First Clinical Comparison of STANDARD F TB-Feron FIA and the QIAreachTM QuantiFERON-TB for Tuberculosis Infection in Tertiary Public Hospital in South America

1. Introduction

Tuberculosis (TB) is an infectious disease that frequently produces respiratory symptoms and is caused by Mycobacterium tuberculosis (MTB) [1]. This infection is transmitted from person to person through airborne droplets expelled by patients with active lung disease (baciliferous) [2]. MTB infection is usually asymptomatic in healthy people [2]; but it can also be presented as an active disease, being the most characteristic symptoms coughing. It’s also frequented to observed mucopurulent sputum and sometimes hemoptysis, chest pain, weakness, weight loss, fever, and night sweats [3]. In 2020, there were at least 9.9 million new infections, with 1.5 million deaths, particularly the most affected group were patients living with HIV (214,000 deaths). In Latin America, PAHO reports estimated 291,000 cases, with 27,000 deaths. The diagnosis of multidrug-resistant TB (MDR-TB) was made in 4,007 cases, but only 89% of these started anti-tuberculosis treatment [4,5].

Approximately 5 to 10% of people infected with Mycobacterium tuberculosis develop the disease during the first 2 to 5 years after infection [6]. Patients who do not develop disease are postulated to be due to a series of pathogen factors and host mechanisms, including the innate immune response that would eliminate the infection without leaving a trace of an immune response (resistance to tuberculosis infection) [6]. In other cases, patients would develop a state of persistent immune response to MTB antigens without clinical evidence of active disease [7]. From this finding of antigenic persistence, it is considered that a quarter of the world population could be infected with M. tuberculosis [8]. Individuals are carriers of TB infection, and it is known as latent tuberculosis infection (LTBI) even if bacterial clearance is achieved, becoming a potential reservoir for active tuberculosis [6].

Currently, in South America, the diagnosis of LTBI continues to be made through the tuberculin skin test (PPD in spanish), also known as the Mantoux tuberculin skin test (TST) [9,10]. However, in the region, a high percentage of the newborn population are vaccinated against Bacillus Calmette–Guérin (BCG) as prevention of complicated tuberculosis (meningitis TB or miliary TB) [11]. According to what was reported by the Ynozente-Lázares et al. among the countries that during 2017 had the highest coverage were Brazil (100%), Venezuela (100%), Argentina (97%) and Guyana (97%), while those with the lowest indicators are Peru (84%) and Paraguay (84%) [12]. In Chile, during 2021, coverage reached 98.4% according to reports from the Ministry of Health. (Preliminary Report on National Immunization Coverage 2021, Period January – December 2021). However, BCG vaccination interferes in the diagnosis of LTBI through TST, this diagnostic technique presents an immunological cross-reactivity between antibodies against the vaccine and antibodies associated with exposure to MTB naturally, so in childhood and adolescence, the use of other diagnostic tests complementary to the TST in suspicion of LTBI [13] would be recommended. Likewise, according to what was stated by Zellweger et al. the TST diagnostic test is the oldest that evaluates LTBI, but it can be associated with various technical errors, has limited positive predictive value, is influenced by BCG vaccination, and various host conditions can reduce skin reactivity [14]. Therefore, current scientific studies have suggested evaluating other diagnostic techniques, such as interferon gamma release assays (IGRA), since these have a higher specificity, are not affected by M. bovis, BCG vaccine, and other environmental mycobacteria. IGRAs use only two M. tuberculosis-specific antigens (ESAT-6 and CFP-10) that are absent from M. bovis and BCG, but present in some environmental Mycobacteria spp. These antigens stimulate the lymphocytes after an incubation process at 37°C, generating interferon gamma, which is only released and detected by diagnostic techniques and only if the patients had previously been exposed to these antigens by contact during their life with M. tuberculosis, currently presenting with LTBI [14]. In the case of the skin test, the TST IC-65 technique is currently used, which was introduced in Romania in 1965 and it has been used as “in vivo” assay for the surveillance of M. tuberculosis infection (diagnosis of LTBI). , the diagnostic kit includes 54 proteins, which were identified through mass spectrometry, among them MPT64, ESAT 6 and CFP 10, antigens recognized in MTB and others that have cross-reactivity against other non-TB Mycobacteria [15].

There are no South American reports comparing diagnostic tests for the study of LTBI in highly complex hospitals, our laboratory group decided to evaluate a new diagnostic test available in the southern cone. The objective of our study was to compare a new immunofluorescence IGRA test against a commercially available IGRA kit, which had previously been compared against other techniques considered gold standard for the determination of interferon in the blood of patients suspected of LTBI. The main difference between the included reagents is that the new diagnostic kit includes a third MTB-specific antigen, TB7.7[16]

2. Materials and Methods

Study design and participants

This prospective and longitudinal study was carried out in the Infectious Diseases/Immunology Department of the Hospital Barros Luco Trudeau, Santiago, Chile, a tertiary care center located in the south of the country's capital, with an assigned population of 1,500,000 users. The study was approved by the ethics committee of the South Metropolitan Health Service, Santiago, and written informed consent was obtained from all study participants.

For this study, subjects classified as close contacts of recently diagnosed (less than 3 months) positive pulmonary TB patients, HIV patients, or who had to receive immunosuppressive drug treatment for an underlying medical condition, particularly methotrexate therapy or biologics, were recruited. Household contacts of patients with pulmonary TB were defined as a large group of family members residing with the pulmonary TB index case in the same household for > 3 months and having a common kitchen arrangement or sleeping together in the same bedroom. Subjects with a history of previously treated TB; HIV; positivity for hepatitis B and C; pregnant women were not excluded from the study. Patients were recruited during the months of September to December 2022.

Inclusion criteria for the group of TST-positive subjects: Positive test greater than 10 mm, being between 18 and 65 years of age, not having been previously diagnosed with TB without treatment or being under treatment for TB during the IGRA sample collection.

A control group was used with 20 users or hospital employees with a low risk of latent tuberculosis, who were asked for a tuberculin test and whose result was negative or <= 9 mm, who were between 18 and 65 years old, had not previously been diagnosed by TB or being treated for TB.

At the time of taking the TST, all study participants had 9 ml of blood drawn (3 ml in 2 tubes with lithium heparin and a 3 ml tube with EDTA) for IGRA studies and complementary tests (CBC). complete with white blood cell count). Clinical and demographic data collected from patients included age, gender, and key parameters of white blood cell counts from the complete blood count. Supplementary Table S1.

Standard F TB-Feron FIA assay

For each participant, 8 mL of blood was obtained, which was transferred to 2 tubes with lithium heparin. In less than 4 hours, the volume was transferred to Standard E TB-Feron Tubes (Nil, TB Ag and the mitogen tube). The antigens evaluated were ESAT-6, CFP-10, TB7.7 (This last one is specific for TB and available only in this commercial technique). After collection, the tubes were incubated at 37 °C for 16 to 24 h. IFN-γ quantification was processed with the STANDARD F2400 equipment (SD Biosensor, Gyeonggi-do, Republic of Korea), an automated FIA (fluorescent immunoassay) device. From sample loading into the cartridge to fluorescence intensity reading and IFN-γ quantification, followed by interpretation, the test is performed automatically within 15 minutes. Results are interpreted according to manufacturer's instructions [17,18].

QIAreach QFT assay

The other previously collected lithium heparin tube was used to draw 1 mL of whole blood from all study participants for transfer directly into the QIAreach QFT Blood Collection Tube (equivalent to QFT-Plus ESAT-6 Antigens tube TB2, CFP-10) and incubated at 37ºC for 16-24 hrs. Plasma samples were centrifuged at 3000 × g for 15 min and analyzed according to the manufacturer's instructions.

A charged eHub was powered on, and the eStick was inserted into the eHub port. Once connected and powered on, the eHub was in ready mode. A total of 150 µL of diluent was added to the processing tube. Next, 150 µL of plasma sample was transferred to the same processing tube. The resulting solution was mixed by pipetting up and down at least 8 times. A total of 150 µL of this mixture was aliquoted from the processing tube into the sample port of the inserted eStick. The trial started automatically, and the status was displayed on the eHub when the mix was detected. At the end of the trial, the test result (+ or -) and the time to result (TTR) were indicated in the eHub screen.

Statistical analysis

Data were statistically analyzed using GraphPad Prism 9.3.1 and presented as No (%) or median (interquartile range) unless otherwise specified. Sensitivity, specificity, and overall agreement (proportion of overall true results) in the Standard F technique were calculated using the QIAreach QFT as the reference standard. A subanalysis was also performed on samples from immunocompromised patients (less than 500 lymphocytes per uL).

Ethical

authorized by the ethics committee of “Servicio de salud metropolitano sur” nº 22/2022

3. Results

In total, 76 patients who were referred for a TST study were analyzed to rule out the suspicion of LTBI. In the sample, the median age was 38 years. (IQR 22-54). 73.68% were women, 93.42% Chilean. 98.68% of the patients did not have immunosuppression secondary to HIV (75/76). And 98.68% were referred due to a requirement to use biological drugs / methotrexate or in a contact study for a positive TB patient in their place of residence.

Table 1. Demographic description of patients studied for TST in the Infectious Diseases/Immunology department. (September to December 2022). (n= 76).

		TST
	Total	Positive	Negative
Age (average)	41,42	42,66	31
Age (median)	38,00	42	36
IQR	22-54	32-55	31-49
Sex (female)	73,68%	60,71%	39,29%
Nationality (chilean)	93,42%	93,33%	93,55%
Foreign	6,58%	6,67%	6,45%

Table 1. Demographic description of patients studied for TST in the Infectious Diseases/Immunology department. (September to December 2022). (n= 76).

		TST
	Total	Positive	Negative
Age (average)	41,42	42,66	31
Age (median)	38,00	42	36
IQR	22-54	32-55	31-49
Sex (female)	73,68%	60,71%	39,29%
Nationality (chilean)	93,42%	93,33%	93,55%
Foreign	6,58%	6,67%	6,45%

Table 2 summarizes the comparison between the new IGRA technique, through immunofluorescence diagnosis. This was compared against the QIAreach technique, which previous studies had compared against the current gold standard (GS), the Quantiferon® TB Gold technique. Because we had the QIAreach technique available to be able to use it in this investigation. Fukushima et al. compared it against Quantiferon® TB Gold [19]

Table 2A shows the sensitivity of STANDARD F TB-Feron FIA. The sensitivity of the new technique was 88.89% and specificity 92.50% in the study population. Since TST was also performed at the hospital, it was decided to compare both diagnostic techniques against this one. Table 2C shows that STANDARD F TB-Feron FIA had an overall sensitivity of 82.86% and a specificity of 80.65%, having an even better specificity than the comparator pattern of 62.50%.

Sensitivity (new technique) 88,89%; Specificity (new technique) 92,50%

Table 2B. Comparison between the new IGRA technique and the Gold standard. Samples collected from HBLT patients (September to December 2022). (n=76).

		TST
		Positive	Negative	Total
QIAreach	Positive	30	15	45
	Negative	6	25	31
	Total	36	40	76

Table 2B. Comparison between the new IGRA technique and the Gold standard. Samples collected from HBLT patients (September to December 2022). (n=76).

		TST
		Positive	Negative	Total
QIAreach	Positive	30	15	45
	Negative	6	25	31
	Total	36	40	76

Sensitivity (new technique) 83,33%; Specificity (new technique) 62,50%

Table 2C. Comparison between the new IGRA technique and the Gold standard. Samples collected from HBLT patients (September to December 2022). ( n=76).

		TST
		Positive	Negative	Total
SD Biosensor	Positive	29	6	35
	Negative	6	25	31
	Total	35	31	66

Table 2C. Comparison between the new IGRA technique and the Gold standard. Samples collected from HBLT patients (September to December 2022). ( n=76).

		TST
		Positive	Negative	Total
SD Biosensor	Positive	29	6	35
	Negative	6	25	31
	Total	35	31	66

Sensitivity (new technique) 82,86%; Specificity (new technique) 80,65%

An analysis by age subgroups was also performed. Separating the samples into a group Under 35 years (34 samples) and greater than or equal to 36 years (42 samples). Table 3A showed a sensitivity of 76.92% and a specificity of 95.24%. In Table 4A, in those over 36 years of age, the sensitivity of the new technique was 95.65% and the specificity 89.47%.

Table 3A. Comparison between the new IGRA technique and the Gold standard. Samples collected from HBLT patients under 36 years of age (September to December 2022). (n=34).

		GS QIAREACH
		Positive	Negative	Total
SD Biosensor	Positive	10	1	11
	Negative	3	20	23
	Total	13	21	34

Table 3A. Comparison between the new IGRA technique and the Gold standard. Samples collected from HBLT patients under 36 years of age (September to December 2022). (n=34).

		GS QIAREACH
		Positive	Negative	Total
SD Biosensor	Positive	10	1	11
	Negative	3	20	23
	Total	13	21	34

Sensitivity (new technique) 76,92%; Specificity (new technique) 95,24%

Table 3B. Comparison between QIAreach and TST. Samples collected from HBLT patients under 36 years of age (September to December 2022) (n=34).

		TST
		Positive	Negative	Total
QIAreach	Positive	10	3	13
	Negative	10	11	21
	Total	20	14	34

Table 3B. Comparison between QIAreach and TST. Samples collected from HBLT patients under 36 years of age (September to December 2022) (n=34).

		TST
		Positive	Negative	Total
QIAreach	Positive	10	3	13
	Negative	10	11	21
	Total	20	14	34

Sensitivity (new technique) 50,00%; Specificity (new technique) 78,57%

Table 3C. Comparison between the new IGRA technique (SD BIOSENSOR) and TST. Samples collected from HBLT patients under 36 years of age, (September to December 2022) (n=34).

		TST
		Positive	Negative	Total
SD Biosensor	Positive	9	2	11
	Negative	11	12	23
	Total	20	14	34

Table 3C. Comparison between the new IGRA technique (SD BIOSENSOR) and TST. Samples collected from HBLT patients under 36 years of age, (September to December 2022) (n=34).

		TST
		Positive	Negative	Total
SD Biosensor	Positive	9	2	11
	Negative	11	12	23
	Total	20	14	34

Sensitivity (new technique) 45,00%; Specificity (new technique) 85,71%

Table 4A. Comparison between the new IGRA technique and the Gold standard. Samples collected from HBLT patients > = 36 years of age, (September to December 2022) (n =42).

		GS QIAREACH
		Positive	Negative	Total
SD Biosensor	Positive	22	2	24
	Negative	1	17	18
	Total	23	19	42

Table 4A. Comparison between the new IGRA technique and the Gold standard. Samples collected from HBLT patients > = 36 years of age, (September to December 2022) (n =42).

		GS QIAREACH
		Positive	Negative	Total
SD Biosensor	Positive	22	2	24
	Negative	1	17	18
	Total	23	19	42

Sensitivity (new technique) 95,65%; Specificity (new technique) 89,47%

Table 4B. Comparison between QIAreach and TST. Samples collected from HBLT patients > = 36 year of age (September to December 2022) (n =42).

		TST
		Positive	Negative	Total
QIAreach	Positive	20	3	23
	Negative	5	14	19
	Total	25	17	42

Table 4B. Comparison between QIAreach and TST. Samples collected from HBLT patients > = 36 year of age (September to December 2022) (n =42).

		TST
		Positive	Negative	Total
QIAreach	Positive	20	3	23
	Negative	5	14	19
	Total	25	17	42

Sensitivity (new technique) 80,00%; Specificity (new technique) 82,35%

Table 4C. Comparison between the new IGRA technique (SD BIOSENSOR) and TST. Samples collected from HBLT patients > = 36 year of age (September to December 2022) (n =42).

		TST
		Positive	Negative	Total
SD Biosensor	Positive	20	4	24
	Negative	5	13	18
	Total	25	17	42

Table 4C. Comparison between the new IGRA technique (SD BIOSENSOR) and TST. Samples collected from HBLT patients > = 36 year of age (September to December 2022) (n =42).

		TST
		Positive	Negative	Total
SD Biosensor	Positive	20	4	24
	Negative	5	13	18
	Total	25	17	42

Sensitivity (new technique) 80,00%; Specificity (new technique) 76,47%

Table 5. Evaluation of gamma interferon levels stimulated by antigens using the novel IGRA technique in patients being studied in the infectious disease department with the TST technique (positive and negative cases).

TST	TST (mm) (median)	leukocytes ug/ml (median)	Leucocitos (ug/ml) (median)	Ag SD Biosensor tube ug/ml (median)
Positive	14	7250	2240,375	0,46
Negative	0	5645	1595,85	0

Table 5. Evaluation of gamma interferon levels stimulated by antigens using the novel IGRA technique in patients being studied in the infectious disease department with the TST technique (positive and negative cases).

TST	TST (mm) (median)	leukocytes ug/ml (median)	Leucocitos (ug/ml) (median)	Ag SD Biosensor tube ug/ml (median)
Positive	14	7250	2240,375	0,46
Negative	0	5645	1595,85	0

Table 5 shows the median number of leukocytes, lymphocytes and gamma interferon release caused by the antigens in the tube of the new IGRA technique under evaluation. The median release of interferon for the positive TST was 0.46 and 0 in the case of the negative TST. Finally, in the case of TST-positive patients, from the subgroup positive for the new IGRA technique, a median of 1.33 ug/ml of gamma interferon was observed when stimulated by the technique's antigens (Data not shown, information available in Supplementary Table S1 (S.1)

4. Discussion

In 2022, the Chilean Ministry of Health generated an update on the tuberculosis management standards [20]. This regulation dictates the action in the country for the diagnosis and treatment of all positive cases for TB, both in public and private hospitals. For the first time, a regulation of the Ministry of Health in Chile considers the study and treatment for LTBI, which is a paradigm shift for the national TB program, the diagnosis must be through TST or IGRA. The new standard considers that the TST is reactive when the induration is 10 mm or more. In the case of patients with HIV and other immunosuppressed patients a TST of 5 mm or more is considered reactive. Reactive TST or positive IGRAs should not be repeated, as the result will persist over time even after LTBI treatment has been performed. These tests cannot differentiate LTBI from tuberculosis disease, so a good clinical and imaging diagnosis is key in case of suspicion of TB disease. This standard also dictates that patients diagnosed with LTBI should receive treatment with rifapentine + isoniazid once a week for 12 weeks. [20]. ITBL study is important for Chile. Since adequate treatment should be given to patients suspected of LTBI, particularly HIV patients, immunosuppressed by biological drugs or close contact of positive TB patients with a diagnosis of less than 2 years, since they are candidates to become ill with Tuberculosis within the next 5 years [6].

Due to this background, it was decided to implement a new diagnostic technique in a highly complex hospital in the southern area of the capital of Chile. Registered population of more than 1.5 million inhabitants, with a TB incidence of about 19,8 per 100 000 persons, higher than other regions of Chile (13,3 per 100.000 persons) [21]. As a research team we considered that it was necessary to perform an IGRA technique beyond the TST. IGRA techniques such as TB-Gold and T-SPOT were not easy to implement in our institution due to the requirements of exclusive technicians for their implementation. Unlike point of care equipment, it is easy to load, such as those evaluated in this study, which allow more than 24 patients to be analyzed within an hour.

The first comparison made in the study was between Standard F TB-Feron FIA and QIAreach™, Table 2 summarizes the comparison with the novel IGRA technique and Table 2A shows the sensitivity of Standard F TB-Feron FIA. The sensitivity of the new technique was 88.89% and specificity 92.50% in the study population. In the population under study, the new technique would provide valid results for the diagnosis, these results would give greater certainty than the TST in the face of clinical suspicion of LTBI or in the case that it is necessary to rule out LTBI to start immunosuppressive treatment. In Chile, with positive results for LTBI, action must be taken following current regulations, therefore, 12 weeks must be treated with anti-tuberculosis drugs. Due to the above, it is necessary to rule out false positive cases, that is, adequate specificity in the new diagnostic technique would ensure that anti-tuberculosis treatments are not indicated, since antimicrobial drugs in prolonged use could generate unwanted adverse drug reactions and alter the intestinal microbiota. Table 2 C shows that Standard F TB-Feron FIA had an overall sensitivity of 82.86% and a specificity of 80.65%, having an even better specificity than the comparator standard 62.50% (QIAreach versus TST). However, the results compared against TST may have been influenced by probable false positive cases in the population under 36 years of age, in whom there could still be an immune response to BCG vaccination (until 2004 there were two vaccination doses, the first within the first month of the newborn and then a second dose at 6 years) [22]. In countries without BCG vaccination, the adolescent and young adult population would not have recognition of antigens against vaccination in the TST test and, therefore, negative results would not have BCG interference. In the case of our study, this could explain the decreased sensitivity (45% in those under 35 years of age vs 80% in those over 36 years of age when comparing Standard F TB-Feron FIA vs TST).

Given this background, it was decided to analyze the data by age subgroups. Separating the samples into a group under 35 years (34 samples) and a group greater than or equal to 36 years (42 samples). In Table 3A (Population under 36 years of age), a sensitivity of 76.92% was observed, lower than that observed in the total number of cases, and a specificity of 95.24%. In Table 4A, 36 years or older, the sensitivity of the new technique was 95.65% and the specificity 89.47%. In this case, it was the greatest success of the new technique when compared to the GS. In table 4 C, Sensitivity of the new technique 80.00% and specificity of 76.47% compared to TST. This increase in sensitivity is probably due to the decrease in TST positive cases in this subgroup, due to the recognition of BCG vaccination antigens in the first group analyzed (under 36 years of age). In table 5, the median of the positive TST group is greater than that of the negative TST group. The median of the positive group was 0.46, a result lower than expected. Therefore, we decided to evaluate the median of the positive TST subgroup together with Standard F TB-Feron FIA with a positive result, whose median was 1.33 ug/ml. This result was unexpected for our group since values greater than 3.39 ug/ml (median) were expected according to the reports described in the literature [23]. It should be noted that in values close to the median 0.46 ug/ml for the antigen tube in the population studied, if the result is negative or indeterminate due to the equation between mitogen and Nil, it is suggested to complement the results with TST or repeat the test in patients at risk of LTBI, such as HIV, other immunosuppression, use of biological drugs, close contact with a positive patient, to avoid false negative results. To resolve this dilemma, we consider it’s necessary to carry out similar studies with other populations worldwide, in places with different prevalence of TB. If the determination of the use of tests that detect results qualitatively was made in the hospital service, it is strongly suggested to carry out a second quantitative test against negative cases in patients with LTBI risk factors.

Limitations: Study in a single center in Santiago de Chile. Only one HIV patient recruited, so it could not be compared with non-HIV immunosuppressed patients. All the patients studied had BCG in the newborn period (within the national immunization program since 1949).

5. Conclusions

IGRA techniques are a new resource in clinical laboratories to be able to make an accurate diagnosis in LTBI in South America, all with a view to eliminating Tuberculosis cases by 2035. In our population, the greatest benefit of this new IGRA available on the laboratory would be observed in people over 36 years of age, where the sensitivity of the technique was like that of GS and with excellent specificity (95.65% and specificity 89.47%). We invite other groups of researchers to publish their data in countries where there is no influence of BCG vaccination, which can interfere with diagnosis through PPD, as occurs in Chile and South America.