Evaluation of Interferon Gamma Release Assay in the Diagnosis of Active and Latent Tuberculosis among Patients and Contacts

.9%, respectively. The total agreement between QFT-GIT and the Mantoux was 71.11%. Positive concordant results (QFT+/TST+) were observed in 86.9%, while negative concordant results (QFT-/TST-) were detected in 37.9% of patients and contacts. Conclusion: The evaluation results of the QFT-GIT with ZN and Mantoux showed high sensitivities but relatively low specificities. The specificity of QFT-GIT was improved by modifying the cut off values to higher levels. Latent tuberculosis infection (LTBI) was likely to occur when both QFT-GIT and TST were positive .


INTRODUCTION
Tuberculosis (TB) is a public health problem that infects about millions of individuals each year.Nowadays, TB is in the list of the top 10 diseases causing deaths globally, and it was ranked above the human immunedeficiency virus (HIV) [1].In 2019, the World Health Organization (WHO) reported that around ten million new TB cases and 1.5 million people died from TB worldwide [1].
In Egypt, tuberculosis is the third predominant communicable disease after hepatitis C and schistosomiasis

Study design:
The study was a cross-sectional design that included 90 participants, forty-five patients with PTB (the patients were included according to their clinical symptoms, ZN stain and mycobacterium culture) , and forty-five close contacts to the confirmed PTB patients with duration of exposure for more than two weeks.The study was conducted in El Maamora Chest Hospital, Alexandria, Egypt.Chart of the study design and main results are shown in (Figure 1).A full questionnaire was filled for each TB patient and another one for their contacts.Each questionnaire contained the patient's data (serial number, robe number, date of entry, name, age, gender, job, and address), chance of TB infection, vaccination with Bacillus Calmette-Guérin (BCG), clinical picture and other diseases (diabetes, hepatitis C and B viruses and Human immunodeficiency virus infection (HIV)).Finally, we performed one year follow up for contacts of TB patients.Informed consent was gained from each subject included in the study.

Sputum for acid-fast bacilli:
Every suspected PTB and contact should submit three sputum samples "on the SPOTearly MORNING -on the SPOT" for microscopy.
The samples were spread over microscopic slides, heat-fixed, stained, and decolorized.Finally, the slides were observed under the microscope for the detection of AFB according to the standard protocol [12,13].

Sputum cultures of mycobacteria:
For every suspected patient with PTB clinical symptoms and abnormal x-ray, an Egg-based Löwenstein-Jensen (LJ) culture was done.Using Petroff's method in concentration and decontamination of sputum samples [13].Observation of cultures was done 48-72 hours after inoculation to detect atypical mycobacterium or any contaminants, any contaminated culture was discarded.Mtb typical colonies are crumbly, rough, non-pigmented, waxy and slow growers appear two to three weeks after inoculation.ZN staining should be performed for the colony with doubtful morphology [13].

Tuberculin skin test (TST):
Mantoux test was performed following the manufacturer procedure of Tuberculin diluted (5 Tuberculin Units/0.1 ml) (Span Diagnostics Ltd-India).In brief, 0.1 ml from the vial was intradermally injected in the forearm.With a black marker pen, a mark was made around the edges of induration before measuring.Then, the induration was measured after 48-72 hours in millimeters.The test was positive when the induration is raised or swelled and measured more than or equal to 5 mm, according to the criteria listed in (Table 1).

QuantiFERON®-TB Gold In-Tube test (QFT-GIT):
QFT test was performed for all patients and contacts as instructed in the leaflet in two stages.First, the whole blood was drawn then incubated for 16 to 24 hours in 37oC, after which, plasma was collected and stored at -80oC.Second, the collected plasma was tested to detect the interferon-γ (IFN-γ) released in response to the peptide antigens by ELISA.

Statistical analysis of the data:
Data were fed to the computer and analyzed using IBM SPSS software package version 20.0 [14].Qualitative data were described using numbers and percentages.Quantitative data were described using mean and standard deviation and median.A Chi-square test was used to compare different groups regarding categorical variables.Correction for chi-square was conducted using Fisher's exact test or Monte Carlo correction when more than 20% of the cells have expected count less than 5.The distributions of quantitative variables were tested for normality using the Shapiro-Wilk test, Kolmogorov-Smirnov test, and D'Agstino test, also Histogram and QQ plot was used for the vision test.Parametric tests were applied only if it reveals normal data distribution.The comparison between two independent populations was done using an independent t-test for normally distributed data.Agreement of the different predictive with the outcome was used and was expressed in sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and accuracy.The significance of the obtained results were judged at the 5% level [15].
The receiver operating characteristic curve (ROC) was plotted to analyze a recommended cutoff, the area under the ROC curve denotes the diagnostic performance of the test.Area more than 50% gives acceptable performance and area about 100% is the best performance for the test.

RESULTS
As presented in (Table 2), there were no statistically significant relations with QFT-GIT results and age, gender, BMI, smoking history, and residence.It found that 95.6 % of active TB patients had cough, 17.8% were diabetic, 2.2% had been infected with HCV, and 2.2% had been infected with HIV.On the other hand, only 4.4% of contacts had cough, and 11.1 % were diabetic.
In this study, 95.6 % out of 45 PTB patients and 42.2% out of 45 contacts were TST positive (Figure 2a).On the other hand, 86.7 % and 68.9% of TB patients and contacts were QFT-GIT positive, respectively, shown in (Figure 2b).The concordant and negative concordant data of QFT-GIT and TST were documented in (Table 3).
The TST sensitivity and specificity to ZN were 100% and 57.1%, respectively.The PPV and NPV of TST among patients and contacts was 67.21% and100.00%,respectively.The overall agreement between TST and ZN was 77.78%.These results were statistically significant (P-value =0.000) (Table 4).
After a one-year follow-up of contacts, we found that two cases had developed active PTB; these were two females with Mantoux test induration ≥ 10 mm and positive QFT-GIT result.

ROC curve analysis for QFT-GIT results:
The ROC curve for QFT-GIT shows that AUC was 0.730, the significance level P (Area=0.5) was 0.0001, and the 95% confidence interval was 0.626 to 0.819.
From the ROC curve, we found that the sensitivity and the specificity of QFT-GIT were 78.33% and 65.52%, respectively, when the cut-off value was >1.53IU/ml as shown in (Figure 3).In the current study, QFT-GIT sensitivity with TST and ZN was relatively high at 85.5% and 85.37%, respectively.The sensitivity results in this study agreed with other reported data in numerous studies [18,19].However, the specificity of QFT-GIT to TST and ZN was 39.3% and 28.6%, respectively.Cho et al. reported relatively low specificity of QFT-GIT at about 57.6% [20].The low specificity in this study may be attributed to the high rate of positive QFT-GIT in contacts that indicated the spreading of LTBI.Besides, ZN stain is negative in all contacts that lower the agreement and accordingly the sensitivity of QFT-GIT with ZN.The overall sensitivity of QFT-GIT was 77.4% that was higher than the sensitivity of TST (63.2%) in PTB patients.While, the overall specificities of the QFT-GIT and TST were 51.2% and 74.4%, respectively.
Studies reported negative agreement results with QFT-/TST+ among contacts and explained these findings as a consequence of BCG vaccination [21,22].However, in this study, high agreement results with QFT+/TST+ were observed in patients and contacts (86% and 84.2%, respectively).Despite most of the patients and contacts were BCG vaccinated but both diagnostic tests in our study were not affected by BCG vaccination.
IGRA is recommended to be used in immunocompromised as HIVinfected individuals as it is not affected by immunosuppression [23].Furthermore, in this study, a positive HIV patient resulted in positive QFT-GIT that supported this finding.
After following up for contacts, two contacts with positive concordant results (QFT+/TST+) had developed active PTB within three months after sampling.Both were diabetic females; their ages 52 and 54 years, and their husbands had been infected with PTB.This finding supported that diabetes mellitus increases the risk of TB infection [24].
The TB incident cases are higher in high-risk groups when both TST and IGRA are positive [25].For that, the approval and availability of IGRA could be a supportive screening test at highrisk contacts for LTBI.In this study, two patients with negative ZN and positive LJ developed positive results with QFT-GIT.For that, we recommend the use of QFT-GIT when the person is clinically suspected of TB but smear-negative or not able to produce sputum.
According to the ROC curve, cut-off points for QFT-GIT wereas chosen to be bigger than 1.53 IU/ml, which was also higher than that stated by the manufacturer 0.35 IU/ml.The difference in cut-offs could be due to the difference in the studied population.A higher value of the cut-off resulted in increasing the specificity without a marked decrease in the sensitivity.This improves the use of QFT for the screening of latent TB in the contact group.Few studies reported the adjustment of the QFT-GIT cut-off value to diagnose active TB [26].

CONCLUSION
From this study, we concluded that QFT-GIT was positive among most patients and contacts.Evaluation of QFT-GIT in relation to TST results showed high sensitivity but relatively low specificity.Furthermore, the specificity of QFT-GIT was improved by modifying the cut off values to higher levels.QFT-GIT assay could be used as an adjunctive diagnostic tool for patients with suspected TB when the conventional diagnostic methods fail.In addition, LTBI was likely to occur when both QFT-GIT and TST were positive.Early identifying and diagnosis of LTBI will aid in the elimination of TB and prevent the spread of resistant Mtb strains.

Ethical considerations:
The Ethical Committee of the High Institute of Public Health, Alexandria University in Egypt approved the study.Our research obeyed the international guidelines of research, of the declaration of Helsinki.

Declaration of competing interest:
The authors declare that there are no conflicts of interest.
Funding: No funding sources.

[ 2 , 3 ]
. There are 12 thousand reported incident cases of TB in Egypt in 2018 [1].In 1993, TB was considered a global health emergency by the WHO.Between 2006 to 2015, the WHO implement a plan to stop TB called (Stop TB Partnership), which aimed to protect 14 million lives [4].After that, a new strategy was launched to end TB by 2030 through decreasing new TB cases by 80% [5].Mycobacterium tuberculosis (Mtb) an acid-fast bacillus transmitted by airborne and causing TB infection.The detection of pulmonary TB depends on the symptoms, chest imaging, detection of acid-fast bacilli (AFB), mycobacteria culturing, and nucleic acid amplification (NAA) techniques [6].El-Maradny and Selim, Afro-Egypt J Infect Endem Dis 2021;11( ):xxx https://aeji.journals.ekb.eg/http://mis.zu.edu.eg/ajied/home.aspx200 Mycobacterium tuberculosis infection without any active TB symptoms is stated as a latent tuberculosis infection (LTBI) [7].Tuberculin is a purified protein derivative (PPD) taken from tubercle bacilli.Tuberculin skin test (TST or Mantoux) is used worldwide and counted as the best method to diagnose LTBI.However, Mantoux is not considered a "gold standard" because of the personal variation in reading the results and the possibility of false results [8,9].Interferon-gamma release assays (IGRAs) are more novel methods to detect LTBI.IGRAs tested the cell-mediated immune reaction from the invitro collected blood sample.The test is using specific mycobacterial antigens (ESAT-6/CFP-10/TB-7.7(p4)) to detect the interferon-gamma (IFN-γ) produced from T cells after exposing to Mtb because of delayed hypersensitivity response [10].The U.S. Food and Drug Administration (FDA) approved QuantiFERON®-TB Gold In-Tube test (QFT-GIT) in the detection of both active and latent tuberculosis [11].The current study compared the ability of QFT-GIT, TST and Ziehl-Neelsen (ZN) stain to detect pulmonary TB (PTB) and screening LTBI in close contacts to TB patients .

Figure ( 1 ):
Figure (1): Flow chart of the study design and baseline patient information.

Figure ( 2 ):Figure ( 3 ):
Figure (2): Charts representing the positive and negative results of a) TST and b) QFT-GIT among TB patients and contacts.

Table ( 2
): Different studied parameters among TB patients and contacts.

Table ( 3
): Different studied parameters among TB patients and contacts.

Table ( 4
): Sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of TST against ZN smear among TB patients and contact group.

Table ( 5
): Sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of QFT-GIT against ZN smear among TB patients and contact group .
Table (6): Sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of QFT-GIT against TST among TB patients and contact group .