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Globally, tuberculosis (TB) was the leading cause of death from a single infectious agent until the coronavirus (COVID-19) pandemic. In , an estimated 10 million people fell ill with TB and a total of 1.5 million people died from the disease. About one-quarter of the global population, almost two billion people, is estimated to be latently infected with Mycobacterium tuberculosis (MTB). Although latent TB infection (LTBI) is asymptomatic and noncontagious, about 5&#;10% of LTBI patients have a lifetime risk of progression to active TB. The diagnosis and treatment of active cases are extremely vital for TB control programs. However, achieving the End TB goal of without the ability to identify and treat the pool of latently infected individuals will be a big challenge. To do so, improved technology to provide more accurate diagnostic tools and accessibility are crucial. Therefore, this chapter covers the current WHO-endorsed tests and advances in diagnostic and screening tests for active and latent TB.
Infection with MTB causes patients to develop one of two disease conditions: latent or active TB ( ). Active TB is caused by the inability of the host immune system to defend against the invading pathogen. In this scenario, the disease is symptomatic due to a recent infection and can spread to others. Alternatively, active TB can be due to a reactivation of a latent infection. In fact, more than 80% of active TB cases nowadays are attributed to reactivation of untreated latent TB in low-incidence countries such as the United States [7] . Latent TB infection (LTBI) is a noninfective state where the bacteria is contained immunologically. In LTBI, the disease is asymptomatic and can&#;t spread to others [8] . Although only 5&#;10% of non-treated LTBI patients may develop active TB, the risk of progression to active TB disease becomes significantly greater in high-risk immunocompromised individuals [9] . Therefore, diagnosis and treatment of LTBI is critical to reduce the rate of TB below the elimination threshold [10] . In this chapter, we review the current World Health Organization (WHO)-endorsed tests and advances in diagnostic and screening tools for active and latent TB.
Tuberculosis (TB), an ancient disease, remains a major global killer. In , Robert Koch reported the successful isolation of the causative agent, Mycobacterium tuberculosis [1] , an aerobic, non-motile, rod-shaped acid-fast bacterium in the family Mycobacteriaceae [2] . Although M. tuberculosis (MTB) is primarily a contagious respiratory pathogen, it can also spread to other parts of the body [3] . As a result, TB claimed the lives of billions over the centuries and continues with 1.5 million deaths in worldwide [4] . The high mortality associated with TB has been documented archeologically, i.e., Pott&#;s disease of the spine, in Egyptian mummies [5] . More recently, molecular studies confirmed the presence of the genetic material of the pathogen in specimens from ancient skeletons [6] .
Tuberculosis disease can occur in various anatomical sites, but can be divided into two main groups, pulmonary TB disease and extrapulmonary disease. Because TB disease predominantly affects the lungs, this form is commonly referred to as pulmonary disease. A person with TB pulmonary disease may present symptoms of hemoptysis, chest pain, unexplained weight loss, night sweats, persistent cough and fever. Although these symptoms are not specific to TB disease, TB should be included in the differential diagnosis especially in geographic regions of high prevalence. The WHO TB diagnostic criteria rely on clinical data vs laboratory testing. Clinical diagnosis may be based on epidemiologic exposure with physical findings, radiographic findings, positive tuberculin skin test or interferon-gamma release assay, analysis of sputum or bronchoscopy specimens, or histopathology analysis [11], [12]. In some cases, bacteriologic confirmation is not possible. In resource limited countries like Kenya, treatment is generally initiated without laboratory evidence that is largely based on clinical symptoms and disease prevalence [13]. Meta-analysis of this approach demonstrated clinical sensitivity and specificity of 61% and 69%, respectively [14]. Mortality rate is double in clinically vs bacteriologically diagnosed patients [13]. Furthermore, empiric treatment could also increase unnecessary administration of TB drugs resulting in increased antimicrobial resistance. Although radiographic anomalies found in screening are inadequate for initiation of treatment, supplemental sputum testing and bronchoscopy improves diagnostic accuracy [15].
Extrapulmonary TB (EPTB) disease symptoms exhibit the same general symptoms of pulmonary disease, i.e., fatigue, unexplained weight loss, night sweats and fever. But additional symptoms may be present depending on disease location within the body [16]. WHO defined EPTB as any bacteriologically confirmed or clinically diagnosed case of TB involving organs other than the lungs and tuberculous intra-thoracic lymphadenopathy (mediastinal and/or hilar) or tuberculous pleural effusion, without radiographic abnormalities in the lungs [17]. Renal TB may present with pyuria [18], back pain is the most common symptom for spinal TB [19], and hoarseness if the larynx is affected [20]. Prevalence factors associated with EPTB include female gender, young age, Asian or African origin and HIV infection [21]. Further discussion will focus on pulmonary TB (PTB) disease.
In patients suspected of PTB disease, testing is initiated by analysis of sputum acid-fast bacillus (AFB) smear and culture. Unfortunately, low- and middle-income countries often have high TB prevalence and rely heavily on outdated diagnostic tests [22]. In , the WHO endorsed nucleic acid amplification and drug resistance testing for TB screening and diagnosis. In , the WHO updated the TB diagnosis guidelines, reported on commercially available nucleic acid amplification test (NAAT) platforms and developed an algorithm for disease identification ( ). NAAT provides accurate and rapid turn-around time and identifies the species and antibiotic susceptibility. Accordingly, the WHO recommends NAAT in addition to existing TB tests such as AFB smear, bacterial culture and antibiotic susceptibility.
Sample type and collection are essential components in obtaining optimal results. Sputum, a thick sticky mucus which accumulates during infection and is expectorated from the respiratory tract, is most appropriate for identification of pulmonary TB disease.
As can be appreciated, analytic sensitivity is dependent on sample type. Sputum, the most common, can be obtained spontaneously by coughing or induction. However, in infants and children spontaneous sputum collection is not generally feasible [23]. Also, about one third of HIV-positive individuals with suspected TB are sputum-scarce [24]. Other more invasive and costly approaches such as bronchoscopic sampling and gastric washing may be utilized as alternative sample types [25]. It should be noted, however, the specimens not indicated in the manufacturer instructions will require validation prior to use.
Sputum induction with nebulized hypertonic saline can help produce adequate sputum for diagnostic testing [26], [27]. In this procedure, the patient inhales the nebulized solution which promotes coughing and enables expectoration of respiratory secretions. Induced sputum tends to be waterier than spontaneously obtained sputum and could be misidentified as saliva. Unfortunately, a side effect of hypertonic solution exposure is bronchoconstriction. In general, induction is an effective alternative and generally produces adequate specimen volume (>1.0&#;mL) for testing [28].
In indicated above, HIV patients tend to be sputum-scarce, so testing for TB in this population can be problematic. Furthermore, HIV patients tend to have lower MTB load in respiratory secretions [29]. A non-invasive oral swab collection was evaluated in TB positive patients with or without HIV co-infection [30]. The study found that PCR signal amplification was weaker in HIV-positive patients. Tongue swabs yielded the strongest signal (vs gum and cheek swabs) and demonstrated a sensitivity and specificity of 78% and 96%, respectively.
Timing of collection is also important. The WHO recommends collecting two sputum samples with at least one being early morning [31]. The morning specimen is preferred because respiratory secretions pool overnight. Smear microscopy has shown that morning sputum samples yielded higher positivity rates for acid fast bacilli vs random samples [32].
In summary, sample type, collection method and timing must be appropriate for the type of assay being employed. Evaluation of diagnostic accuracy is necessary for different sample types and is obligatory.
Microbial confirmation of pulmonary TB is vital to reduce empiric antibiotic use that potentially contributes to multi-drug resistance. Microscopic analysis of sputum smear is a quick and easy to assess bacteriologic evidence of mycobacteria. Acid-Fast staining, i.e., Ziehl-Neelsen, Kinyoun and Auramin-Rhodamine Fluorochrome, takes advantage of the mycolic acids present in the outer membrane of acid-fast mycobacteria.
Ziehl-Neelsen and Kinyoun methods use carbolfuchsin. The main difference is the use of heat in the former while no heat and higher dye concentration is used in the latter. Acid-Fast bacillus stain red when exposed to carbolfuchsin even after washing with acid alcohol. Slides can be analyzed using standard bright-field microscopy. Auramine O and rhodamine B are fluorescent dyes that also bind mycolic acids. Stained acid-fast organisms are identified by fluorescence microscopy which requires specific filters for proper visualization. Acid-fast bacteria fluoresce yellow-orange.
Both fluorescence microscopy and Ziehl-Neelsen staining had 100% specificity using the Xpert MTB/RIF as the reference test [32]. In addition, the former demonstrated higher sensitivity (84.5% vs 54.8%) and shorter reading time.
Smear microscopy is a fast and simple approach for detecting mycobacteria. Although fluorescence microscopy demonstrated better diagnostic values, cost was substantially higher. Smear results depend on bacterial burden, samples with low bacterial burden may be missed. Positive smears require further testing for speciation. In addition, other mycolic acid containing species, i.e., Rhodococcus, Nocardia and Corynebacterium, can be incorrectly identified as mycobacteria [33]. As such, smear microscopy is generally insufficient and further testing is needed to differentiate MTB from other mycobacteria and other species with mycolic acids. Conventional selective bacterial culture can be subsequently used to identify the mycobacterial species.
MTB is slow growing. Due to the challenges of cultivation, the use of one liquid and one solid media is recommended to maximize recovery [34]. A two year study published in compared four culture media that included Lowenstein-Jensen (LJ), Middlebrook (7H10), Petragnani and ribonucleic acid (RNA) media and found that LJ and RNA media were best for routine practice [35]. The recommended media for isolating MTB includes egg- or agar-based media supplemented with green malachite and Middlebrook broths or solid media [36]. Culture typically requires 3&#;8 weeks to detect growth from clinical samples.
Although bacterial culture is considered the gold standard, contamination with other bacteria and fungi could undermine its effectiveness [37]. Samples are processed using N-acetyl-L-cysteine-sodium hydroxide, a common chemical used for decontamination, prior to culturing. The use of selective media such as LJ medium is most common, especially in resource limited settings. Addition of antibiotics to LJ media dramatically decreases contamination (32&#;2.3%) when supplemented with Selectatab-MB [38].
Drug susceptibility of MTB isolate is performed by observing growth or metabolic inhibition in a medium containing an anti-tuberculosis drug. Unfortunately, manual culturing extends resulting time especially in high TB burden countries. However, newer techniques are now available to shorten detection of growth inhibition. The most widely used US FDA-cleared automated systems for rapid detection of mycobacteria using liquid media are BacT/ALERT 3D (Biomerieux), BACTEC MGIT (Becton Dickinson) and VersaTREK (Thermo Fisher). These employ automated continuous monitoring of carbon dioxide (CO2) production or oxygen (O2) consumption to detect MTB. BACTEC and VersaTrek are also FDA-cleared for susceptibility testing of the MT complex (MTBC). Due to the possibility of instrument negative mycobacterial growth, some manufacturers recommend visual inspection [39].
BACTEC MGIT 960, a non-radiometric version of BACTEC 460 and fully automated system, detects growth by measuring O2 consumption in the culture vial via an O2-sensitive fluorescent indicator [40]. Using this approach, mean detection time improved from 38.6 to 21.4 days for abscess samples vs those cultured in LJ media [41]. Smear negative sputum, bronchial aspirate and pleural fluid had higher positivity rate (16.2% vs 9.8%) and shorter detection time (11.1 vs 30.7 days) when BACTEC MFIT was compared to LJ culture [42]. A 3-year Massachusetts General Hospital study reported that found that 10% of all cultures yielded mycobacteria using BACTEC MGIT. Visual inspection of instrument-negative samples revealed colony-like clumps at the bottom of the tube and hence recommended supplementary procedures after 42 days incubation. This included comparing AFB smears, visual inspection and performing an additional AFB smear for the colony-like clumps.
The BacT/ALERT system utilizes a colorimetric CO2 sensor. Parallel testing of the BacT/ALERT and BACTEC MGIT 960 systems demonstrated the latter had shorter detection time (13.5 vs 25.2 days) [43], lower contamination rate [44] and higher sensitivity (100% vs 66.6%) [43]. The authors suggested that longer incubation times may improve recovery using BacT/ALERT system. VersaTREK monitors headspace pressure due to O2 consumption to detect bacterial growth. An MTB susceptibility study that compared the VersaTREK and BACTEC MGIT 960 reported 100% concordance for isoniazid, rifampin and ethambutol and 97% concordance for pyrazinamide [45].
One difficulty in containing and managing TB is patient follow-up compliance that disrupts continuity of care and promotes disease spread. The ability to decrease bacterial culture time is important to stop TB spread by enabling earlier use of anti-tuberculosis medication. Unfortunately, culturing has limitations. Positive-smear culture-negative specimens underscore the possibility of non-cultivable MTB. In addition, MTB is also prone to transitioning to a dormant non-replicative phenotype believed the source of latent TB infection [46]. Clinical and experimental studies indicate that the composition of MTB is likely heterogenous [47]. Active replicating bacilli are killed effectively by first-line drugs such as isoniazid while dormant MTB may not be eliminated. The latter further underscores the limitation of culturing as a diagnostic test.
Nucleic acid tests can be used in symptomatic patients with AFB positive or negative culture results. NAAT does not replace microbiologic tests and is considered complementary.
Nucleic acid testing (NAT) identifies a species or subspecies of an organism by detecting the genetic make-up, i.e., ribonucleic acid (RNA) or deoxyribonucleic acid (DNA). NATs coupled with PCR, i.e., nucleic acid amplification testing (NAAT), enables trace or ultra-low detection.
In , the WHO endorsed the Xpert MTB/RIF assay (Cepheid, USA) for TB testing [48]. It is the most commonly used test worldwide (>130 countries) [49]. In , the US FDA approved the Xpert MTB/RIF for TB diagnosis [4]. Although this assay simultaneously detects MTBC and its susceptibility to rifampicin, it does not detect resistance to isoniazid. The Xpert MTB/RIF utilizes nested real-time PCR for the qualitative detection of the MTB complex and RIF resistance [50]. The WHO Consolidated Guidelines on Tuberculosis Module 3 recommends that Xpert MTB/RIF be used in adults and children with signs and symptoms of pulmonary TB as an initial diagnostic test rather than smear microscopy/culture and phenotypic drug susceptibility testing [51]. The diagnostic algorithm using NAAT as an initial diagnostic test pending culturing is shown ( ). Although NAAT increases diagnostic specificity, sensitivity is still too low to rule out disease, especially in paucibacillary cases [52]. The predictive value in smear positive specimens is 95%. However, false-negative NAAT can occur in the presence of PCR inhibitor(s) [53]. Rapid turn-around time of NAAT and AFB is necessary for diagnosis and the prompt initiation of therapy pending culture results. An algorithm that includes AFB, NAAT and bacterial culture is shown ( ). NAAT results should always be assessed in respect to other diagnostic tests, clinical symptoms and disease prevalence [54].
Open in a separate windowA Taiwanese study where NAAT using Enhance Amplified MTB Direct Test [E-MTD] (Gen-Probe, USA) was used long-term (>20&#;y) showed that NAAT was useful in identifying MTB and excluding infections caused by nontuberculous mycobacteria [55]. After inclusion of NAAT in the testing protocol, the study showed a decrease in unnecessary isolations and treatment of suspected cases of PTB especially among patients with non-PTB, i.e., those infected with hepatitis B and C. Therefore, NAAT may be useful in regions with high hepatitis B and C prevalence. The authors reported that the sensitivity, specificity, positive predictive value, and negative predictive value of NAAT were 99.0%, 92.3%, 99.0% and 92.3%, when compared to culture, respectively.
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An independent study in South Africa, i.e., a country with high TB burden, compared four commercial platforms (Abbott RealTime MTB and MTB rifampicin and isoniazid resistance assays (RIF/INH), Hain Lifescience FluoroType MTBDR assay, BD Max MDR-TB assay, and the Roche cobas MTB and MTB-RIF/INH) for detecting MTBC and resistance to rifampicin and isoniazid. They concluded that the clinical performance of these methods are similar to WHO-recommended molecular TB and MDR-TB assays [56]. The average limit of detection, defined as number of genomes/mL at the 95% hit rate was , 322, 826, and for Xpert MTB/RIF, Abbott RealTime MTB, BD Max MDR-TB, Roche cobas MTB and Hain Lifescience FlouroType MTBDR, respectively.
Due to the expense of NAAT, it is important to use sample types that offer the best diagnostic performance. Meta-analysis for the Xpert MTB/RIF assay concluded that expectorated sputum provided the best diagnostic performance, i.e., 90% sensitivity and 98% specificity, in adults [57]. Sensitivity and specificity for bronchoalveolar lavage and induced sputum was 87% and 91% and 86% and 97%, respectively. In the pediatric population, however, gastric aspiration was superior to expectorated sputum, induced sputum and nasopharyngeal aspirates.
Various genes are targeted by NAAT. Commercially available assays and the target genes are listed ( ). The MTBC gene targets are IS, protein antigen B (pab), rpoB and IS. IS, an insertion element, can be found multiple times in the MTB genome and is considered a sensitive diagnostic target. IS, thought to be found exclusively within the members of the MTBC, is also present in the genome of mycobacterium smegmatis [58]. The pab gene encodes a 38&#;kDa protein specific for MTB [59]. Rifampicin resistance is detected through the mutations in rpoB gene, which codes for the Beta subunit of the RNA polymerase, while detection of katG gene mutations, coding for the catalase peroxidase, is used to identify isoniazid resistance [60]. Inh A gene, which codes for 2-trans-enoyl-acyl carrier protein reductase [61], was identified as important for isoniazid and ethionamide resistance [62].
Advantages of NAAT include multiplex capability to increase specificity without dramatic increase in analytical run time and the ability to amplify low copy genomes to improve sensitivity. Despite these advances, the use of laboratory-bound instrumentation limits remote testing in geographic regions with high TB burden.
A better approach in TB testing would enable point-of-care for immediate isolation and initiation of therapy thus mitigating loss of patient contact especially in remote areas.
For example, isothermal amplification methods enable nucleic acid detection at constant temperature thus eliminating the constraint of thermal cycling. These include loop-mediated isothermal amplification, strand displacement amplification, helicase-dependent amplification, and recombinase polymerase amplification. All provide analytical sensitivity and specificity comparable to traditional thermal cycling.
In point-of-care molecular assays, choosing the appropriate enzyme is critical for isothermal amplification-based MTB detection. Helicase-dependent amplification unwinds the double-stranded DNA target thus eliminating the need for heat denaturation and thermal cycling. A proof-of-concept study reported that isothermal amplification with a single primer for IS in MTB had a limit of detection of 2.5&#;×&#;105 copies/µL [63]. A loop mediated isothermal amplification study that used an inexpensive device reported an analytical sensitivity of 10 genomic copies [64]. A non-NAAT point-of-care test using lateral flow immunoassay to detect urinary mycobacterial lipoarabinomannan demonstrated improved sensitivity for TB in those coinfected with HIV [51].
The development of decentralized TB test devices has received much commercial interest. For example, Cepheid has developed a battery operated GeneXpert Omni system for Xpert cartridge designed for remote low-throughput settings. This system demonstrated high concordance with the traditional Xpert MTB/RIF assay (99.5%; 198/199) [65]. Technologies based on microfluidic devices, extensively used in diagnostic testing [66], [67], likely provide innovative solutions applicable to TB [68].
Future development of these novel tools will overcome geographic, economic and resource limitations as part of the WHO End TB strategy [69].
Examinations of clinical specimens (e.g., sputum, urine, or cerebrospinal fluid) are of critical diagnostic importance. The specimens should be examined and cultured in a laboratory that specializes in testing for M. tuberculosis. Contact your state TB program for information about public health laboratory services for your area.
Treatment generally should not be delayed while waiting for bacteriologic results if TB disease is the presumptive diagnosis. Treatment can be started as soon as specimens have been collected or even while specimens are being collected if a patient is very ill. Consultation with a TB expert is recommended for deciding when to start treatment in relation to specimen collection.
Optimal bacteriologic examination has five parts:
Patients presumed to have pulmonary TB disease may cough up sputum (phlegm) into a sterile container for processing and examination.
Other sputum specimen collection methods include inducing sputum, bronchoscopy, and gastric washing. Health care providers should use precautions to control the spread of TB bacteria during sputum collection procedures.
In patients who have presumed extrapulmonary TB disease, the way specimens are obtained depends on the part of the body affected.
Specimens are smeared onto a glass slide and stained so that they can be examined for acid-fast bacilli (AFB) under a microscope. M. tuberculosis complex organisms are one kind of AFB. Smear examination is a quick procedure, and results should be available within 24 hours of specimen collection.
When AFB are seen in a smear, they are counted and classified as 4+, 3+, 2+ or 1+, according to the number of AFB seen. The greater the number, the more infectious the patient.
Negative smears do not exclude TB disease. The AFB in a smear may be organisms other than M. tuberculosis.
Nucleic acid amplification (NAA) tests are used to amplify DNA and RNA segments to rapidly detect M. tuberculosis DNA in specimens in just hours, compared to a week or more for detection of TB bacteria in culture. CDC recommends that NAA testing be performed on at least one respiratory specimen from each patient with symptoms of pulmonary TB disease for whom:
The Xpert MTB/RIF assay is an NAA test that simultaneously detects and identifies M. tuberculosis complex detects genetic mutations that can predict resistance to rifampin (RIF), one of the most effective drugs used to treat TB. A sputum sample is mixed with a sterilizing reagent provided with the assay, and a cartridge containing the mixture is placed in the GeneXpert machine.
NAA test and Xpert MTB/RIF assay results can help guide health care provider's decisions for patient therapy and isolation; however, they do not replace the need for AFB smear, culture, growth-based drug susceptibility testing, and genotyping. Health care providers and laboratories should ensure patient specimens are available for all recommended mycobacterial testing.
Culturing the specimen means growing the mycobacteria on solid or in liquid media. All specimens should be cultured, regardless of whether the smear is positive or negative. Culture is the gold standard for laboratory confirmation of TB disease.
Specimen culture is important for TB genotyping, a laboratory-based approach used to analyze the genetic material (e.g., DNA) of M. tuberculosis. TB genotyping results, when combined with epidemiologic data, help identify persons with TB disease involved in the same chain of recent transmission.
Drug susceptibility tests should be done when a patient is first found to have a positive culture for M. tuberculosis. These tests will determine which drugs will be effective in a combination regimen for treating TB disease.
Molecular Detection of Drug Resistance (MDDR) assays allow rapid detection of drug resistance through the detection of genetic mutations associated with resistance. Respiratory specimens from patients with risk factors for drug-resistant TB disease, AFB smear positive results or NAA test positive results should be sent for molecular drug resistance testing immediately.
CDC's MDDR service is available nationally and free of charge through state public health laboratories.
Growth-based drug susceptibility testing can be done using a liquid medium or a solid medium method. Liquid medium methods are faster than solid media methods for determining susceptibility to first-line TB medications.
The results of both growth-based and molecular drug susceptibility test should inform health care providers' choices of the appropriate drugs for treating each patient.
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