5 курс / Пульмонология и фтизиатрия / Clinical_Tuberculosis_Friedman_Lloyd_N_,_Dedicoat

Identifying how laboratory cross-contamination occurred can be further complicated by the need to refer specimens and isolates from clinical settings to commercial and reference laboratories for additional testing.

Laboratorians and TB control program staff can use genotyping data to monitor for and detect potential false-positive culture results. In countries with universal genotyping services, prospective strain typing of M. tuberculosis isolates can facilitate detection. If available, an isolate from a specimen collected on a different date can be submitted for repeat genotyping when contamination is suspected to determine if the patient’s first and second genotyping results differ from one another. Unusual genotyping results, such as matches to reference strains used for proficiency testing or quality control that are not transmitted in the community (e.g., H37Rv), may indicate that a result is either falsely positive or has resulted from an occupational laboratory exposure. Genotyping results should also be reviewed if there is no alternative explanation for an unusual increase in isolates with the same drug-resistance pattern or an unexpected increase in the percentage of culture-positive results.

False-positive investigations are a multidisciplinary endeavor that should involve laboratorians, clinicians, and other TB program staff. Clinical indicators of false positivity include a single positive culture from multiple specimens collected for a new patient, prolonged incubation time with late appearing growth (liquid media) or scanty growth (solid media), the absence of clinical or radiographic findings consistent with TB, and another confirmed diagnosis that is consistent with the patient’s clinical presentation. Indistinguishable genotyping results provide evidence for false positivity if isolates from different patients or reference strains were recently processed in the same laboratory or different patients’ specimens were collected recently in the same facility. Reference laboratories can routinely monitor for clustering of isolates from source laboratories. Many laboratories have also developed policies and procedures to minimize and detect M. tuberculosis cross-contamination.

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INTRODUCTION

Globally, in high burden settings, recent transmission, including reinfection, accounts for most incident TB clinical cases, compared to reactivation of remote latent infection.1 Ongoing transmission, therefore, especially in congregate settings, is driving the TB epidemic.2 In low-burden settings, however, where transmission is infrequent, reactivation of latent infection is relatively more important, and treatment of latent infection has been a strategy for TB elimination.3 Even under low-burden settings, it is argued, most disease results within a year or so of transmission, and the risk from old infection may be overestimated.1

Importance of the undiagnosed case or undiagnosed drug resistance

This chapter will review current understanding on Mycobacterium tuberculosis (Mtb) transmission and its control, with emphasis on the importance of transmission from persons with unsuspected TB, or unsuspected drug resistance, not on effective therapy.5 Although effective therapy has long been known to rapidly stop transmission, conventional TB infection control efforts tend to focus on known TB patients, most of whom are on effective therapy and not likely to be sources of transmission.6,7 Guidelines and practice need to shift from a focus on known cases started on effective therapy to finding unsuspected cases in congregate settings through screening, followed by rapid molecular diagnosis and drug susceptibility testing, leading to prompt, effective treatment.8 This re-focused, intensified administrative approach to TB transmission control has been packaged as an easy-to-remember

acronym, “F-A-S-T,” Find cases Actively, Separate temporarily, and Treat effectively, based on molecular drug susceptibility testing.8 The acronym emphasizes the need to reduce time from symptom reporting to effective treatment. In crowded congregate settings, however, such as registration areas and ambulatory clinics, effective screening for unsuspected TB may not be feasible. There, administrative measures (triage, appointments) to reduce crowding, and air disinfection through natural ventilation and upper room germicidal lamps are important interventions.9,10 Likewise, respiratory protection for healthcare workers is less important for encounters with known TB patients on effective treatment—where they are most commonly used—and more likely to be effective for encounters with patients at high risk for undiagnosed TB or undiagnosed drug resistance.11 Good examples are caring for patients with undiagnosed respiratory infection in the emergency room, or patients undergoing bronchoscopy for lung infiltrates where TB is prevalent.

Airborne spread

TB pathogenesis begins through person-to-person transmission, almost exclusively by the airborne route, a relatively unusual transmission route among human pathogens, shared primarily with some respiratory viruses.12 Airborne Mtb infection requires host susceptibility to the extremely small dose of Mtb, carried as droplet nuclei (the 1–5 µm dried residua of larger droplets) able to traverse the structural barriers of the upper respiratory track to reach the peripheral lung where pulmonary alveolar macrophages (PAMs) reside.13 PAMs are both a first line of innate immunity and, paradoxically, the target cell for initial Mtb replication. To accomplish this, Mtb has evolved specific mechanisms for blocking PAM

intracellular killing. Airborne transport is probably facilitated by Mtb’s uniquely thick, hydrophobic cell wall, able to protect this preferentially intracellular pathogen from the stresses of aerosolization, dehydration in room air, transport, and rehydration in the humid respiratory track of a new host.14 Barer has described a “fat and lazy” spore-like phenotype that may be preparatory to aerosolization.15 Exactly what microbial gene-expression signatures are necessary to facilitate airborne transport remain largely unknown, but may be important to elucidate. For example, the rapid impact of effective treatment on transmission, discussed later in this chapter, well before sputum smear and culture conversion, might be due to pharmacologic interference with specific microbial adaptive mechanisms.16

Particles small enough to be inhaled deep into the lung settle very slowly in still air and can remain suspended indefinitely in occupied rooms where air is rarely stagnant due to body heat, air infiltration, and other factors.17 The 1–5 µm size of infectious droplet nuclei is defined not by organism size (0.5 × 1.0 µm), per se, but by the size of the entire particle containing one, or at most several (estimated 2–3) Mtb organisms, but also dried solute, mucous, and cellular debris found in respiratory lining liquid.18 High airflow in the relatively narrow upper airways and trachea, generated by cough and other forced respiratory maneuvers, shears off relatively large droplets that evaporate rapidly into droplet nuclei once expelled, depending on fluid viscoelasticity and ambient humidity.17 Thinner lung lining fluid may be easier to aerosolize than viscous sputum. Quiet breathing can also generate droplet nuclei as alveoli pop open with inhalation, but the contribution of this mechanism to TB transmission is unknown.19 A hallmark of chronic TB disease is lung cavitation, the result of caseation necrosis of a granuloma and erosion into an airway. With cavities containing an estimated 108 Mtb organisms, expectorated sputum may contain tens of thousands of detectable Mtb organisms per cubic millilitre in various states of viability and infectiousness. Aerosolization by cough and other respiratory maneuvers into room air results in a variety of respiratory droplets and droplet nuclei—the majority of which, it is believed, contain damaged, dead, or dying organisms, including microbial fragments of DNA from Mtb, other microorganisms, and respiratory lining cells. Molecular testing of ambient air samples in an unventilated small chamber occupied by an infectious TB patient reveal orders of magnitude more Mtb DNA copies than the estimated numbers of viable, infectious droplet nuclei that actually reach the alveolus and result in sustained infection.20,21 Thus, there is a steep cascade from large numbers of particles generated, many of which are dead or dying, to fewer and fewer particles that reach the distal lung and remain both viable and infectious—and succeed in causing a sustained infection.20

Unable to define the exact number of infectious droplet nuclei that must be inhaled to result in infection in a host, Wells applied the word “quanta,” a general term referring to the smallest amount of almost anything—in this case, the smallest infectious dose. For an individual infection, the lowest infectious dose (or quanta) of Mtb, cannot be less than one intact, viable, infectious Mtb organism, but nor can it be more than can be associated with a single airborne infectious droplet nucleus. Unlike multifocal pointsource histoplasmosis infection, for example, initial (primary)

TB infection in humans (and naturally infected experimental animals) is rarely more than a single lung focus—usually in better ventilated dependent (lower) regions of lungs. This strongly implies a single infectious droplet nucleus. By definition, the clinical foci of infection recognized by x-ray or at autopsy likely represent only the successful host−pathogen interactions from the pathogen perspective, leading to persistent infection manifest by detectable adaptive inflammatory responses. Unknown are the number of host−pathogen interactions that occurred where the host (i.e., macrophage) wins the initial encounter, resulting in complete microbial clearance, leaving little or no measurable evidence (by current methods) of infection or an innate immune response, much less any evidence of any aborted adaptive immune responses.22,23

Understanding that inhaling particles is probabilistic, Wells introduced Poisson’s law of small chances and defined a “quanta” of TB infection on a population basis—the average dose necessary to infect 63% of a population—the remaining exposed susceptible hosts escaping infection by chance. The estimated source strength from human outbreaks and human-to-guinea-pig experiments (see subsection “Source factors” and Figure 6.8), ranges from 1.25 infectious quanta per hour (qph), to 13 qph for an infectious office worker, to 60 qph for a case of laryngeal TB, and approximately 250 qph for a bronchoscopy-related TB case.24–26

Room air concentrations of infectious quanta vary with dilution of in-room volume, and from ventilation and die-off over time, but estimates from Riley’s classic 2-year human-to-guinea- pig transmission study are as low as 1 infectious quanta in 11,000 ft3 (311 m3) of air.27,28 These low levels of contagion, however, were sufficient to explain the rate at which student nurses became infected on hospital TB wards in the pre-chemotherapy era.29 The more infectious a particular airborne pathogen for a particular host, the less role chance plays, for example, initial measles exposure almost always results in human infection.

Measuring infectiousness

TB is routinely diagnosed in sputum by smear microscopy, culture, and by molecular detection of Mtb. Smear microscopy will detect about half of the organisms that will grow in sputum culture—and the newest molecular methods detect about the same as does liquid culture. However, it has not been possible to grow Mtb sampled from ambient room air on selective media due to overwhelming numbers of fast-growing competing environmental organisms. As noted previously, while molecular methods can detect nucleic acids, their viability and infectivity remain unknown. Coughing into a sterilized air-sampling cylinder, directly onto agar surfaces or into liquid media (cough aerosol sampling system, CASS), incorporating the host’s ability to cough and generate aerosol, has allowed source strength quantification that has been shown to correlate better with household Mtb spread than does sputum smear or culture alone.30 Because it is technically challenging, CASS remains a research tool. In low burden settings, accurate estimates of infectiousness of a source case requires testing human contacts for infection by tuberculin skin testing or IGRA. Under high-burden settings, however, pre-existing tuberculin hypersensitivity from vaccination or previous mycobacterial infection limits the utility of contact tracing.

Because of far fewer variables, for research purposes, the gold standard method for measuring human infectiousness remains quantitative animal studies, where hundreds of highly vulnerable guinea pigs breath the exhaust air from an experimental TB ward and are tested monthly for new infection.22,26,31

Although human-to-animal transmission experiments go back to the late nineteenth century, the first quantitative human- to-guinea-pig (H-GP) transmission study, envisioned by Wells and conducted by Riley in the late 1950s and the early 1960s, proved beyond doubt that Mtb was routinely spread via the air, that patients varied greatly from one another in infectiousness, and that effective treatment had an immediate and profound impact on transmission.26 More recently, Escombe conducted similar studies in Peru, focused on an HIV co-infected patient population, and proved the effectiveness of upper room germicidal UV (GUV) air disinfection (see Figure 6.1 and subsection “Upper room GUV”).32 In South Africa, Nardell, Stoltz, and colleagues have used the same model for more than 14 years (Figure 6.1)—major findings include the rapid impact of effective treatment, and new regimens, on transmission of multi-drug resistant (MDR)/extensively drug-resistant (XDR)-TB, the effectiveness of surgical masks on patients, the effectiveness of upper room GUV air disinfection (with dosing specifications), the ineffectiveness of selected room air cleaning (filtration) devices, and importantly, new insights into the early events of infection in the guinea-pig model, demonstrating frequent transient infections, with spontaneous clearance, as well as reinfection. These findings are discussed further, below.6,22,33,34

TB primary infection (and reinfection), as noted above, follows inhalation of viable and infectious Mtb-containing droplet nuclei small enough (1–5 µm) to reach and impact surfaces in the peripheral lung patrolled by alveolar macrophages.13 Macrophage recognition and phagocytosis of Mtb triggers a series of host innate and metabolic defense mechanisms aimed at killing the microbe at

that stage, and initiating specific adaptive immunity, should it be necessary. But phagocytosis of Mtb also triggers a series of microbial virulence factors intended to evade host defenses (see subsections “Infection: Transient and sustained” and “Host factors”) and, indeed, to allow the organisms to replicate rather than die within and, ultimately, outside of host cells.13 Stimulation of intracellular macrophage signaling also impacts the cytokine environment thereby modifying the strength of adaptive immunity, leading to Mtb survival and tolerance (granuloma formation, necrosis, latency) over time. Thus, the early innate host responses to Mtb are essential for preventing sustained TB infection as well as for developing effective adaptive immunity should infection persist.23,35

Development of active (clinical) TB, therefore, represents a failure of host defenses at several levels, but critically, failure of the earliest response to prevent sustained infection from relatively rare, individual Mtb-containing droplet nuclei. Given the natural variability in host innate immunity and microbial virulence, a spectrum of host−pathogen outcomes is inevitable, but poorly documented for TB. Not well described in the literature, transient TB infection occurs—the best documented example of which— albeit not usually resolved at the earliest stage of infection due to an extremely large microbial dose—is BCG vaccination, where despite clear evidence of local response to live bacteria at the injection site, measurable systemic hypersensitivity is almost always transient (months to many years) following complete clearance of this attenuated strain.36

Infection: Transient and sustained

Despite a clear understanding that initial TB infection entails innate immune responses to one or, at most, several viable Mtb organisms able to reach the alveoli in a single focus, most experimental laboratory aerosol challenge models employ brief (10–20 min) exposures to 10–50 CFUs of cultured organisms to assure infection