Utility of monocyte parameters in diagnosing Sputum Smear Negative Pulmonary TB

Abstract

Background - Smear-negative TB (SNTB) may remain undetected for a long duration and new bio-markers are needed for diagnosis. Our study investigated monocyte parameters in this regard.

Thirty cases each of SNTB, sputum positive cases and healthy controls were included. Full blood count and immune-phenotyping for ‘monocyte parameters’ by flow cytometry were performed using CD45, CD16, CD14, CD64 and HLA-DR The percentage of monocyte subsets and MFI of CD64 and HLA- DR noted and data statistically compared.

The monocyte to lymphocyte ratio, percentage of intermediate monocytes, expression of CD64 on total as well as intermediate monocytes were significantly elevated in cases with SNTB as compared to healthy controls (p value, 0.07, 0.07, 0.001, 0.001 respectively). The cut off value for MFI CD64 on total and intermediate monocyte was >18 and >19.6 respectively.

Thus monocyte parameters can serve as useful biomarkers for diagnosing tuberculosis in the sputum negative group.

Keywords

Pulmonary TB Smear negative Diagnosis Flow cytometry Intermediate monocytes Monocyte lymphocyte ratio MFI CD64

Introduction

Pulmonary tuberculosis (TB) is one of the most common infectious disease worldwide.¹

Microscopic examination of sputum smear, a routinely used investigation for diagnosis, has poor sensitivity as it requires 5,000–10,000 bacilli per ml of sputum.^2–4 However, persons in contact with such patients are at a risk of infection and subsequent development of disease.^5,6 Patients with smear-negative TB (SNTB) are responsible for 10–20% of disease transmission.^7–9 The revised RNTCP guidelines recommend upfront chest radiograph (CXR) for sputum-negative cases followed by a cartridge-based nucleic acid amplification test (CBNAAT) for confirmation if CXR findings are suggestive of TB.¹⁰ However, both these tests may not be diagnostic in all cases. Moreover, CBNAAT is limited by availability and is largely inaccessible even at large centres. Thus, in the absence of rapid and accurate diagnostic tools for SNTB, newer biomarkers remain an absolute necessity.

Monocytes are cells of the innate immune system which play a pivotal role in the fight against tubercle bacilli by transforming into specialized epithelioid macrophages. It is well known that both monocytes and lymphocytes levels increase in TB. On the basis of flow cytometric expression of surface CD14 and CD16 antigens, monocytes can be subdivided into three functionally and phenotypically distinct subsets, viz. classical, non-classical and intermediate. While classical monocytes are the predominant population in peripheral blood and important for initial control of infection, the intermediate monocytes play an important role in later stages. Perturbations in the balance between the three subsets have been observed in various conditions such as obesity and cardiovascular diseases, as well as TB. A few preliminary studies have reported expansion of CD16 + monocytes, i.e. the non-classical and intermediate monocytes in TB. This expansion may be useful in diagnosing as well as defining severity of the disease.^11,12 Further studies on monocytes found overexpression of CD64 and the Fc receptor of IgG to be useful to distinguish TB patients from healthy controls.^13,14 Likewise, a decrease in the expression of HLA-DR on the surface of monocytes was also reported.¹⁵

However, these data are limited with respect to the Indian population where TB still remains rampant and sputum negative TB difficult to diagnose. Therefore, we aimed to explore the utility of monocyte parameters :- the absolute number of monocytes, monocyte to lymphocyte ratio, monocyte subpopulations, and the surface expression of CD64 and HLA-DR in differentiating patients with sputum smear negative tuberculosis from healthy controls.

Methods

Our case control study was conducted in a tertiary-care hospital in Delhi over a period of eighteen months between August 2018 and January 2020. Following approval by the Institutional Ethics Committee, patients were included in the study after an informed consent process.

Our study enrolled 30 subjects each in SNTB, sputum-positive tuberculosis patients and healthy controls. All patients who were >18 years of age and had clinically, radiologically, or microbiologically proven pulmonary tuberculosis according to the Revised National Tuberculosis Control Program (RNTCP) guidelines were included.¹⁰ Patients already on anti-tubercular treatment, those who had a relapse, or treatment failure and those who had defaulted treatment in the past were excluded. Patients with primary or secondary immune deficiency, diabetes and coronary artery disease were also excluded from the study.

Baseline demographic (age, sex), clinical (complaints, presentation, symptoms, past history, vaccination, comorbidity profile) and laboratory (radiological, hematological) information was recorded for all participants. All participants underwent a tuberculin sensitivity (Mantoux) test and the diameter of the skin induration was recorded. Induration of >10 mm was considered positive.

Peripheral venous blood (3ml) was drawn under aseptic condition and collected in an EDTA vial. Of this, 1ml was used for a full blood count and 2 ml for flow cytometry. The former was achieved using an automated analyzer (ABX Micros 60). Monocyte to lymphocyte ratio (MLR) was calculated by dividing the absolute number of monocytes by the absolute number of lymphocytes. These absolute numbers were calculated as follows- First total leucocyte count (TLC) provided by the analyser was noted. Then the smears were stained with Leishman’s stain and 200 leucocytes were counted and percentage of monocytes and leucocytes out of the total leucocytes were noted. The absolute numbers of these cells ie leucocytes and monocytes were then calculated by multiplying their respective percentages with the TLC.

Single tube flow-cytometry analysis was carried out for sub-populations and analysis of expression of CD64 on the surface of total and intermediate monocytes with an unstimulated tube containing only CD45. Whole blood was taken in each tube and diluted with sheath fluid to together make up 500 µL (such that each tube, i.e. 500 µL, would contain one million leucocytes). The sample was processed by a stain-lyse-wash method. The following antibodies were used: FITC- CD16 (clone 3G8); PE - CD 45 (clone J33); ECD - CD 64 (clone22); PC5 - CD HLA-DR (clone Immun357); PC7 - CD14 (clone RMO52).

The tubes were acquired on a 5-colour dual laser Beckman Coulter FC 500 flow cytometer using cytometry List Mode Data (LMD) acquisition. Viability gate (Forward v. Side scatter) was used to exclude debris. Neutrophils, monocytes and lymphocytes were gated separately on the basis of forward, side scatter and CD45. The monocyte population was further gated using CD14 and CD64. CD14 (vertical axis) vs CD16 (horizontal axis) dot plot was used to distinguish classical (CD14 + +, CD16-); intermediate (CD14 + +, CD16 + ) and non-classical monocytes (CD14 + , CD16 + ). Figure 1 shows the separation of different monocyte populations in a case of SNTB.

Figure 1.

Flow cytometric analysis showing separation of different monocyte populations on CD14 vs CD16 dot-plot.

The percentage of total monocytes and their individual subsets respectively, and the mean flourescence intensity of CD64 and HLA- DR on total and intermediate monocytes were noted.

Data were compiled into a spreadsheet and analysis was conducted using Stata software (version 13.0, Stata Inc USA), computing summary statistics as mean ( + standard deviation), median (inter-quartile range) for continuous variables and frequency (percentages) for categorical variables as appropriate. Independent continuous variables were compared in different groups using analysis of variance (ANOVA) with Bonferroni multiple-comparison test Categorical variables were compared between groups using the χ² test.

Univariate linear regression models were used to compare differences between sputum-positive and negative groups using the control group as baseline. Co-efficients were computed after adjusting for age and sex. In addition, we developed a receiver operator characteristic (ROC) curve to determine the best cut-off values for discriminating between sputum-negative and control subjects.

Sputum smear positivity was defined as either

At least two initial sputum smear positive for Acid fast bacilli (AFB), or

One sputum examination positive for AFB along with radiological features consistent with active pulmonary TB, or

One sputum positive for AFB along with culture positive for AFB.

Sputum smear negativity was defined as - Smear negative for acid-fast bacilli with tuberculin positive patients with symptoms and radiograph compatible with tuberculosis.¹

Results

The mean age of study subjects was 33.5 ± 11.3 years with a similar age distribution in the sputum positive, sputum negative and healthy controls. The baseline comparison of the demographic, clinical and hematological parameters between three groups is presented in Table 1. It was observed that radiological features and Mantoux test were less useful in identifying SNTB as compared with a positive sputum examination. Amongst the haematological parameters, MLR had discriminatory value in differentiating SNTB from those without illness (p value, p, 0.01) (Figure 2).

Figure 2.

Box plots showing comparison of monocyte parameters (monocyte lymphocyte ratio, % intermediate monocytes, MFI CD64 on total and intermediate monocytes) between SNTB and healthy controls.

Table 1.

Baseline comparison nof cases and control groups.

Characteristic	Sputum negative N = 30	Sputum positive N = 30	Healthy controls N = 30	Total N = 90
Demographic and clinical parameters
Age (years)	36.5 ± 12.4	31.1 ± 11.4	32.7 ± 9.5	33.5 ± 11.3
Male sex	16 (53.3%)	18 (60%)	26 (86.7%)	60 (66.7%)
Cough with expectoration	28	29	0	57
Fever	22	25	0	47
Weight loss	23	24	0	47
Breathlessness	21	16	0	37
Radiological features consistent with TB (10)	19	30	1	50
Mantoux Test	3 (10%)	9 (30%)	0	12 (13.3%)
Haematological parameters
Haemoglobin g/l	116 ± 28	122 ± 29	108 ± 30	115 ± 29
Total WBC count (/L)	10.9 ± 4.5 x10⁹	8.28 ± 2.6 x10⁹	8.6 ± 3.4 x10⁹	9.3 ± 3.7 x10⁹
Platelet count (/L)	2.41 ± 1.53 x10⁹	2.84 ± 2.23 x10⁹	2.48 ± 1.8 x10⁹	2.58 ± 1.9 x10⁹
ALC (/L)	2.10 ± 1.01 x10⁹	2.43 ± 1.17 x10⁹	1.97 ± 0.97 x10⁹	1.91 ± 0.8 x10⁹
ANC (/L)	6.78 ± 2.94 x10⁹	8.07 ± 3.52 x10⁹	5.95 ± 1.99 x10⁹	6.31 ± 2.7 x10⁹
Eosinophil count (/L)	0.16 ± 1.04 x10⁹	0.15 ± 0.11 x10⁹	0.15 ± 0.09 x10⁹	0.16 ± 0.1 x10⁹
Monocyte (/L) count	0.25 ± 0.16 x10⁹	0.26 ± 0.18 x10⁹	0.25 ± 0.14 x10⁹	0.24 ± 0.2 x10⁹
MLR	0.88 ± (1.2)	0.6 ± (0.6)	0.26 ± 0.26	0.58 ± 0.83

Legend for Table 1:.

Data have been presented as mean (± standard deviation) or number (percentage) as appropriate.

List of abbreviations: WBC, white blood cells; ALC, absolute lymphocyte count; ANC, absolute neutrophil count; Eo, Eosinophil; MLR, monocyte to lymphocyte ratio.

Flow cytometry was used to study the subpopulation of monocytes, expression of CD64 surface antigen and of HLA- DR antigen on the surface of monocytes. The results of the flow-cytometric analysis are presented in Table 2. The MFI of CD64 on total monocyte in negative cases was significantly elevated (p = 0.011). The MFI of CD64 on intermediate monocyte between sputum negative and control (p < 0.001).

Table 2.

Sub-population of monocytes, expression of CD64 and HLA-DR surface antigens.

Sub-population of Monocyte	Sputum negative	Sputum positive	Healthy controls	Total
Classical monocytes (% of total monocytes)	65.6 ± 21.2	57 ± 25.8	69.4 ± 25.6	64 ± 24.6
Intermediate monocytes (% of total monocytes)	13.8 ± 15.3	17.3 ± 13.4	8 ± 7.4	13 ± 12.9
Non-classical monocytes (% of total monocytes)	9.8 ± 9	16.7 ± 18.7	9.1 ± 16.5	11.9 ± 15.5
CD16 + monocyte (% of total monocytes)	23.6 ± 16.6	34.0 ± 23.1	17.1 ± 18.2	24.9 ± 20.5
Expression of CD64 surface Ag
MFI CD64 on Total monocyte	56.7 ± 38.0	41.6 ± 37.0	21.1 ± 22.5	39.8 ± 36.0
MFI CD64 on Intermediate monocyte	69.3 ± 41.5	50.9 ± 46.2	17.4 ± 20.3	45.9 ± 43.1
Expression of HLA- DR
MFI of HLA – DR on Total monocytes	16.4 ± 26.4	22 ± 23.1	20.9 ± 15.5	19.8 ± 22
MFI of HLA – DR on Intermediate monocytes	47.8 ± 44.1	69.5 ± 92.5	53.5 ± 77.4	56.9 ± 73.9

Legend for Table 2:.

Data have been presented as mean ( + standard deviation) or median (IQR) as appropriate.

List of abbreviations: CD, cluster differentiation; MFI, mean fluorescence intensity; IQR, interquartile range; ANOVA, analysis of variance.

MFI of HLA-DR on total monocyte in sputum negative cases as compared to controls was insignificant (p = 0.711). The MFI of HLA-DR on intermediate monocyte in sputum negative cases as compared to controls was insignificant (p= 0.952).

Flow cytometry showed that, in the sputum positive group, the intermediate (17.3 vs 8.0; p, 0.002) and non-classical monocytes (16.7 vs 9.1; p, 0.09) were significantly elevated compared to healthy controls. However, in sputum negative group, only the intermediate monocytes were found elevated (13.8 + 15.2) compared to healthy individuals (8.0 + 7.4; p, 0.07) (Figure 2).

The expression of CD64 was seen to have a significant elevation in both sputum positive and negative cases as compared to healthy controls. No difference was found however in HLA-DR expression in the three groups. Thus a regression analysis was done for CD64 expression. (Table 3), which showed expression of CD64 on total as well as intermediate monocytes was found to be significantly elevated in both sputum negative group (20.5; p, 0.02) and sputum positive group (35.7; p, <0.001), as compared to controls as a baseline category. The difference in expression was significant after adjusting for age and sex.

Table 3.

Regression analysis for expression of CD64 surface Ag on total and intermediate monocytes .

	Total monocytes		Intermediate monocytes
	Univariate analysis	Multivariable analysis	Univariate analysis	Multivariable analysis
Age	−0.8 (p, 0.02)*	−0.8 (p, 0.018)*	−0.5 (p, 0.2)	(p, 0.2)
Male sex	−10.3 (p, 0.2)	(p, 0.7)	−17.2 (p, 0.07)	(p, 0.9)
Healthy controls	-	-		-
Sputum negative	20.5 (p, 0.02)*	24.2 (p, 0.007)*	33.6 (p, 0.001)*	34.8 (p, 0.001)
Sputum positive	35.7 (p, <0.001)*	35.4 (p, <0.001)*	51.9 (p, <0.001)*	50.7 (p, <0.001)

Legend for Table 3: This table presents the results of a multivariable linear regression analysis. Healthy controls were used as the baseline category against which the sputum negative and the sputum positive cases were compared.

*indicates values found significant with p value <0.05.

We found MFI CD64 on total and on intermediate monocyte were significantly elevated in the sputum negative cases as compared to healthy individuals. Hence, a ROC curve was plotted to determine optimum cut off values (Figure 3). This was found to be >18 with sensitivity of 76.6% and specificity of 73.3% for total monocytes, and >19.6 with sensitivity of 83.3% with specificity of 83.3% for intermediate monocytes.

Figure 3.

ROC curves for monocyte parameters in SNTB cases.

Discussion

Human blood monocyte can be divided into three different on the basis of expression of CD14 and CD16 cell surface receptors. ‘Classical’ monocytes express CD14 but no CD16, and ‘Non-classical’ low CD14 and strong CD16. Further analysis identifies another subset of CD14⁺ CD16⁺ monocytes which also expresses CCR5 and shows intermediate expression of receptors divergently expressed in the other two subsets, viz. CCR2 and CX3CR1. This additional subset is classified as an ‘Intermediate monocyte’.¹⁶ During injury or inflammation, classical monocytes are rapidly recruited to invade the inflamed tissue and contribute to the immunological response by recognizing and removing micro-organism and dying cells by secreting pro-inflammatory cytokines such as IL-1β and TNF-α.¹⁶ The intermediate monocytes are recruited at a later stage of infection and are mainly associated with APC presentation, high secretion of pro-inflammatory cytokines, wound healing and micro-organism recognition whereas the non-classical monocyte displays patrolling behaviour and constantly surveys the endothelium as part of an innate local surveillance.¹⁶

Previous studies also found that patients with pulmonary TB showed increased CD16 + monocytes, which normalized after treatment.¹¹ It is postulated that this increase is also linked to the severity of the disease.¹⁷ The intermediate subset of monocytes is found to be elevated in association with many inflammatory and auto-immune conditions apart from TB.^18,19 The bone marrow in patients with TB is enriched with intermediate monocyte-like cells.²⁰ Where TB is co-infected with HIV, the increase in such cells is associated with higher mortality and thus bacterial persistence.²¹ We found the same also to hold true for SNTB.

In our study, CD64 was overexpressed on total monocyte as well as intermediate monocyte in patients with SNTB and values were useful in discriminating them from healthy controls. CD64 is an fc gamma receptor expressed on the surface of activated neutrophils and monocytes. It plays an important role in phagocytosing IgG opsonized bacteria, inducing reactive oxygen species (ROS) production, triggering antibody dependent cytotoxicity (ADCC) and clearing the immune complex.^22,23 It also facilitates control of M. tuberculosis by stimulating the respiratory burst in intracellular mononuclear phagocytes.²⁴ Expression of CD64 on total monocytes, measured as MFI, was useful in discriminating patients with active TB from healthy controls.¹³ The novel finding in our study was the overexpression of CD64 specifically in SNTB, which has not been reported before.

An increase in MLR in these patients is associated with change of gene transcription in monocytes with an impairment in anti-mycobacterial functions.^25,26

We also found that radiological findings and Mantoux test could not reliably diagnose TB, especially in cases with SNTB as they were present only in 19 (63.33%) and 10 (33.33%) of SNTB respectively. This is important because, according to the revised RNTCP guidelines, an upfront chest radiograph is advised in all cases of SNTB and a CBNAAT is carried out only if the radiological findings are suggestive.¹⁰ There are therefore large numbers of SNTB cases where chest radiography is not suggestive of TB and these cases are likely to be missed using the current protocol.

A limitation of our study was the small number of cases in each group; also follow up of patients after induction of ATT to assess change in monocyte parameters was not possible.

Conclusion

We concluded that expression of CD64 on total and intermediate monocytes, expansion of intermediate monocytes and the monocyte lymphocyte ratio may serve as independent predictors of tuberculosis especially in the sputum negative group which may be difficult to diagnose in the absence of AFB, a positive TST or radiological findings despite of a strong clinical suspicion of Tuberculosis.

Footnotes

Conflict of interest

The authors declare that there are no conflicts of interest

Funding

The study was funded by Delhi University as a part of an intramural research grant.

ORCID iD

Richa Gupta

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