Nucleic Acid Amplification Techniques

Tuberculous otitis media and mastoiditis, or tuberculous otomastoiditis, is a rare but well-described infectious process occasionally affecting individuals in the United States but more frequently seen in countries where tuberculosis is endemic. Infection may be primary and occur through mucus aspirated through the Eustachian tube. Alternatively, organisms may secondarily infect the nasopharynx when expectorated from the lungs and, less frequently, may be hematogenously spread. Chronic otorrhea and hearing loss are common symptoms, and extensive perforation of the tympanic membranes and facial nerve paralysis are routinely described. Diagnosis is made by direct culture of Mycobacterium tuberculosis, although more recently, molecular techniques have been used. Successful treatment of tuberculous otomastoiditis routinely involves surgical intervention combined with prolonged antituberculosis therapy.

In 1981, two reports from the west and east coasts of the United States marked the beginning of the acquired immunodeficiency syndrome (AIDS) epidemic by the lethal, immunosuppressant retrovirus, human immunodeficiency virus (HIV). Just four years later, in 1985, Kary Mullis and his colleagues described the polymerase chain reaction (PCR), which contributed to an explosion of research on HIV and AIDS. The story begins with an overview of the molecular genetics revolution, which was fundamental to what ultimately became molecular diagnostic virology. Key to management of infected individuals has been determination of viral load, detected by nucleic acid amplification techniques such as the PCR. Prior to the explosive commercial development of PCR and other nucleic acid amplification tests, certain molecular nucleic acid methods were explored for clinical diagnostic virology. Diagnostic virology played a central role in patient management, in pharmacotherapy, and in researching the epidemiology of circulating viral infections. Likewise, management of infections with viruses such as HIV, cytomegalovirus (CMV), hepatitis B virus (HBV), and hepatitis C virus (HCV) has been significantly improved. The current trend is to devise user-friendly and walk-away molecular tests allowing more clinical laboratories to offer viral diagnostic services. From transmission studies of yellow fever with human volunteers to the application of high-throughput sequencing, diagnostic virology sits astride the confluence of advances in science and the challenges presented by human disease.

While the number of Haemophilus species described greatly exceeds the number of human pathogens, eight species affecting humans currently included in this genus are H. influenzae, H. aegyptius, H. ducreyi, H. pittmaniae, H. parainfluenzae, H. haemolyticus, H. parahaemolyticus, and H. paraphrohaemolyticus. Aggregatibacter aphrophilus, Aggregatibacter paraphrophilus, and Aggregatibacter segnis were formerly included in the genus Haemophilus but have recently been reclassified into the genus Aggregatibacter (formerly Actinobacillus) based on molecular taxonomy. In the absence of the recently reclassified species, significant genetic diversity still exists in the Haemophilus genus. Haemophilus influenzae may be found as part of the commensal bacterial flora of the mucosal surfaces of the upper respiratory tracts (URT) of many healthy individuals. Several molecular methods, including 16S rRNA gene sequencing, polymerase chain reaction (PCR), and fluorescence in-site hybridization have been described in the literature as being effective tools for the species identification of Haemophilus when performed on organisms recovered in culture. Other molecular methods for typing have also been applied to Haemophilusspecies. In one study evaluating the performance of intergenic dyad sequence (IDS)-PCR with 69 NTHi isolates, the assay demonstrated 65 different banding patterns that were epidemiologically classified as fingerprints similar to those obtained by pulsed-field gel electrophoresis (PFGE).

Borreliosis, in the form of louse-borne relapsing fever (LBRF), has been the cause of massive epidemics as recently as the early 1900s. Tick-borne relapsing fever (TBRF) and Lyme borreliosis (LB) are caused by over a dozen Borrelia species and were first described about 100 years ago. There is a high prevalence of B.afzelii among human skin isolates from Europe, whereas isolates from cerebrospinal fluid (CSF) in Europe are most often B.garinii. A few studies have reported the detection of Borrelia species (B.valaisiana, B.spielmanii, and B. lusitaniae) in patient samples in Europe. The genomes of the borreliae are unusual among prokaryotes in having a small linear chromosome of approximately 1,000 kb and both linear and circular plasmids. Two colinear plasmids (lp54 and cp26) seem to belong to the basic genome inventory of the Borrelia species that causes Lyme disease. The ecological components that maintain Borrelia species in nature are quite diverse and are spread throughout the world. This chapter talks about collection, transport, and storage of specimens. Direct microscopic visualization of borreliae in clinical samples is applicable only to cases of relapsing fever. The chapter describes molecular techniques and immunological techniques for identifying Borrelia species, and explains about the serologic test. The antimicrobial susceptibility of Borrelia species has been studied intensively in vitro. Standard methods for the determination of the minimal bactericidal concentration have not been established. Clinical criteria (case history and clinical findings) are decisive factors in the diagnosis and ordering of microbiological laboratory testing.

This chapter analyses methods focused on conventional cell cultures, classical serologic techniques, and light and electron microscopy. Novel detection methods have permitted the diagnosis of multiple respiratory viruses in a single multiplex PCR test. Quantitative monitoring of viral load in blood has become more widely applied due to implementation of real-time PCR techniques. As tests become more sensitive, low levels of clinically irrelevant or nonviable viruses may be detected and can be misleading to clinicians. Similarly, interpreting the clinical relevance of multiple viral pathogens in the same sample when relative quantification is not available is problematic. Laboratories need to choose which tests to offer. Selecting the appropriate test will depend on the viruses sought, sample site, clinical presentation, clinical purpose, patient characteristics, and disease prevalence. Laboratories must recognize the uses and also the limitations of each test in order to guide clinicians in interpreting the results. Due to the speed of methodological change and the continuing discovery of new viruses and new therapies, keeping abreast of the most recent literature is strongly recommended.

This chapter focuses on Epstein-Barr virus (EBV) that is a member of the Herpesviridae and belongs to the subfamily Gammaherpesvirinae, replicating in epithelial cells and establishing long-term latency in lymphocytes like its closest human-pathogenic relative, human herpesvirus 8. EBV nuclear antigen 1 (EBNA1), EBNA2, EBNA3 (also referred to as EBNA3a), EBNA4 (EBNA3b), EBNA5 (EBNALP), EBNA6 (EBNA3c), and latent membrane proteins (LMP1, -2A, and -2B) may be expressed in B cells. Four types of B-cell latency have been defined, based on various levels of expression of the latency-associated proteins. During lytic replication more than 70 proteins are expressed, including the virus capsid antigens (VCA) and the early antigens used in diagnostics. Nucleic acid detection techniques (NAT) might be applied to blood, cerebrospinal fluid (CSF), or biopsy samples. A wide variety of detection methods are available, ranging from in situ hybridization on frozen or paraffin sections through cytohybridization on cell suspensions, dot blot hybridization, Southern blot hybridization, and nucleic acid amplification techniques (NAAT). The most commonly used clinical diagnostic tools for direct detection of EBV are NAAT. Quantitative real-time PCR is the most popular method today for EBV monitoring in patients at risk for EBV-associated disorders. Individual viral isolates can be characterized by molecular techniques on the basis of polymorphism of the EBNA1 and EBNA3 genes. Patients with X-linked lymphoproliferative syndrome have typically a high viral load and do not develop EBNA antibodies. The ultimate diagnosis, however, is based on genetic analysis for a variety of mutations in the SH2D1A gene domain.

Real-time-based platforms currently offer numerous advantages over conventional nucleic acid amplification techniques (NAATs) such as higher speed, less handling of PCR products, decreased risk of false-positive results due to carryover contamination, and the capability to quantify results. This chapter reviews some updates on the detection by NAAT of some relevant individual agents alone, and associated with clinical syndromes, with emphasis on the detection of respiratory agents, particularly the atypical pathogens Mycobacterium pneumoniae, Chlamydophila pneumoniae, Legionella spp., and Bordetella pertussis but also Streptococcus pneumoniae, for which also quantitative real-time amplification is discussed. Given the many alternative amplification protocols proposed for some applications, such studies are clearly needed. Although such studies are often lacking, new-generation molecular techniques are gradually replacing tissue culture and even conventional PCRs as the gold standard for the diagnosis of respiratory infections and for the detection of particular individual bacterial pathogens, as is further reviewed in this chapter. A recent development is the application of PCR for detecting the family of Haemophilus integrating conjugative elements among antibiotic-resistant Haemophilus influenzae type b directly to cerebrospinal fluid (CSF) to diagnose Haemophilus influenzae type b meningitis and predict the organism's susceptibility, irrespective of culture results. The potential of pyrosequencing after broad-range PCR was successfully investigated by Jordan for the identification of bacterial pathogens responsible for sepsis in neonates.

Approximately one-third of the world’s population is thought to harbor latent forms of tuberculosis and the resultant huge reservoir for future disease. The recent increase in multidrug resistant disease and a dangerous interplay between tuberculosis and human immunodeficiency virus (HIV) infection continues to pose serious challenges for health care in the years to come. M. tuberculosis is transmitted by the inhalation of small droplet nuclei, which are aerosolized infectious particles generated by coughing, talking, and sneezing. A minority of patients with tuberculosis, who are started on highly active antiretroviral therapy (HAART) experience immune reconstitution disease (IRD) due to rapid restoration of the immune system with an increased response to mycobacterial antigens. To be valuable as a surrogate endpoint, a biomarker should measure an event that is directly involved in pathogenesis or protection and should change early during treatment. Early bactericidal activity (EBA) is used in the early evaluation of new tuberculosis drugs and is based on the rate of fall of colony-forming unit counts of M. tuberculosis (log10cfu/day) in overnight sputum samples that are collected before and on alternate days of treatment, up to day 14. A host of nonspecific markers have been described that appear promising as biomarkers for tuberculosis. The extent of the tuberculosis problem that exists today represents an affront to modern society. New tools are urgently needed and only multidisciplinary approaches that include all sectors of healthcare providers and researchers stand a chance to address the problem effectively.

The incidence of tuberculosis has increased worldwide, resulting in an increased incidence of tuberculous lymphadenitis. Tuberculous infection in the parotid gland usually develops following infection of intraparotid lymph nodes. As the capsule of the infected lymph tissue breaks down, parotitis may ensue. Historically, lymph node and parotid tuberculosis was caused mainly by Mycobacterium bovis, but nearly all tuberculous lymphadenitis is now due to M. tuberculosis. Symptoms associated with tuberculous lymphadenitis depend largely on the location of involved nodes. Differential diagnosis of tuberculous lymphadenitis or parotitis is extensive. Tuberculin skin testing is the most definitive noninvasive diagnostic procedure, yielding positive results in more than 90% of persons with tuberculous lymphadenitis. When diagnosis of tuberculous lymphadenitis remains in doubt, biopsy material must be submitted for histology, culture, and potentially PCR. Cytologic findings identify granulomatous changes in 50 to 80% of patients with tuberculous lymphadenitis, but acid-fast bacilli are identified in only 30 to 60% and cultures are positive for only 20 to 80%. A recent systematic review found that nucleic acid amplification tests for tuberculous lymphadenitis produce highly variable and inconsistent results, precluding the determination of clinically meaningful results. Management of tuberculous lymphadenitis and parotitis involves appropriate use of antituberculous chemotherapy with the judicious use of surgical excision in a minority of patients. There are no published trials of therapy for parotitis, so guidelines for lymphadenitis should be followed. Treatment involves the use of combination antituberculous chemotherapy, with occasional need for surgical excision.