Indeterminate Quantiferon test result in a case of active tuberculosis
The patient was found to be sputum Acid Fast Bacilli (AFB) positive and the TB PCR done on the sputum sample was reported as positive for M. tuberculosis complex. AFB cultures were positive too and the patient was started on anti-tuberculosis treatment. Various tests to rule out other infectious causes like the respiratory viral panel (Respiratory Syncytial Virus (RSV), Influenza A and B), Legionella urinary antigen and Streptococcus pneumoniae antigen were found to be negative. Peripheral blood smear did not show any hemoparasites. The HIV, HBV and HCV tests conducted were also negative.
Tuberculosis (TB) is prevalent amongst the human population since ancient history. It is still endemic in some parts of the world like the developing countries of Africa and Asia. It is commonly caused by the Mycobacterium tuberculosis, which is one of the eight mycobacteria forming the M. tuberculosis complex (MTBC). The eight Mycobacteria in this complex are M. tuberculosis, M. bovis, M. africanum, M. microti, M. pinnipedii, M. caprae, M. mungi and M. canetti . Most of these species have been shown to cause disease in humans, though majority of the TB cases are caused by M. tuberculosis. The specific mycobacterial strain was not reported in the present case.
Tuberculosis infections are of continued concern due to the susceptibility by patients with HIV- acquired immune-deficiency syndrome (HIV-AIDS) and with the emergence of multiple drug resistant strains . According to World Health Organization's (WHO's) Global Tuberculosis Report of 2019, TB is one of the top 10 causes of death worldwide and is the leading cause of death from a single infectious agent . In 2018, there were about 10 million people who fell ill with tuberculosis world over.3 In the United States total tuberculosis cases in 2018 were approximately 9000 and the total TB incidence was recorded as 3 per 100,000 population [3,4].
Tuberculosis infection is acquired by inhalation of infectious droplet nuclei, which are released by a patient with active pulmonary tuberculosis on coughing, sneezing, talking, singing etc. Tuberculosis can occur as a primary infection or a secondary infection in the lungs. When a patient is infected by the tubercular bacilli for the first time, it's called a primary infection. The bacilli after inhalation of the infectious droplet nuclei, reach the alveoli, where they are engulfed by the alveolar macrophages. While these macrophages try to kill the mycobacteria, the latter try to evade the bactericidal action using various mechanisms. The macrophages with engulfed bacteria are also stimulated to release numerous cytokines and chemokines, which play a role in attracting other immune cells such as T cells, neutrophils, monocytes to the site of infection. The macrophages with engulfed mycobacteria, travel to the regional lymph nodes, where they prime the CD4+ and CD8+T cells. In latent tubercular infection a small percentage of the tubercle bacilli enter the blood stream and travel to different parts of the body, but at each site they are contained by the macrophages and other immune cells. These immune cells form granulomas to contain the tubercular bacilli. Such patients have dormant bacilli, but do not have an active infection [5,6].
When a patient's immunity is suppressed as with medications (e.g. steroids, immunosuppressants), acquired immunodeficiency, or with age, there can be a reactivation of the disease or there can be a re-infection. This results in secondary tuberculosis. Apical cavitary lung lesions are common in secondary pulmonary tuberculosis and are one of the characteristic signs of active tuberculosis. Tuberculosis infection can also be extra-pulmonary and can infect organs like the brain, kidneys, pleura, larynx, oral cavity, bones, joints and gonads.
Immunity to M. tuberculosis is primarily mediated by CD4+ T cells, which stimulate macrophages through secretion of interferon gamma, to kill the mycobacteria. CD8+ T cells have also been found to play an important role in controlling TB infection by secreting Interferon gamma and directly killing TB infected cells. They can also lyse the intra-cellular TB bacilli. The CD8+ T cells have been found to play a stronger role in active tuberculosis.
TB Diagnostic Testing
Screening for latent TB generally occurs via the tuberculin skin test or an interferon gamma release assay. Symptomatic patients with cough, fever and weight loss and those with positive screening tests are followed up with a chest X-ray and those with findings of upper lobe infiltration and cavitation, are said to have signs of tuberculosis. For confirmation testing, sputum is sent for acid fast bacilli (AFB) staining and for culture, as was done in the present case. Positive culture is the definitive diagnosis of tuberculosis; however, it takes 2-6 weeks usually on the conventional solid media (Lowenstein Jensen media). Other methods are being employed to reduce the time for detection such as the BACTEC Mycobacteria Growth Indicator Tube Systems (MGIT), in which mycobacteria are grown in a liquid broth and the growth is detected by the consumption of oxygen. A fluorescent compound embedded in the tube sensitive to the presence of oxygen detects changes in oxygen levels. After the MGIT turns positive, an AFB stain is performed to ensure AFB bacilli are present. If positive, a DNA chemiluminescent probe (Accuprobe) is performed to identify MTBC and MAI (Mycobacterium avium intercellulare) complex. These probes are highly sensitive and specific, and can be resulted the same day of MGIT positivity. These probes can also be performed from colonies on solid media. There are the newer molecular methods like the TB PCR. All TB screening and diagnostic tests need to be interpreted carefully in conjunction with the presenting clinical signs and symptoms.
TB Screening Tests: Applications and limitations
Tuberculin skin test and Quantiferon are the frequently used screening tests for Mycobacterium tuberculosis. Any of these, if found positive are followed by chest X-ray and sputum AFB and cultures to rule out or confirm an active tuberculous infection. Tuberculin skin test (TST) is based on the type IV hypersensitivity reaction to intra-dermal injection of purified protein derivative. It is performed by intradermal injection of 0.1 mL of purified protein derivative, and then the size of induration is measured after 48-72 hours. It is non-specific and cannot differentiate between the species of Mycobacteria. It is less expensive than Quantiferon and convenient in large screening settings, however, it cannot be used in patients with prior BCG vaccine and can also give false positive results in patients previously tested with TST. Quantiferon is an Interferon Gamma Release Assay and is based on the principle that previously exposed individuals have primed memory T cells, which on re-exposure to specific TB antigens are stimulated to release Interferon-gamma, which is then quantitatively assessed using an enzyme linked immunoassay. It does not require a second visit from the patient to read the results, unlike in TST where the patient has to return after 72 hours for interpretation of the induration at the site of injection. Also, the BCG vaccination or repeat testing do not affect Quantiferon results 
In Quantiferon TB Gold PLUS test, patient's blood is collected and put in four tubes (in order), the Nil tube (Grey cap), the TB antigen 1 tube (Green cap), the TB antigen 2 tube (Yellow cap) and the Mitogen tube (Purple cap), mixed well to dissolve all antigens attached to sides of the wall of test tubes, and incubated at 37 degrees Celsius for 16-24 hours, after which the amount of Interferon Gamma released is measured using enzyme linked immunosorbent assay. The incubation step is important for the primed T cells to be activated by the TB antigens to release interferon gamma .
The Nil tube is the negative test control and contains no additive. It is used to determine if the patient has any pre-existing immune response that could produce a false positive result. In order for the test to be considered valid, the Nil tube must have a value ≤ 8.0 IU/mL. The TB antigen 1 tube is coated with the long peptides of M. tuberculosis specific antigens, ESAT-6 (Early Secreted Antigenic Target 6) and CFP-10 (Culture Filtrate Protein 10) which stimulates CD4+ T cells in those previously exposed to TB. The TB antigen 2 tube contains the long and short peptides of ESAT-6 and CFP-10 to stimulate both CD4+ T cells and CD8+ T cells. CD8+ T cells are thought to have a strong response in patients with active tuberculosis. Moreover, in HIV positive patients, who have an attenuated CD4+ T cell response, the CD8+ response can be used to assess the tubercular infection [9-11]. Therefore, addition of the TB2 antigen tube in the Quantiferon TB Gold Plus test enables the detection of both active and latent tuberculosis, and it also aids in the diagnosis of TB infection in HIV positive patients with low CD4+ counts. For a test to be positive, the TB antigen tube value minus the Nil tube value must be ≥ 0.35 IU/mL. Lastly, the Mitogen tube is the Positive control tube that contains a mitogen (phytohaemogglutin-P), which is a non-specific stimulator of T-cells. It is used to ensure that the patient has a healthy immune response capable of producing interferon gamma and serves as a control for correct blood handling and incubation. It is used to detect false-negative readings. The mitogen tube must have a gamma interferon value ≥ 0.5 IU/mL higher than the value of the Nil tube, for the positive control and the test to be valid. This indicates that the mitogen control, as the positive control, is functioning correctly.
While there are many advantages of Quantiferon over the TST  as discussed above, it can yield indeterminate results. Indeterminate results are obtained when either the nil tube (negative control) reads >8.0 IU/mL, or the mitogen tube (positive control) reads < 0.5 IU/mL above that for the Nil tube.
There are various causes for indeterminate results. Lack of incubating the samples properly (such as not incubating the sample at all or over incubating) is an important factor. Tubes are incubated for 16-24 hours, in order for the primed T cells to be activated by the TB antigens to release interferon gamma. Storage of filled blood collection tubes outside the recommended temperature range (22°C ± 5°C) prior to 37°C ± 1°C incubation are other factors that can cause indeterminate results. Inadequate mixing of blood collection tubes is also an important cause for a false negative test, as the TB antigens attached to the test tube walls will not mix with the blood and won't be able to stimulate T cells. Presence of heterophile antibodies in the patient sample and intrinsic IFN-γ secretion can result in a false positive result which can be caught in the Nil tube. Recent patient illness, recent vaccinations, lymphocytes responding indiscriminately (recent patient bouts with poison ivy, rheumatoid arthritis, etc.), lack of response to the mitogen phytohaemagglutinin (occurs in less than 1 in 1,000 patients), compromised mitogen transport tubes, compromised immune status of the individual being tested due to medications (like steroids, immune inhibitor drugs, acquired or inherited immunodeficiencies), insufficient lymphocytes, inability of the patient's lymphocytes to generate IFN-γ are other causes for invalid/ indeterminate Quantiferon results .
In the present case, we got an indeterminate result on Quantiferon, with Mitogen tube and both the TB antigen tubes having a result of zero, although the negative control appeared to have worked well. This indicates a likely technical issue with the test as the positive control failed, while the negative control worked. Amongst the various causes of indeterminate results discussed above, possible causes in the present case could range from faults with the sample processing, transport to lab, irregularities with temperatures during storage, irregularities with incubation temperatures, duration of incubation or insufficient lymphocytes. The lack of response to phytohaemagglutinin can also be a possibility. In patients with medication related or innate immune response suppression, the TB antigen tubes would give some value, even if a low one. We expect some response in the TB antigen tubes in this patient, being an active TB case, even if muted due to a low lymphocyte count.
There are various studies on this subject. A study conducted in Worcestor, USA on 196 patients with 107 having inflammatory bowel disease and 89 with rheumatoid arthritis, concluded that a significant number of patients with IBD when on higher doses of steroids gave indeterminate Quantiferon results . Another study from Seoul, Korea concluded that Quantiferon results are frequently indeterminate in elderly patients with active tuberculosis, and suggested caution to be taken while interpreting Quantiferon results in patients with low immunity, chronic diseases, or severely diseases and elderly patients . A study from Japan emphasized the occurrence of indeterminate Quantiferon in immunosuppressed and elderly individuals .
This case emphasizes the importance of the clinical presentation of the patient, and the fact that Quantiferon test needs to be interpreted keeping all the clinical features and presentation of the patient in mind, and not as a stand-alone screening test. This patient had a presentation of cough, fever, night sweats, weight loss for 2 months, had a few episodes of hemoptysis, was coming from an endemic area with history of frequent visits to hospitals in her country. Moreover, the chest X-ray findings were very suggestive of TB. Therefore, it was important to proceed with further TB testing in this patient despite an indeterminate Quantiferon test. The interpretation of Quantiferon test must be done in accordance with the clinical setting of a patient.
Contributed by Davsheen Bedi, MD and Sarah Wheeler, PhD, FACB, CC (NRCC)