eMedicine Specialties > Pediatrics: General Medicine > Infectious Disease

Tuberculosis: Differential Diagnoses & Workup

Author: Vandana Batra, MD, Pediatrician, Department of Pediatrics, Division of General Pediatrics/Primary Care, Nemours Pediatrics
Coauthor(s): Jocelyn Y Ang, MD, Assistant Professor, Department of Pediatrics, Division of Infectious Diseases, Children's Hospital of Michigan and Wayne State University
Contributor Information and Disclosures

Updated: Oct 30, 2008

Differential Diagnoses

Workup

Laboratory Studies

• Making the diagnosis of tuberculosis (TB) in children is extremely challenging because of the difficulty in isolating M tuberculosis. Definitive diagnosis of tuberculosis depends on isolation of the organism from secretions or biopsy specimens.
• Despite innovations in rapid diagnosis, many of the classic diagnostic tools remain useful and continue to be used in the evaluation of patients with tuberculosis.
• Detection and isolation of the mycobacterium are accomplished as follows:
  • The initial step is to obtain appropriate specimens for bacteriologic examination. Examination of sputum, gastric lavage, bronchoalveolar lavage, lung tissue, lymph node tissue, bone marrow, blood, liver, cerebrospinal fluid (CSF), urine, and stool may be useful, depending on the location of the disease.
  • Gastric aspirates are used in lieu of sputum in very young children (<6 y) who usually do not have a cough deep enough to produce sputum for analysis.
    • Using the correct technique for obtaining the gastric lavage is important because of the scarcity of the organisms in children compared to adults. An early morning sample should be obtained before the child has had a chance to eat or ambulate because these activities dilute the bronchial secretions accumulated during the night.
    • Initially, the stomach contents should be aspirated, and then a small amount of sterile water injected through the orogastric tube. This aspirate should also be added to the specimen.
    • Because gastric acidity is poorly tolerated by the tubercle bacilli, neutralization of the specimen should be performed immediately with 10% sodium carbonate or 40% anhydrous sodium phosphate. Even with careful attention to detail and meticulous technique, the tubercle bacilli can be detected in only 70% of infants and in 30-40% of children with disease.
  • Sputum specimens may be used in older children. Nasopharyngeal secretions and saliva are not acceptable. In older children, bronchial secretions may be obtained by the stimulation of cough by an aerosol solution of propylene glycol in 10% sodium chloride.
  • Bronchoalveolar lavage may also be used to provide bronchial secretions for detection of tubercle bacilli.
  • Decontamination of other microorganisms in the specimens obtained may be performed by the addition of sodium hydroxide, usually in combination with N -acetyl-L -cysteine. Other body fluids (eg, CSF, pleural fluid, peritoneal fluid) can also be centrifuged; the sediment can be stained and evaluated for presence of acid-fast bacilli (AFB). CSF smear results are positive in fewer than 10% of patients in some series. Enhancement of the yield may be possible by staining any clot that may have formed in standing CSF specimens, as well as using the sediment of a centrifuged specimen. Increased yield may also be obtained from cisternal or ventricular fluid.
  • Obtain overnight urine specimens in the early morning. Send immediately for analysis because the tubercle bacilli poorly tolerate the acidic pH of urine.
• Staining of the specimen is as follows:
  • Because M tuberculosis is an AFB, staining of AFB provides preliminary confirmation of the diagnosis.
  • Staining can also give a quantitative assessment of the number of bacilli being excreted (eg, 1+, 2+, 3+). This can be of clinical and epidemiologic importance in estimating the infectiousness of the patient and in determining the discontinuation of respiratory isolation. However, for reliably producing a positive result, smears require approximately 10,000 organisms/mL. Therefore, in early stages of the disease or in children in whom the bacilli in the respiratory secretions are sparse, the results may be negative. A single organism on a slide is highly suggestive and warrants further investigation.
  • A significant drawback of AFB smears is that they cannot be used to differentiate M tuberculosis from other acid-fast organisms such as other mycobacterial organisms or Nocardia species.
  • Conventional methods include the Ziehl-Neelsen staining method. The Kinyoun stain is modified to make heating unnecessary. Fluorochrome stains, such as auramine and rhodamine, are variations of the traditional stains. The major advantage of these is that slides can be screened faster because the acid-fast material stands out against the dark, nonfluorescent background. However, fluorochrome-positive smears must be confirmed by Ziehl-Neelsen staining.
• Conventional growth techniques are as follows:
  • Culture of mycobacterium is the definitive method to detect bacilli. It is also more sensitive than examination of the smear. Approximately 10 AFB/mm of a digested concentrated specimen are sufficient to detect the organisms by culture.
  • Another advantage of culture is that it allows specific species identification and testing for recognition of drug susceptibility patterns. However, because M tuberculosis is a slow-growing organism, a period of 6-8 weeks is required for colonies to appear on conventional culture media.
  • Conventional solid media include the Löwenstein-Jensen medium, which is an egg-based medium, and the Middlebrook 7H10 and the 7H11 media, which are agar-based media. Liquid media (eg, Dubos oleic-albumin media) are also available, and they require incubation in 5-10% carbon dioxide for 3-8 weeks. These media usually have antibacterial antibiotics, which are slightly inhibitory for tubercle bacilli.
• Modern approaches in diagnosis are as follows:
  • Because mycobacteria require 6-8 weeks for isolation from conventional media, automated radiometric culture methods (eg, BACTEC) are increasingly used for the rapid growth of mycobacteria. The methodology uses a liquid Middlebrook 7H12 medium that contains radiometric palmitic acid labeled with radioactive carbon 14 (¹⁴ C). Several antimicrobial agents are added to this medium to prevent the growth of nonmycobacterial contaminants. Production of¹⁴ CO₂ by the metabolizing organisms provides a growth index for the mycobacteria. Growth is generally detected within 9-16 days.
  • Another rapid method for isolation of mycobacteria is SEPTICHEK. This nonradiometric approach has a biphasic broth-based system that decreases the mean recovery time versus conventional methods.
  • Mycobacterial growth indicator tubes (MGITs), which presently are used as a research tool, have round-bottom tubes with oxygen-sensitive sensors at the bottom. MGITs indicate microbial growth and provide a quantitative index of M tuberculosis growth.
• Identification of species is as follows:
  • M tuberculosis can be reliably differentiated from other species on the basis of culture characteristics, growth parameters, and other empiric tests. M tuberculosis produces heat-sensitive catalase, reduces nitrates, produces niacin, and grows slowly. Serpentine cording is demonstrated on smears prepared from the BACTEC system.
  • Addition of p -nitro-acetyl-amino-hydroxy-propiophenone (NAP) inhibits the growth of M tuberculosis complex (including M bovis and M africanum) but does not inhibit growth of other mycobacteria. This provides the basis for the NAP differentiation test.
  • Chromatographic analysis of mycobacterial cell wall lipids can provide further speciation. The most useful approaches include gas-liquid chromatography and high-performance liquid chromatography (HPLC). The unique mycolic acid pattern associated with the species can be detected by the chromatographic separation of the ester. A significant drawback of these methods is the requirement of bacterial colonies grown in conventional solid media, a process that takes at least 3 weeks. However, the recent combination of HPLC with fluorescence detection has made the method more sensitive, thus BACTEC broth culture can be used instead of conventional solid media. This may make the method comparable to the NAP and AccuProbe tests. The expense of the initial equipment limits the availability of HPLC.
• Nucleic acid probes are used as follows:
  • Because biochemical methods are time-consuming and laborious, nucleic acid hybridization using molecular probes has become widely accepted. Commercially available probes, including the AccuProbe technology, help advance identification of the M tuberculosis complex. Sensitivity and specificity approach 100% when at least 100,000 organisms are present.
  • The basic principle is the use of a chemiluminescent, ester-labeled, single-strand DNA probe. A luminometer is used to assess the chemiluminescence.
  • Positive test results should be reported as M tuberculosis complex because the probe does not reliably differentiate between M tuberculosis and other members of the complex (eg, M bovis). Final identification to species level is required because pyrazinamide should not be included in the treatment regimen if the isolate is M bovis.
  • Niacin production, nitrate reduction, pyrazinamidase, and susceptibility to thiophene-2-carboxylic acid hydrazide can help differentiate between M bovis and M tuberculosis.
• Polymerase chain reaction (PCR) and other amplification tests are used as follows:
  • Nucleic acid amplification allows the direct identification of M tuberculosis in clinical specimens, unlike the nucleic acid probes, which require substantial time for bacterial accumulation in broth culture.
  • The US Food and Drug Administration (FDA) has approved 2 tests, the amplified M tuberculosis direct test and the AMPLICOR M tuberculosis test. The AMPLICLOR test targets the DNA. The most commonly used target sequence for the detection of M tuberculosis has been the insertion sequence IS6110. The amplified M tuberculosis direct test is an isothermal transcription-mediated amplification that targets RNA.
  • Although amplification techniques are promising tools for the rapid diagnosis of tuberculosis, several caveats remain. Contamination of samples by products of previous amplification and the presence of inhibitors in the sample may lead to false-positive or false-negative results.
  • Although the sensitivity and specificity of the nucleic acid techniques in smear-positive cases exceed 95%, the sensitivity of smear-negative cases varies from 40-70%. Thus, discordance between the acid-fast smear result and the nucleic acid amplification techniques requires careful clinical appraisal and judgment.
• T-SPOT Tb and QuantiFERON-TB Gold, interferon-gamma release assays (IGRAs), are 2 blood tests recently available to aid in the diagnosis of tuberculosis.⁹
  • The QuantiFERON-TB Gold has recently been approved by the FDA as an in vitro diagnostic test and the CDC has published a guideline for its use. The test is an enzyme-linked immunoassay (ELISA) that basically detects the presence of interferon gamma release protein (IFN-g) from the blood of sensitized patients when incubated with the early secretory antigenic target-6 (ESAT6) and culture filtrate protein 10 (CFP10) peptides. The test is as sensitive as, and more specific than, the tuberculin skin test (TST) and has been recommended as a screening tool in all situations in which the tuberculin skin test has been used (ie, for diagnosing disease as well as infection).
  • Other advantages include the fact that no return visits are needed to read the skin tests and no boosting occurs due to the tests.
  • Initial studies are underway to determine if these tests can be used to monitor response to therapy.
• M tuberculosis drug susceptibility is determined as follows:
  • Because of the emergence of multidrug-resistant (MDR) organisms, determination of the drug susceptibility panel of an isolate is important so that appropriate treatment can be ensured.
  • Numerous chromosomal mutations are associated with drug resistance. Genotypic methods now being evaluated to identify these mutations include DNA sequencing, solid phase hybridization, and PCR–single-strand combination polymorphism analysis.
  • Mutations of the catalase peroxidase gene katG, the inhA gene involved in fatty acid biosynthesis, the ahpc gene, and the oxyR gene have been identified as major determinants for isoniazid (INH) resistance.
  • Resistance to rifampin is determined by mutations in the rpoB gene encoding the beta subunit of the RNA polymerase.
  • Phenotypic susceptibility assays, which remain experimental, use mycobacteriophages to type the mycobacteria grown in the presence of antituberculous agents.
• Serology is as follows:
  • M tuberculosis increases the levels of antibody titers in the serum.
  • No available serodiagnostic test for tuberculosis has adequate sensitivity and specificity for routine use in diagnosing tuberculosis in children.

Imaging Studies

• Chest radiography
  • Chest radiography is a classic diagnostic tool when evaluating patients for pulmonary tuberculosis.
  • Initial studies include posteroanterior and lateral views. Apical-lordotic and oblique views may be helpful if further evaluation of the extent of lung involvement is indicated (eg, patients with apical lesions or extensive hilar adenopathy).
  • If pleural effusion is present, lateral decubitus views aid in the determination of the nature of effusion (ie, free moving, loculated).
• CT scanning and MRI
  • CT scanning and MRI are not routinely indicated when chest radiography findings are unremarkable.
  • However, in patients with pulmonary tuberculosis, these imaging studies can help demonstrate hilar lymphadenopathy, endobronchial tuberculosis, pericardial invasion, and early cavitations or bronchiectasis.

Other Tests

• The TST is a widely used diagnostic test for evaluation of patients who have symptoms of tuberculosis or in whom infection with M tuberculosis is suspected. Although the sensitivity and the specificity of the TST are less than 100%, no better diagnostic test is widely available.
• The American Academy of Pediatrics (AAP) has issued the following guidelines for pediatric testing:¹⁰
  • Immediate skin testing is indicated for the following children:
    • Those who have been in contact with persons with active or suspected tuberculosis
    • Immigrants from countries in which tuberculosis is endemic (eg, Asia, Middle East, Africa, Latin America) or children with travel histories to these countries
    • Those who have radiographic or clinical findings suggestive of tuberculosis
  • An annual TST is indicated for the following children:
    • Children who are infected with HIV or those living in a household with persons infected with HIV
    • Incarcerated adolescents
  • Testing at 2-year to 3-year intervals is indicated if the child has been exposed to high-risk individuals including those who are homeless, institutionalized adults who are infected with HIV, users of illicit drugs, residents of nursing homes, and incarcerated adolescents or adults.
  • Testing when children are aged 4-6 years and 11-16 years is indicated for the following children:
    • Children without risk factors residing in high-prevalence areas
    • Children whose parents emigrated from regions of the world with a high prevalence of tuberculosis or who have continued potential exposure by travel to the endemic areas and/or household contact
  • Performing an initial TST before the initiation of immunosuppressive therapy is recommended in any patient.
• Administration of TST is as follows:
  • The recommended TST is the Mantoux test. The dosage of 0.1 mL or 5 TU purified protein derivative (PPD) should be injected intradermally into the volar aspect of the forearm using a 27-gauge needle. A detergent called Tween 80 to prevent loss of efficacy on contact and adsorption by glass stabilizes the PPD. A wheal should be raised and should measure approximately 6-10 mm in diameter.
  • Skilled personnel always should read the test 48-72 hours after administration. Measure the amount of induration and not erythema. This should be measured transverse to the long axis of the forearm.
  • Multiple puncture tests (eg, tine test, Heaf test) lack sensitivity and specificity and hence are not recommended.
• The CDC and the AAP have provided recommendations on the size of the induration created by the TST that is considered a positive result and indicative of disease. The TST is interpreted on the basis of rule of 5 mm, 10 mm, and 15 mm.
  • Induration of 5 mm or more is considered a positive TST result in the following children:
    • Children having close contact with known or suspected contagious cases of the disease, including those with household contacts with active tuberculosis whose treatment cannot be verified before exposure
    • Children with immunosuppressive conditions (eg, HIV) or children who are on immunosuppressive medications
    • Children who have an abnormal chest radiography finding consistent with active tuberculosis, previously active tuberculosis, or clinical evidence of the disease
  • Induration of 10 mm or more is considered a positive TST result in the following children:
    • Children who are at a higher risk of dissemination of tuberculous disease, including those younger than 5 years or those who are immunosuppressed because of conditions such as lymphoma, Hodgkin disease, diabetes mellitus, and malnutrition
    • Children with increased exposure to the disease, including those who are exposed to adults in high-risk categories (eg, homeless, HIV infected, users of illicit drugs, residents of nursing homes, incarcerated or institutionalized persons); those who were born in or whose parents were born in high-prevalence areas of the world; and those with travel histories to high-prevalence areas of the world
  • Induration of 15 mm or more is considered a positive TST result in children aged 5 years or older without any risk factors for the disease.
• False-positive reactions and false-negative results can have various causes.
  • False-positive reactions often are attributed to asymptomatic infection by environmental nontuberculous mycobacteria (due to cross-reactivity).
  • False-negative results may be due to vaccination with live-attenuated virus, anergy, immunosuppression, immune deficiency, or malnutrition. Other factors that may cause a false-negative result include improper administration (eg, subcutaneous injection, injection of too little antigen), improper storage, and contamination. PPD has been recognized to have an initial false-negative rate of 29%.
• The following are important when administering the TST to prior recipients of bacille Calmette-Guérin (BCG) vaccine:
  • Immunization with BCG is not a contraindication to the TST. BCG vaccination is used in many parts of the world, especially in developing countries.
  • Differentiating tuberculin reactions caused by vaccination with BCG versus reactions caused by infection with M tuberculosis is difficult. History of contact with a person with contagious tuberculosis or emigration from a country with a high prevalence of tuberculosis suggests that the positive results are due to infection with M tuberculosis. However, multiple BCG vaccinations may increase the likelihood that the positive TST result is due to BCG vaccination. The positive reactivity caused by BCG vaccination generally wanes with the passage of time. With the administration of TST, this positive tuberculin reactivity may be boosted.
  • A prior BCG vaccination does not affect interpretation of a TST result for a person who is symptomatic or in whom tuberculosis is strongly suspected.

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