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Hindawi Publishing CorporationISRN PulmonologyVolume 2013, Article ID 256148, 8 pageshttp://dx.doi.org/10.1155/2013/256148

Clinical StudyBlood Cells and Interferon-Gamma Levels Correlation in LatentTuberculosis Infection

Iukary Takenami,1 Camila Loureiro,2 AlmérioMachado Jr.,2, 3

Krisztina Emodi,4 LeeW. Riley,4 and Sérgio Arruda1, 2

1 Advanced Laboratory of Public Health, Oswaldo Cruz Foundation, Gonçalo Moniz Research Center,40296 710 Salvador, BA, Brazil

2 Bahia School of Medicine and Public Health, 40290 000 Salvador, BA, Brazil3Hospital Especializado Octávio Mangabeira, Secretariat of Health of Bahia State, 40320 350 Salvador, BA, Brazil4 Division of Infectious Diseases and Vaccinology, School of Public Health, University of California, Berkeley, CA 94720, USA

Correspondence should be addressed to Iukary Takenami; [email protected]

Received 20 November 2012; Accepted 12 December 2012

Academic Editors: A. Altraja and N. P. Juffermans

Copyright © 2013 Iukary Takenami et al. is is an open access article distributed under the Creative Commons AttributionLicense, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properlycited.

e Mycobacterium tuberculosis (M. tb) infection is largely spread in world’s population. Most infected individuals develop latenttuberculosis infection (LTBI). Tuberculin skin test (TST) and interferon-gamma release assays (IGRAs) are the available tests todetect the infection. It has been reported that some individuals take a longer period of time to develop the infection than otherswith the same exposure level. It is suggested that the innate immunity, in which neutrophils have an important protective role, isresponsible for this.Many hematologic abnormalities have been described as common �ndings during severe disease. To investigateif these changes are related to LTBI development and if they interfere in TST and IFN-𝛾𝛾 production, we recruited 88 householdcontacts of tuberculosis (TB) pulmonary patients and compared blood cell counts with these tests’ results.erewere no statisticallysigni�cant changes in hemoglobin, hematocrit, platelets, global leukocyte, neutrophils, basophils, eosinophils, typical lymphocytes,atypical lymphocytes, andmonocytes counts between infected and noninfected individuals. Also, there was no correlation betweenTST or IGRA and blood cell counts. ese results suggest that blood cell counts are not LTBI markers and do not interfere in TSTresults or IFN-𝛾𝛾 levels obtained by IGRA.

1. Introduction

Tuberculosis (TB) continues to be a public health problemworldwide. e disease is one of the most important causesof death from infectious diseases, and the infection withMycobacterium tuberculosis (M. tb) is also disseminatedthroughout world’s population [1]. Bra�il has noti�ed thehighest number of TB cases in America for the last 10 years.In 2010, there were estimated 85,000 new cases of TB, andapproximately 6,700 of them occurred in Bahia state [1, 2].

e transmission occurs by inhalation of droplets con-taining the bacilli from pulmonary TB individuals, mainlyfor their household contacts (HHCs) who are the mostexposed to the infection. Only 5%–10% of infected indi-viduals develop active TB, and the remaining individuals

will develop latent tuberculosis infection (LTBI). e latencyrepresents a balance between intracellular bacilli survival andhost defense [3]. However, considering that the reactivationof latent infection is related to a high percentage of casesand signi�cantly increases the transmission, diagnosing andtreating people with LTBI is crucial to control the diseaseworldwide [4].

e most common test to detect LTBI is the tuberculinskin test (TST) that basically consists in measuring thedelayed-type hypersensitivity reaction to puri�ed proteinderivate (PPD). However, this test has a low speci�city dueto the possibility of cross-reactivity to Bacillus Calmette-Guérin (BCG) or exposure to nontuberculous mycobacterialstrains from the environment [5–7]. Detection of surrogatemarkers that allow the infection diagnosis has been pursued.

2 ISRN Pulmonology

Recently, a new sensitive and more speci�c test that has beendeveloped to diagnose the infection is known as interferon-gamma release assays (IGRAs). is blood-based methodevaluates the T-cell response to bacilli antigens, includingESAT-6, CFP-10, and TB7.7 [6, 8].

It is well documented that HIV-positive individuals havean increased risk of developing active TB, and HIV infectionmay have some in�uence on IGRA’s performance [9, 10].On the other hand, it has been suspected that some exposedindividuals take longer to be infected by M. tb comparedto others despite similar exposure levels. It is probablyassociated to the possibility that the innate immunity mayclear the infection before the induction of the acquired T-cell response [11]. Also, many hematological abnormalities,such as leucocytosis, monocytosis, or anemia, have beenobserved in TB patients, especially in severe disease, andmaybe valuable markers to diagnosis [12–14]. However, there isstill a lack of available information about the relation betweenthe hematological status and the infection occurrence aercertain exposure to pulmonary TB patients (index cases). Inaddition, the new IGRA test is based on the IFN-𝛾𝛾 release bylymphocytes, which depends on a complex cell interaction.us, this current study aim is to study possible hematologi-cal changes in household contacts thatmay be related to LTBIdevelopment and their in�uence upon IFN-𝛾𝛾 levels.

2. Materials andMethods

2.1. Survey Population. Eighty-eight TB household contactswere identi�ed from thirty-eight patients with pulmonarytuberculosis (index cases). TB patients were recruited fromHospital Especializado Octávio Mangabeira (HEOM), Sal-vador, Bahia, Brazil, from March to August of 2007. Allparticipants signed a formal written consent, approved bythe Ethics Committee for Human Research, CEP-FIOCRUZnumber 92/2006. All TB index cases (ICs) and householdcontacts (HHCs) were evaluated at baseline with standard-ized questionnaires, and HHCs were submitted to TSTand chest radiograph. Blood samples for hematologic tests,IFN-𝛾𝛾 assays, and HIV screening were obtained from alldonors at study entry. Household contacts were de�ned asindividuals who (1) had lived with the index case for atleast 15 days, (2) had apparent clinical health, and (3) hadnormal chest radiograph. Exclusion criteria were active TBin the past and HIV seropositivity. According to the TSTresults, they were classi�ed into two groups: TST-negativeand TST-positive. Individuals with induration ≥10mm wereconsidered infected (TST-positive).

2.2. Tuberculin Skin Test (TST). Aer all blood samples werecollected for the hematological tests and IGRA, a TST wascarried out by the Mantoux procedure with 2TU of RT23puri�ed protein derivate (RT23PPD) (Staten Serum Institute,Copenhagen, Denmark). Reading was performed aer 72hours using the ball-point method [15].

2.3. Hematological Tests. Peripheral venous blood sampleswere collected in tubes containing ethylenediamine tetra

acetic acid (EDTA). Hematological tests were carried outusing commercially available kits and done in the lab ofHEOM using routine laboratory process.

2.4. Cell Culture and IFN-𝛾𝛾 Levels. e IGRA used was awhole blood assay, the QuantiFERON-TB Gold In-Tube kit(QuantiFERON-TB Gold In-Tube, Cellestis Ltd., Carnegie,Australia), performed according to the manufacturer’s rec-ommendations. Cells were cultured in vitro with speci�cantigens: ESAT-6, CFP-10, and TB7.7. Samples were thenincubated for 16–24 hours, centrifuged, and its supernatantwere collected and stored in −80∘C until use. IFN-𝛾𝛾 levelsin culture supernatants were assessed by ELISA. A positivetest was de�ned as IFN-𝛾𝛾 blood concentration ≥0.35 interna-tional units (IUs)/mL [16].

2.5. Statistical Analysis. Correlations were established byMannWhitney𝑈𝑈 test. Statistical differences were consideredsigni�cant when 𝑃𝑃 𝑃 𝑃𝑃𝑃𝑃. All statistical tests and graphswere performed by GraphPad Prism 5.0 (GraphPad soware,San Diego, CA).

3. Results

3.1. Household Contacts’ Characteristics. Out of 38 TB pul-monary index cases, 88 household contacts were identi�ed, inan average of 2.31 contacts per index case.irty-eightHHCswere considered uninfected (TST-negative), while 42 wereconsidered infected (TST-positive) with the TST conversion.Eight HHCs were excluded from analysis because they didnot return for TST measurement. Agreement between theTST and IGRA was 57.1%. Out of the 42 TST-positiveindividuals, 24 had also a positive IGRA test (Figure 1).

Among all contacts, 56 (63.64%) were women. emean ± SD age was 2𝑃8 ± 1𝑃8 years for 0–5 years oldindividuals, 11𝑃4±3𝑃3 for 6–15 years old ones, and 36𝑃1±13𝑃9for those who were older than 15 years. Most HHCs weremulatto (34.1%) or black (50%). BCG scar was present in 62HHCs (70.4%). In relation to the number of people/home,39 (44.3%) HHCs lived in a house with one to three people,28 (31.8%) with four to �ve people, and 21 (23.9%) livedwith six or more people. Seventy-six HHCs (86.4%) had noprevious contact when traced, and the higher mean exposuretime was found in 12 HHCs with exposure time ≥7 months.Most HHCs were �rst-degree relatives of the index case(68.9%) (Table 1).

3.2. Hematological Pro�le. Comparisons of hematology tests’values are shown in Table 2 for both TST-negative and TST-positive and in Table 3 for both IGRA-negative and IGRA-positive individuals, respectively. Comparing the hematologyresults of 80 TST-negative and TST-positive individuals,the median values were similar for hemoglobin, hemat-ocrit, and platelets. Global leukocyte, neutrophils, basophils,eosinophils, typical lymphocytes, atypical lymphocytes, andmonocytes counts were also similar for TST-negative andTST-positive (Table 2).

Similar to TST-negative and TST-positive comparisons,there were no signi�cant differences in the median values

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Index cases

Household contacts identification, invitation

Did not return for TST measurement

n = 38

n = 88

n = 08 (9%)

<10 mmn = 38 (47.5%)

≥10 mmn = 42 (52.5%)

IGRA-negativen = 33 (86.8%)

IGRA-positive

n = 05 (13.2%)

IGRA-negative

n = 18 (42.9%)

IGRA-positive

n = 24 (57.1%)

F 1: Flow chart showing the distribution of TST and IGRA result. TST: tuberculin skin test; IGRA: interferon-gamma release assay.

T 1: Demographic characteristics of household contacts ofpulmonary tuberculosis patients included in the study (𝑛𝑛 𝑛 𝑛𝑛).

Characteristic 𝑛𝑛 (%) Mean ± SDAge — 28.3 ± 16.9Gender

Male 32 (36.4) —Female 56 (63.6) —

Self-de�nitionWhite 11 (12.5) —Mulatto 30 (34.1) —Black 44 (50.0) —Refused to classify 03 (3.4) —

BCG scar presenceYes 62 (70.4) —No 26 (29.6) —

Index case �rst-degree relativeYes 60 (68.2) —No 28 (31.8) —

Previous contact tracingYes 12 (13.6) —No 76 (86.4) —

Exposure time0-1 month — 0.85 ± 0.342–6 months — 3.65 ± 1.56≥7 months — 12

Household per home1–3 39 (44.3) —4-5 28 (31.8) —≥6 21 (23.9) —

BCG: Bacille Calmette-Guérin; SD: Standard deviation.

for hemoglobin, hematocrit, platelets, global leukocyte, neu-trophils, basophils, eosinophils, typical lymphocytes, atypical

lymphocytes, andmonocytes counts between IGRA-negativeand IGRA-positive individuals (Table 3).

3.3. Neutrophil-Lymphocyte and Monocyte-LymphocyteRatios. Comparing neutrophil-lymphocyte (N/L) andmonocyte-lymphocyte (M/L) ratios, which are consideredsensitive detectors for tuberculosis infection [17], to IFN-𝛾𝛾production levels, there were no linear correlations betweenIFN-𝛾𝛾 levels and these ratios (Figures 2(a) and 2(b)).Apparently, for higher numbers of neutrophils in relationto lymphocytes, there is a slight reduction of IFN-𝛾𝛾 levelsdetected by IGRA.On the other hand, IFN-𝛾𝛾 levels increased,but not signi�cantly, when M/L ratio is higher.

3.4. Correlation between IFN-𝛾𝛾 Production and Blood Cells.To determine the correlation between IFN-𝛾𝛾 levels and cellcounts, we individually compared each cell count to IFN-𝛾𝛾production levels. ere were no signi�cant differences inmonocytes, eosinophils, basophils, and typical lymphocytescounts compared to different IFN-𝛾𝛾 production (Figures 3(a),3(b), 3(d), and 3(e)). However, at higher amounts of IFN-𝛾𝛾, the number of neutrophils slightly decreased. us, therewas a negative linear correlation between IFN-𝛾𝛾 productionand neutrophils counts, but it does not reach statisticalsigni�cance (Figure 3(c)).

4. Discussion

is is the �rst study that evaluated the correlation betweenhematological pro�le, TST, and IGRA in latent tuberculosisinfected individuals. Different authors have compared IGRAtest to TST in LTBI diagnosis in household contacts, but noone has analyzed the in�uence of the hematology in thesetests.

As IGRA is based on the stimulation of sensitized T-lymphocytes followed by measurement of interferon-𝛾𝛾 pro-duced by CD4 T-cells, we excluded HIV-positive individuals

4 ISRN Pulmonology

T 2: Hematological tests and TST result. Median and range of the values for hematological tests and TST result of household contacts(𝑛𝑛 𝑛 𝑛𝑛).

Variable TST < 10mm TST ≥ 10mm𝑃𝑃

Median Range Median RangeHemoglobin (g/dL) 13.80 10–19.8 13.70 11.6–15.8 0.686Hematocrit (%) 39.70 33.5–59.9 40.60 34.5–87.4 0.992Platelets (mm3) 289.000 169.000–532.000 305.500 172.000–441.000 0.458Global (mm3) 6.950 2.200–13.600 6.450 4.100–15.900 0.372Neutrophils (mm3) 3.743 1.204–8.094 3.558 1.377–7.204 0.689Basophils (mm3) 26.35 0–228 22.20 0–206 0.693Eosinophils (mm3) 192.30 0–1.406 290.50 0–1.919 0.221Typical lymphocytes (mm3) 2.424 0.240–5.572 2.147 0.806–13.106 0.058Atypical lymphocytes (mm3) 0 0–17.50 0 0–59.85 0.630Monocytes (mm3) 536.20 65–2.033 522.50 153–1.113 0.859MannWhitney𝑈𝑈 test. TST: tuberculin skin test.

T 3: Hematological tests and IGRA result. Median and range of the values for hematological tests and IGRA result among householdcontacts of patients recently diagnosed as pulmonary TB (𝑛𝑛 𝑛 𝑛𝑛).

Variable IGRA-negative IGRA-positive𝑃𝑃

Median Range Median RangeHemoglobin (g/dL) 13.55 10.5–19.8 13.85 10–15.6 0.301Hematocrit (%) 39.45 31.1–87.4 40.75 34.5–45.9 0.184Platelets (mm3) 293.500 169.000–532.000 302.500 172.000–441.000 0.878Global (mm3) 6.950 2.200–13.600 6.650 4.500–15.900 0.467Neutrophils (mm3) 3.882 1.204–8.100 3.562 1.472–5.928 0.570Basophils (mm3) 26.95 0–228 17.90 0–126 0.217Eosinophils (mm3) 206.9 0–1.704 306.9 0–1.919 0.178Typical lymphocytes (mm3) 2.403 868–9.520 2.191 1.093–3.672 0.190Atypical lymphocytes (mm3) 0 0–50 0 0–18 0.989Monocytes (mm3) 542.0 65–2.033 505.70 153–1.113 0.280MannWhitney𝑈𝑈 test. IGRA: interferon-gamma release assay.

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Mo

no

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s/ly

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F 2: Correlation between IFN-𝛾𝛾 production (IGRA test) and ratios. (a) Neutrophils/lymphocytes ratio; 𝑟𝑟2 𝑛 𝑛.𝑛𝑛9; 𝑃𝑃 𝑛 𝑛.𝑃 (ns). (b)Monocytes/lymphocytes ratio, 𝑟𝑟2 𝑛 𝑛.𝑛4; 𝑃𝑃 𝑛 𝑛.𝑛𝑃 (ns). IGRA: interferon-gamma release assay.

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0.01 0.1 1 10 100

IFN-γ (IU/mL)

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F 3: Correlation between IFN-𝛾𝛾 production (IGRA test) and blood cell counts (mm3). (a) Monocytes, 𝑟𝑟2 = 0.005; 𝑃𝑃 = 0.52𝑃 (ns). (b)Eosinophils, 𝑟𝑟2 = 0.0𝑃4; 𝑃𝑃 = 0.2𝑃0 (ns). (c) Neutrophils, 𝑟𝑟2 = 0.03; 𝑃𝑃 = 0.𝑃03 (ns). (d) Basophils, 𝑟𝑟2 = 0.00𝑃; 𝑃𝑃 = 0.𝑃22 (ns). (e) Typicallymphocytes, 𝑟𝑟2 = 0.005; 𝑃𝑃 = 0.4𝑃2 (ns).

from this study. Recent studies have shown that there is astrong correlation between low CD4 T-cell count and a lowmitogen response, and it has implications for LTBI diagnosis.In patients with low CD4 cell count, there was an increasedproportion of both indeterminate and false negative IGRAresults [10, 18, 19]. Furthermore, our objective was to identifyany hematological change directly related to LTBI infection inhousehold contacts and possible in�uence on IFN-𝛾𝛾 levels.

It was e�pected to �nd no great di�erences in periph-eral blood. At a very early phase of infection, there is aformation of just a few bacilli and granuloma. So, thereare no speci�c circulating blood cells. Some changes wereobserved in circulating blood cells during disease. It has beendemonstrated that hematological abnormalities are detectedin severe pulmonary and in disseminatedTB.Anemia,mono-cytosis, basophilia, leukocytosis, leucopenia, and “leukemoid

6 ISRN Pulmonology

reactions” are some of these abnormalities found [12–14, 20].Otherwise, a recent study demonstrated that many patientswith TB presented normal blood in�ammatory markers [21].So, it is possible to consider that in a very initial phaseof M. tb infection, the blood cells pro�le may not re�ectthe characteristical changes that appear during the infectionprogress to disease [17]. Our study is in agreement with thisidea. ere were no signi�cant differences between hema-tology data by comparing either TST-negative versus TST-positive or IGRA-negative versus IGRA-positive individuals.is result suggests that the hematological pro�le has nointerference in both TB infection diagnostic tests.

Some studies have shown that the N/L andM/L ratios arereliable detectors of tuberculosis infection [17, 22]. We didnot �nd any relation between these ratios and IFN-𝛾𝛾 levelsobtained by IGRA, despite a slightly negative in�uence ofneutrophils in IFN-𝛾𝛾 amounts. Apparently, IFN-𝛾𝛾 increasedin individuals with high M/L ratios.

As neutrophils showed a negative effect on IFN-𝛾𝛾 release,they may be important in TB infection control. ese cellsare persistently recruited and accumulated in the sites ofinfection, with a T-cell-independent peak on the �rst day anda T-cell-dependent peak aer 8–15 days and lasting until theend of the infection [23]. However, neutrophil protective rolein host response to mycobacteria remains unclear. Multiplefunctional roles have been suggested, like alternative antigenpresentation for T-cells, release of chemokines, and inductionof granuloma formation [24–28]. ere is no consensusabout neutrophil antimicrobial activity. Some in vitro studieshave demonstrated that neutrophils may be activated bysoluble mycobacterial antigens and are capable of controllingmycobacteria growth in cultures [29, 30]. Other studieshave evidenced a cell-cell cooperation of neutrophils andmacrophages. It was shown that neutrophil componentsincreased in vitro antimycobacterial activity of peritonealmacrophages [31]. Recently, it has been demonstrated thatapoptotic neutrophils may be phagocytized by macrophagesinfected with mycobacteria, and thereby these macrophagesacquire neutrophil antimicrobial peptides [32]. In contrast,the inability of human neutrophils to killM. tb independentlyof cytokine activation was reported and also that the M. tbbacilli were not found in association with neutrophils [23,33].

A recent work demonstrated an inverse relation betweenM. tb infection risk and peripheral neutrophil count. AfricanTB contacts were more susceptible to develop the infectionbecause they had lower neutrophil counts and lower serumconcentrations of HNP1-3 and lipocalin 2 than South Asianand white individuals [11]. In our study, we did not observesigni�cant differences in neutrophil counts related to TSTand IGRA results, and it was not related to ethnicity. Itis known that both IFN-𝛾𝛾 and IL-17 producing T-cells areinduced inmycobacterial infection and that IL-17 is an activerecruiter of neutrophils to infectious foci. It has been shownthat IFN-𝛾𝛾 controls the induction and proliferation of IL-17 producing cells [34]. Maybe IL-17 levels and neutrophilcounts are increased in the acute phase of infection and thatan increased IFN-𝛾𝛾 production may limit the expansion and

production of IL-17 producing cells in periphery and therebydecrease neutrophils counts in a latter phase.

Although there was no correlation between TST or IGRAand blood cell counts, these results should be interpretedcarefully. As there is no gold standard for the diagnosis ofLTBI, the TST is used as a standard diagnostic tool to assessTB infection in Brazil. TST may have a low speci�city amongBCG-vaccinated persons [35] as in this study population.However, we previously demonstrated in another study usingthe same population that prior BCG vaccination did notin�uence the outcome of TST results [36]. Furthermore,both tests do not discriminate recent from past TB infection.us, TB infection is not uncommon among people who livein high-transmission setting such as Salvador, Brazil. It ispossible that the same study participants were exposed toM.tb in the past and that these results may not re�ect a recentinfection, contributing to the lack of correlation between TSTor IGRA and blood cells count.

Finally, the effect of LTBI on peripheral blood cells turn-over, especially lymphocytes and monocytes, has been ham-pered by the lack of data in the literature. e large variationin T-cell turnover was reported in HIV infection. In thisstudy, it was postulated that decreased production andperipheral turnover cells cause T-helper cell depletion [37].e clinical consequences of increased cell turnover couldbe measured by anemia and hypermetabolism leading tofatigue, weakness, weight loss, and anorexia. No clinical signswere reported in this LTBI population. us, we believethat the blood cells turnover rate can be restricted to thesite of infection and might be relevant in progression to TBdisease; however, this cannot be detected in peripheral bloodof infected individuals.

5. Conclusion

In summary, there was no signi�cant interference of hem-oglobin, hematocrit, and platelets values, as well asmonocyte,eosinophil, basophil, lymphocyte, and neutrophil countson TST or IFN-𝛾𝛾 production. Neutrophil-lymphocyte andmonocyte-lymphocyte ratios were not related to IFN-𝛾𝛾 levels.It is not known yet if neutrophils have direct bactericidal orother immunological functions in mycobacterium infection.e negative linear correlation between INF-𝛾𝛾 levels andneutrophil counts supports that IFN-𝛾𝛾 increasing levels arerelated to neutrophil decreasing counts. In conclusion, bloodcell counts are not LTBI markers and do not in�uence TST orIFN-𝛾𝛾 production in LTBI.

Con�ict o� �nte�ests

None of the authors of this paper have a con�ict of interests.

Acknowledgments

e authors acknowledge Raildes Maria do Espírito Santofrom HEOM laboratory for her technical assistance withhematological tests. ey also thank EBMSP students fortheir contribution with medical questionnaire interviews.

ISRN Pulmonology 7

Financial support was provided byNIHFogarty InternationalCenter 5U2RTW 006885 and the Bahia State Research Sup-port Foundation (FAPESB).

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