detection of spliced mRNA from human bocavirus 1 in clinical samples from children with respiratory tract infections

Detection of human bocavirus mRNA in respiratory secretions correlates with high viral load and concurrent diarrhea

Evaluation of three rapid diagnostic tests for the detection of human infections with Plasmodium knowlesi

A new single-step PCR assay for the detection of the zoonotic malaria parasite Plasmodium knowlesi

Rapid molecular detection of Lujo virus RNA

Molecular surveillance and phylogenetic analysis of Old World arenaviruses in Zambia

Genetic diversity among Bolivian arenaviruses

Chapare virus

Emergence of a new pathogenic Ehrlichia species, Wisconsin and Minnesota

Complete genome sequence of Ehrlichia muris strain AS145T, a model monocytotropic Ehrlichia strain

In vitro transcribed RNA molecules for the diagnosis of pandemic 2009 influenza A(H1N1) virus by real-time RT-PCR

Using high-throughput sequencing to leverage surveillance of genetic diversity and oseltamivir resistance: a pilot study during the 2009 influenza A (H1N1) pandemic

Candidatus Neoehrlichia mikurensis infection identified in 2 hematooncologic patients: benefit of molecular techniques for rare pathogen detection

First case of human "Candidatus Neoehrlichia mikurensis" infection in a febrile patient with chronic lymphocytic leukemia

Severe fever with thrombocytopenia syndrome

Fever with thrombocytopenia associated with a novel bunyavirus in China

Real-time reverse transcription-PCR assay panel for Middle East respiratory syndrome coronavirus

Respiratory tract samples, viral load, and genome fraction yield in patients with middle East respiratory syndrome

The genesis and source of the H7N9 influenza viruses causing human infections in China

Chapare virus, a newly discovered arenavirus isolated from a fatal hemorrhagic fever case in Bolivia

Completion of the Lassa fever virus sequence and identification of a RING finger open reading frame at the L RNA 5 0 End

Molecular characterization of Nipah virus, a newly emergent paramyxovirus

Nucleotide sequence of the Barmah Forest virus genome

Complete genome sequencing and genetic characterization of Alkhumra hemorrhagic fever virus isolated from Najran, Saudi Arabia

Complete genomic DNA sequence of the East Asian spotted fever disease agent Rickettsia japonica

Candidatus Neoehrlichia mikurensis infection identified in 2 hematooncologic patients: benefit of molecular techniques for rare pathogen detection

Emergence of a new pathogenic Ehrlichia species, Wisconsin and Minnesota

Complete genome sequence of Ehrlichia muris strain AS145T, a model monocytotropic Ehrlichia strain

Fever with thrombocytopenia associated with a novel bunyavirus in China

The genesis and source of the H7N9 influenza viruses causing human infections in China

The long and winding road leading to the identification of the hepatitis C virus

Discovery of the hepatitis C virus

Temporal and geographic evidence for evolution of Sin Nombre virus using molecular analyses of viral RNA from Colorado

Naturally occurring Sin Nombre virus genetic reassortants

Novel respiratory viruses: what should the clinician be alert for

Identification of a novel coronavirus in patients with severe acute respiratory syndrome

A novel coronavirus associated with severe acute respiratory syndrome

Coronavirus as a possible cause of severe acute respiratory syndrome

Characterization of a novel coronavirus associated with severe acute respiratory syndrome

Comprehensive detection and identification of human coronaviruses, including the SARS-associated coronavirus, with a single RT-PCR assay

Rapid and sensitive detection of multiple genes from the SARScoronavirus using quantitative RT-PCR with dual systems

Detection of Bartonella species in the blood of veterinarians and veterinary technicians: a newly recognized occupational hazard?

Bartonella henselae infections in solid organ transplant recipients: report of 5 cases and review of the literature

Sensitive multiplex PCR assay to differentiate Lyme spirochetes and emerging pathogens Anaplasma phagocytophilum and Babesia microti

Anaplasma phagocytophilum-a widespread multi-host pathogen with highly adaptive strategies

Intravascular persistence of Anaplasma platys, Ehrlichia chaffeensis, and Ehrlichia ewingii DNA in the blood of a dog and two family members

Detection of medically important Ehrlichia by quantitative multicolor TaqMan real-time polymerase chain reaction of the dsb gene

Emerging and reemerging tick-transmitted rickettsial and ehrlichial infections

Development of a molecularrespiratory syndrome (SARS-CoV): a general methodology for detecting rapidly mutating viruses

Molecular epidemiology of severe acute respiratory syndromeassociated coronavirus infections in Taiwan

Characterizing 56 complete SARS-CoV S-gene sequences from Hong Kong

The large 386-nt deletion in SARS-associated coronavirus: evidence for quasispecies?

Keeping pace with parvovirus B19 genetic variability: a multiplex genotype-specific quantitative PCR assay

Genetic diversity among Bolivian arenaviruses

Detection of spliced mRNA from human bocavirus 1 in clinical samples from children with respiratory tract infections

Geographical and genospecies distribution of Borrelia burgdorferi sensu lato DNA detected in humans in the USA

Next-generation sequencing of HIV-1 RNA genomes: determination of error rates and minimizing artificial recombination

Evolutionary history of the Clostridium difficile pathogenicity locus

Genome sequence of human herpesvirus

Diverse sources of C. difficile infection identified on wholegenome sequencing

Genetic diversity between and within the arenavirus species indigenous to western Venezuela

Variability in minimal genomes: analysis of tandem repeats in the microsporidia Encephalitozoon intestinalis

Comparative analyses of Legionella species identifies genetic features of strains causing Legionnaires inverted question mark disease

Genetic characterization and phylogeny of Sabia virus, an emergent pathogen in Brazil

Molecular surveillance and phylogenetic analysis of Old World arenaviruses in Zambia

Comparative population genomics of the Borrelia burgdorferi species complex reveals high degree of genetic isolation among species and underscores benefits and constraints to studying intra-specific epidemiological processes

Genetic analysis among environmental strains of Balamuthia mandrillaris recovered from an artificial lagoon and from soil in

Eighth major clade for hepatitis delta virus

Comparative genomics of cryptosporidium

Termini of all mRNA species of Marburg virus: sequence and secondary structure

Whole genome PCR scanning reveals the syntenic genome structure of toxigenic Vibrio cholerae strains in the O1/O139 population

Molecular characterization of human T-cell lymphotropic virus type 1 full and partial genomes by Illumina massively parallel sequencing technology

Genetic stability among temporally and geographically diverse isolates of Barmah Forest virus

Gain and loss of multiple functionally related, horizontally transferred genes in the reduced genomes of two microsporidian parasites

Nucleotide sequence analysis of isolates of human T-lymphotropic virus type 1 of diverse geographical origins

Intragenomic sequence variation of the ITS-1 region within a single flow-cytometry-counted Cyclospora cayetanensis oocysts

Genomic diversity and virulence characterization of historical Escherichia coli O157 strains isolated from clinical and environmental sources

Recombination drives genome evolution in outbreak-related Legionella pneumophila isolates

Phylogeny of Rickettsia spp. inferred by comparing sequences of 'gene D', which encodes an intracytoplasmic protein

Lineage-specific virulence determinants of Haemophilus influenzae biogroup aegyptius

Using highthroughput sequencing to leverage surveillance of genetic diversity and oseltamivir resistance: a pilot study during the 2009 influenza A(H1N1) pandemic

Marburgvirus genomics and association with a large hemorrhagic fever outbreak in Angola

Sequence analysis of the Hendra virus nucleoprotein gene: comparison with other members of the subfamily Paramyxovirinae

Prevalence and molecular characterization of Cyclospora cayetanensis

Continuing challenges in influenza

The burden of influenza-associated critical illness hospitalizations

In vitro transcribed RNA molecules for the diagnosis of pandemic 2009 influenza A(H1N1) virus by real-time RT-PCR

Development and validation of a clinical decision rule for the diagnosis of influenza

Low Prevalence of non-subtype B HIV-1 strains in the Texas prisoner population

Identification of human immunodeficiency virus type 1 non-B subtypes and antiretroviral drug-resistant strains in United States blood donors

Genetic characterization of diverse HIV-1 strains in an immigrant population living in New York City

Genetic diversity among human immunodeficiency virus-1 non-B subtypes in viral load and drug resistance assays

A real time PCR platform for the simultaneous quantification of total and extrachromosomal HIV DNA forms in blood of HIV-1 infected patients

Understanding HIV infection for the design of a therapeutic vaccine. Part I: Epidemiology and pathogenesis of HIV infection

A "big data" approach to HIV epidemiology and prevention

Consensus proposals for a unified system of nomenclature of hepatitis C virus genotypes

Hepatitis C virus genotypes and viral concentrations in participants of a general population survey in the United States

The epidemiology of hepatitis C infection in the United States

HCV genotype and subtype distribution of patient samples tested at University of Texas Medical Branch in

Hepatitis C virus NS3 protease genotyping and drug concentration determination during triple therapy with telaprevir or boceprevir for chronic infection with genotype 1 viruses, southeastern France

Drug resistance of a viral population and its individual intrahost variants during the first 48 hours of therapy

Development of a high-throughput pyrosequencing assay for monitoring temporal evolution and resistance associated variant emergence in the hepatitis C virus protease codingregion

Infrequent development of resistance in genotype 1-6 hepatitis C virus-infected subjects treated with sofosbuvir in phase 2 and 3 clinical trials

Comparison of real-time PCR and a microimmunofluorescence serological assay for detection of Chlamydophila pneumoniae infection in an outbreak investigation

In silico synteny based comparative genomics approach for identification and characterization of novel therapeutic targets in Chlamydophila pneumoniae

HCV RNA quantification with different assays: implications for protease-inhibitor-based response-guided therapy

High-resolution hepatitis C virus (HCV) subtyping, using NS5B deep sequencing and phylogeny, an alternative to current methods

Real-time reverse transcriptase PCR assay for improved detection of human metapneumovirus

Human metapneumovirus viral load is an important risk factor for disease severity in young children

HIV-1 genotypic resistance testing on low viral load specimens using the Abbott ViroSeq HIV-1 Genotyping System

Comparison of real-time PCR and antigen assays for detection of hepatitis E virus in blood donors

Lyme disease: diagnostic issues and controversies

Detection of bacterial agents in Amblyomma americanum (Acari: Ixodidae) from Georgia, USA, and the use of a multiplex assay to differentiate Ehrlichia chaffeensis and Ehrlichia ewingii

Human monocytotropic ehrlichiosis, Missouri

Development of a panel of recombinase polymerase amplification assays for detection of biothreat agents

INFECTIOUS DISEASE

Development and evaluation of a panel of filovirus sequence capture probes for pathogen detection by nextgeneration sequencing

Survey of culture, goldengate assay, universal biosensor assay, and 16S rRNA gene sequencing as alternative methods of bacterial pathogen detection

DNA microarray for detection of gastrointestinal viruses

Metagenomic analysis of viruses in feces from unsolved outbreaks of gastroenteritis in humans

Multiplex PCR assays for the detection of Vibrio alginolyticus, Vibrio parahaemolyticus, Vibrio vulnificus, and Vibrio cholerae with an internal amplification control

Sequencing the human microbiome in health and disease

The human virome: new tools and concepts

Metagenomics for pathogen detection in public health

Genomics and metagenomics in medical microbiology

Strengthening the Reporting of Molecular Epidemiology for Infectious Diseases (STROME-ID): an extension of the STROBE statement

Rapid oligonucleotide suspension array-based multiplex detection of bacterial pathogens