Role of QuantiFERON-TB Gold,Interferon Gamma Inducible Protein-10 and Tuberculin Skin Test in Active Tuberculosis Diagnosis

Background

The measurement of Interferon gamma or Interferon gamma inducible protein (IP)-10 in antigen stimulated blood samples is suggested as an alternative method for latent tuberculosis (TB) diagnosis. Nonetheless, their role in active TB diagnosis, particularly in TB endemic settings is yet to be defined. In this study, the sensitivities and specificities of Interferon gamma release assay (IGRA), IP-10 assay and tuberculin skin test (TST) in detecting active TB cases were assessed in human immunodeficiency virus (HIV) sero-negative TB patients and healthy controls respectively.

Methods/Principal Findings

A total of 177 adult TB patients and 100 healthy controls were included for this study. QuantiFERON-TB Gold In-tube (QFT-IT) method was used to analyze the sensitivity and specificity of IGRA. QFT-IT, IP-10 and TST yielded the diagnostic sensitivities of 90.6% (95%CI: 86.3%–94.9%), 92.5% (95%CI: 88.6%–96.4%) and 68.9% (95%CI: 60.6%–77.2%) and specificities of 55% (95% CI: 35.2%–54.8%), 48% (95% CI: 38.2%–57.8%) and 75.5% (95% CI: 66.8%–84.2%), respectively. The extent of pulmonary involvement or presence of diabetes mellitus did not appear to influence the sensitivities of any of these tests. The combination of any of the two tests among QFT-IT, IP-10 and TST showed >98% sensitivity among smear negative cases and particularly the combination of IP-10, TST and smear microscopy showed 100% sensitivity, however, the specificity was decreased to 44.8%.

Conclusions/Significance

QFT-IT and IP-10 were highly sensitive in detecting active TB cases. The combination with TST improved the sensitivity of QFT-IT and IP-10 significantly. Although the higher sensitivity of combination of QFT-IT/IP-10 and TST may be useful in active TB diagnosis, they are limited by their poor specificity due to the high prevalence of latent TB in our settings.     

Modern Lineages of Mycobacterium tuberculosis Exhibit Lineage-Specific Patterns of Growth and Cytokine Induction in Human Monocyte-Derived Macrophages

Background

Strains of Mycobacterium tuberculosis vary in virulence. Strains that have caused outbreaks in the United States and United Kingdom have been shown to subvert the innate immune response as a potential immune evasion mechanism. There is, however, little information available as to whether these patterns of immune subversion are features of individual strains or characteristic of broad clonal lineages of M. tuberculosis.

Methods

Strains from two major modern lineages (lineage 2 [East-Asian] and lineage 4 [Euro-American]) circulating in the Western Cape in South Africa as well as a comparator modern lineage (lineage 3 [CAS/Delhi]) were identified. We assessed two virulence associated characteristics: mycobacterial growth (in liquid broth and monocyte derived macrophages) and early pro-inflammatory cytokine induction.

Results

In liquid culture, Lineage 4 strains grew more rapidly and reached higher plateau levels than other strains (lineage 4 vs. lineage 2 p = 0.0024; lineage 4 vs. lineage 3 p = 0.0005). Lineage 3 strains were characterized by low and early plateau levels, while lineage 2 strains showed an intermediate growth phenotype. In monocyte-derived macrophages, lineage 2 strains grew faster than lineage 3 strains (p<0.01) with lineage 4 strains having an intermediate phenotype. Lineage 2 strains induced the lowest levels of pro-inflammatory TNF and IL-12p40 as compared to other lineages (lineage 2: median TNF 362 pg/ml, IL-12p40 91 pg/ml; lineage 3: median TNF 1818 pg/ml, IL-12p40 123 pg/ml; lineage 4: median TNF 1207 pg/ml, IL-12p40 205 pg/ml;). In contrast, lineage 4 strains induced high levels of IL-12p40 and intermediate level of TNF. Lineage 3 strains induced high levels of TNF and intermediate levels of IL-12p40.

Conclusions

Strains of M. tuberculosis from the three major modern strain lineages possess distinct patterns of growth and cytokine induction. Rapid growth and immune subversion may be key characteristics to the success of these strains in different human populations.     

A three-way comparison of tuberculin skin testing, QuantiFERON-TB gold and T-SPOT.TB in children

Background

There are limited data comparing the performance of the two commercially available interferon gamma (IFN-γ) release assays (IGRAs) for the diagnosis of tuberculosis (TB) in children. We compared QuantiFERON-TB gold In Tube (QFT-IT), T-SPOT.TB and the tuberculin skin test (TST) in children at risk for latent TB infection or TB disease.

Methods and Findings

The results of both IGRAs were compared with diagnosis assigned by TST-based criteria and assessed in relation to TB contact history. Results from the TST and at least one assay were available for 96 of 100 children. Agreement between QFT-IT and T-SPOT.TB was high (93% agreement, κ = 0.83). QFT-IT and T-SPOT.TB tests were positive in 8 (89%) and 9 (100%) children with suspected active TB disease. There was moderate agreement between TST and either QFT-IT (75%, κ = 0.50) or T-SPOT.TB (75%, κ = 0.51). Among 38 children with TST-defined latent TB infection, QFT-IT gold and T-SPOT.TB assays were positive in 47% and 39% respectively. Three TST-negative children were positive by at least one IGRA. Children with a TB contact were more likely than children without a TB contact to have a positive IGRA (QFT-IT LR 3.9; T-SPOT.TB LR 3.9) and a positive TST (LR 1.4). Multivariate linear regression analysis showed that the magnitude of both TST induration and IGRA IFN-γ responses was significantly influenced by TB contact history, but only the TST was influenced by age.

Conclusions

Although a high level of agreement between the IGRAs was observed, they are commonly discordant with the TST. The correct interpretation of a negative assay in a child with a positive skin test in clinical practice remains challenging and highlights the need for longitudinal studies to determine the negative predictive value of IGRAs.     

Development of a One-Step Probe Based Molecular Assay for Rapid Immunodiagnosis of Infection with M. tuberculosis Using Dried Blood Spots

Background

Antigen specific release of IP-10 is the most promising alternative marker to IFN-γ for infection with M. tuberculosis. Compared to Interferon-γ release assays (IGRA), IP-10 is released in high levels enabling novel approaches such as field friendly dried blood spots (DBS) and molecular detection.

Aim

To develop a robust IP-10 based molecular assay for the diagnosis of infection with M. tubercuolsis from whole blood and DBS.

Method

We developed a one-step probe based multiplex RT-qPCR assay for detecting IP-10 and IFN-γ mRNA expression from whole blood and DBS samples. The assay was validated and applied for the diagnosis of M. tuberculosis infection in DBS samples from 43 patients with confirmed TB, 13 patients with latent TB and 96 presumed uninfected controls. In parallel, IP-10 and INF-γ levels were measured in Quantiferon (QFT-TB) plasma supernatants.

Results

IP-10 mRNA upregulation was detectable at 4 hours after stimulation (6 fold upregulation) peaking at 8 hours (108 fold upregulation). IFN-γ expression occurred in concert but levels were lower (peak 6.7 fold upregulation). IP-10 gene expression level was significantly higher in patients with tuberculosis (median 31.2, IQR 10.7–67.0) and persons with latent tuberculosis infection (LTBI) (41.2, IQR 9.8–64.9) compared to healthy controls (1.6, IQR 1.1–2.4; p<0.0001). The IP-10 mRNA and protein based tests had comparable diagnostic accuracy to QFT-TB, sensitivity (85% and 88% vs 85%) and specificity (96% and 96% vs 97%, p = ns.).

Conclusion

We developed a rapid, robust and accurate molecular immunodiagnostic test for M. tuberculosis infection. By combining DBS based sample acquisition, mail or currier based sample transport with centralized molecular detection, this immunodiagnostic test concept can reduce the local technological requirements everywhere and make it possible to offer highly accurate immunodiagnostic tests in low resource settings.     

Combination of Cytokine Responses Indicative of Latent TB and Active TB in Malawian Adults

Background

An IFN-γ response to M. tuberculosis-specific antigens is an effective biomarker for M. tuberculosis infection but it cannot discriminate between latent TB infection and active TB disease. Combining a number of cytokine/chemokine responses to M. tuberculosis antigens may enable differentiation of latent TB from active disease.

Methods

Asymptomatic recently-exposed individuals (spouses of TB patients) were recruited and tuberculin skin tested, bled and followed-up for two years. Culture supernatants, from a six-day culture of diluted whole blood samples stimulated with M. tuberculosis-derived PPD or ESAT-6, were measured for IFN-γ, IL-10, IL-13, IL-17, TNF-α and CXCL10 using cytokine ELISAs. In addition, 15 patients with sputum smear-positive pulmonary TB were recruited and tested.

Results

Spouses with positive IFN-γ responses to M. tuberculosis ESAT-6 (>62.5 pg/mL) and TB patients showed high production of IL-17, CXCL10 and TNF-α. Higher production of IL-10 and IL-17 in response to ESAT-6 was observed in the spouses compared with TB patients while the ratios of IFN-γ/IL-10 and IFN-γ/IL-17 in response to M. tuberculosis-derived PPD were significantly higher in TB patients compared with the spouses. Tuberculin skin test results did not correlate with cytokine responses.

Conclusions

CXCL10 and TNF-α may be used as adjunct markers alongside an IFN-γ release assay to diagnose M. tuberculosis infection, and IL-17 and IL-10 production may differentiate individuals with LTBI from active TB.     

IFNγ Response to Mycobacterium tuberculosis,Risk of Infection and Disease in Household Contacts of Tuberculosis Patients in Colombia

Objectives

Household contacts (HHCs) of pulmonary tuberculosis patients are at high risk of Mycobacterium tuberculosis infection and early disease development. Identification of individuals at risk of tuberculosis disease is a desirable goal for tuberculosis control. Interferon-gamma release assays (IGRAs) using specific M. tuberculosis antigens provide an alternative to tuberculin skin testing (TST) for infection detection. Additionally, the levels of IFNγ produced in response to these antigens may have prognostic value. We estimated the prevalence of M. tuberculosis infection by IGRA and TST in HHCs and their source population (SP), and assessed whether IFNγ levels in HHCs correlate with tuberculosis development.

Methods

A cohort of 2060 HHCs was followed for 2–3 years after exposure to a tuberculosis case. Besides TST, IFNγ responses to mycobacterial antigens: CFP, CFP-10, HspX and Ag85A were assessed in 7-days whole blood cultures and compared to 766 individuals from the SP in Medellín, Colombia. Isoniazid prophylaxis was not offered to child contacts because Colombian tuberculosis regulations consider it only in children under 5 years, TST positive without BCG vaccination.

Results

Using TST 65.9% of HHCs and 42.7% subjects from the SP were positive (OR 2.60, p<0.0001). IFNγ response to CFP-10, a biomarker of M. tuberculosis infection, tested positive in 66.3% HHCs and 24.3% from the SP (OR = 6.07, p<0.0001). Tuberculosis incidence rate was 7.0/1000 person years. Children <5 years accounted for 21.6% of incident cases. No significant difference was found between positive and negative IFNγ responders to CFP-10 (HR 1.82 95% CI 0.79–4.20 p = 0.16). However, a significant trend for tuberculosis development amongst high HHC IFNγ producers was observed (trend Log rank p = 0.007).

Discussion

CFP-10-induced IFNγ production is useful to establish tuberculosis infection prevalence amongst HHC and identify those at highest risk of disease. The high tuberculosis incidence amongst children supports administration of chemoprohylaxis to child contacts regardless of BCG vaccination.     

Response prediction in chronic hepatitis C by assessment of IP-10 and IL28B-related single nucleotide polymorphisms

Background

High baseline levels of IP-10 predict a slower first phase decline in HCV RNA and a poor outcome following interferon/ribavirin therapy in patients with chronic hepatitis C. Several recent studies report that single nucleotide polymorphisms (SNPs) adjacent to IL28B predict spontaneous resolution of HCV infection and outcome of treatment among HCV genotype 1 infected patients.

Methods and Findings

In the present study, we correlated the occurrence of variants at three such SNPs (rs12979860, rs12980275, and rs8099917) with pretreatment plasma IP-10 and HCV RNA throughout therapy within a phase III treatment trial (HCV-DITTO) involving 253 Caucasian patients. The favorable SNP variants (CC, AA, and TT, respectively) were associated with lower baseline IP-10 (P = 0.02, P = 0.01, P = 0.04) and were less common among HCV genotype 1 infected patients than genotype 2/3 (P<0.0001, P<0.0001, and P = 0.01). Patients carrying favorable SNP genotypes had higher baseline viral load than those carrying unfavorable variants (P = 0.0013, P = 0.029, P = 0.0004 respectively). Among HCV genotype 1 infected carriers of the favorable C, A, or T alleles, IP-10 below 150 pg/mL significantly predicted a more pronounced reduction of HCV RNA from day 0 to 4 (first phase decline), which translated into increased rates of RVR (62%, 53%, and 39%) and SVR (85%, 76%, and 75% respectively) among homozygous carriers with baseline IP-10 below 150 pg/mL. In multivariate analyses of genotype 1-infected patients, baseline IP-10 and C genotype at rs12979860 independently predicted the first phase viral decline and RVR, which in turn independently predicted SVR.

Conclusions

Concomitant assessment of pretreatment IP-10 and IL28B-related SNPs augments the prediction of the first phase decline in HCV RNA, RVR, and final therapeutic outcome.     

Longitudinal analysis of QuantiFERON-TB Gold In-Tube in children with adult household tuberculosis contact in South Africa: a prospective cohort study

Background

QuantiFERON-TB Gold In Tube (QFT-GIT) is a tool for detecting M. tuberculosis infection. However, interpretation and utility of serial QFT-GIT testing of pediatric tuberculosis (TB) contacts is not well understood. We compared TB prevalence between baseline and 6 months follow-up using QFT-GIT and tuberculin skin testing (TST) in children who were household contacts of adults with pulmonary TB in South Africa, and explored factors associated with QFT-GIT conversions and reversions.

Method

Prospective study with six month longitudinal follow-up.

Results

Among 270 enrolled pediatric contacts, 196 (73%) underwent 6-month follow-up testing. The 6-month prevalence estimate of MTB infection in pediatric contacts increased significantly from a baseline of 29% (79/270, 95%CI [24–35]) to 38% (103/270, 95% CI [32–44], p<0.001) using QFT-GIT; prevalence increased from a baseline of 28% (71/254, 95%CI [23–34]) to 33% (88/263, 95%CI [21–32], p = 0.002) using TST. Prevalence estimates were influenced by thresholds for positivity for TST, but not for QFT-GIT. Among 134 children with a negative or indeterminate baseline QFT-GIT, 24 (18%) converted to positive at follow-up; conversion rates did not differ significantly when using more stringent thresholds to define QFT-GIT conversion. Older age >10 years (AOR 8.9 95%CI [1.1–72]) and baseline TST positivity ≥5 mm (AOR 5.2 95%CI [1.2–23]) were associated with QFT-GIT conversion. Among 62 children with a positive baseline QFT-GIT, 9 (15%) reverted to negative; female gender (AOR 18.5 95%CI [1.1–321]; p = 0.04] was associated with reversion, while children with baseline positive TST were less likely to have QFT-GIT reversion (AOR 0.01 95%CI [0.001–0.24]).

Conclusion

Among pediatric contacts of adult household TB cases in South Africa, prevalence estimates of TB infection increased significantly from baseline to 6 months. Conversions and reversions occurred among pediatric TB contacts using QFT-GIT, but QFT-GIT conversion rates were less influenced by thresholds used for conversions than were TST conversion rates.     

Variation of Mycobacterium tuberculosis Antigen-Specific IFN-γ and IL-17 Responses in Healthy Tuberculin Skin Test (TST)-Positive Human Subjects

Objective

To determine the variation of IFN-γ and IL-17 responses to M. tuberculosis antigens in healthy TST+ humans.

Methods

We isolated peripheral blood mononuclear cells from 21 TST+ healthy adults, stimulated them with phytohemagglutinin (PHA), PPD, Ag85B, ESAT-6, and live M. bovis BCG, and assayed IFN-γ and IL-17 secretion by ELISA in supernatants after 24 or 72 hours of incubation respectively.

Results

As in other studies, we found a wide range of IFN-γ responses to M. tuberculosis antigens; the variation significantly exceeded that observed in the same donors to the polyclonal T cell stimulus, phytohemagglutinin (PHA). In addition, we assayed IL-17 secretion in response to the same stimuli, and found less subject-to-subject variation. Analysis of the ratio of IFN-γ to IL-17 secretion on a subject-to-subject basis also revealed a wide range, with the majority of results distributed in a narrow range, and a minority with extreme results all of which were greater than that in the majority of subjects. The data suggest that study of exceptional responses to M. tuberculosis antigens may reveal immunologic correlates with specific outcomes of M. tuberculosis infection.

Conclusion

Variation of IFNγ and IFN-γ/IL-17 responses to mycobacterial antigens exceeds that of responses to the polyclonal stimulus, PHA, in TST positive healthy humans. This indicates a quantitative spectrum of human immune responses to infection with M. tuberculosis. Since the outcome of human infection with M. tuberculosis varies greatly, systematic study of multiple immune responses to multiple antigens is likely to reveal correlations between selected immune responses and the outcomes of infection.     

Evaluation of the Diagnostic Potential of IP-10 and IL-2 as Biomarkers for the Diagnosis of Active and Latent Tuberculosis in a BCG-Vaccinated Population

Background

The Mycobacterium tuberculosis (Mtb)-specific T-cell interferon gamma release assays (IGRAs) are useful in detecting Mtb infection but perform poorly at distinguishing active tuberculosis disease (ATB) and latent tuberculosis infection (LTBI). This study is aimed at evaluating additional cytokines as biomarkers besides interferon-gamma (IFN-γ) to improve the identification of ATB and LTBI.

Methodology/Principal Findings

Sixty-six patients with ATB, 73 household contacts (HHC) of ATB patients and 76 healthy controls (HC) were recruited to undergo QuantiFERON TB GOLD in-tube assay (QFT) and the enzyme-linked immunosorbent assay (ELISA) where the release of IFN-γ, IFN-γ inducible protein 10 (IP-10), Interleukin 2 (IL-2) and Tumor Necrosis Factor-α (TNF-α) was determined in the whole blood with or without antigen-stimulation. The positive rates of the QFT, IP-10 and IL-2 tests were 86.4%, 89.4% and 86.4% for the ATB group with no difference between them (p>0.05). However, QFT in combination with IP-10 and IL-2 significantly increased the detection rate to 95.5% in the ATB group (p = 0.03) and the indeterminate rate of all samples decreased from 2.3% (5/215) to 0.4% (1/215). The un-stimulated level of IP-10 was significantly higher in the HHC than the ATB and HC groups. The IP-10 responses were strongly associated with extended Mtb exposure time and the degree of smear-positivity of the index cases. The IL-2/IFN-γ ratio in the antigen-stimulated plasma could discriminate LTBI from ATB with a sensitivity of 77.2% and a specificity of 87.2%.

Conclusion

The increased Mtb-specific antigen-stimulated expression of IP-10 and IL-2 may be useful for detecting both ATB and LTBI. Combining the QFT with IP-10 and IL-2 could increase the detection accuracy of active TB over the QFT alone.     

Monitoring Procalcitonin in Febrile Neutropenia: What Is Its Utility for Initial Diagnosis of Infection and Reassessment in Persistent Fever?

Background

Management of febrile neutropenic episodes (FE) is challenged by lacking microbiological and clinical documentation of infection. We aimed at evaluating the utility of monitoring blood procalcitonin (PCT) in FE for initial diagnosis of infection and reassessment in persistent fever.

Methods

PCT kinetics was prospectively monitored in 194 consecutive FE (1771 blood samples): 65 microbiologically documented infections (MDI, 33.5%; 49 due to non-coagulase-negative staphylococci, non-CNS), 68 clinically documented infections (CDI, 35%; 39 deep-seated), and 61 fever of unexplained origin (FUO, 31.5%).

Results

At fever onset median PCT was 190 pg/mL (range 30–26''800), without significant difference among MDI, CDI and FUO. PCT peak occurred on day 2 after onset of fever: non-CNS-MDI/deep-seated-CDI (656, 80–86350) vs. FUO (205, 33–771; p<0.001). PCT >500 pg/mL distinguished non-CNS-MDI/deep-seated-CDI from FUO with 56% sensitivity and 90% specificity. PCT was >500 pg/ml in only 10% of FUO (688, 570–771). A PCT peak >500 pg/mL (1196, 524–11950) occurred beyond 3 days of persistent fever in 17/21 (81%) invasive fungal diseases (IFD). This late PCT peak identified IFD with 81% sensitivity and 57% specificity and preceded diagnosis according to EORTC-MSG criteria in 41% of cases. In IFD responding to therapy, median days to PCT <500 pg/mL and defervescence were 5 (1–23) vs. 10 (3–22; p = 0.026), respectively.

Conclusion

While procalcitonin is not useful for diagnosis of infection at onset of neutropenic fever, it may help to distinguish a minority of potentially severe infections among FUOs on day 2 after onset of fever. In persistent fever monitoring procalcitonin contributes to early diagnosis and follow-up of invasive mycoses.     

Protective immunity to Mycobacterium tuberculosis infection by chemokine and cytokine conditioned CFP-10 differentiated dendritic cells

Background

Dendritic cells (DCs) play major roles in mediating immune responses to mycobacteria. A crucial aspect of this is the priming of T cells via chemokines and cytokines. In this study we investigated the roles of chemokines RANTES and IP-10 in regulating protective responses from Mycobacterium tuberculosis (M. tb) 10 kDa Culture Filtrate Protein-10 (CFP-10) differentiated DCs (CFP10-DCs).

Methods and Findings

Infection of CFP10-DCs with mycobacteria down-modulated RANTES and IP-10 levels. Pathway specific microarray analyses showed that in addition to RANTES and IP-10, mycobacteria infected CFP10-DCs showed reduced expression of many Th1 promoting chemokines and chemokine receptors. Importantly, T cells co-cultured with RANTES and IP-10 conditioned CFP10-DCs mediated killing of mycobacteria from infected macrophages. Similarly, T cells recruited by RANTES and IP-10 conditioned CFP10-DCs mediated significant killing of mycobacteria from infected macrophages. IFN-gamma treatment of CFP10-DCs restored RANTES and IP-10 levels and T cells activated by these DCs mediated significant killing of virulent M. tb inside macrophages. Adoptive transfer of either RANTES and IP-10 or IL-12 and IFN-gamma conditioned CFP10-DCs cleared an established M. tb infection in mice. The extent of clearance was similar to that obtained with drug treatment.

Conclusions

These results indicate that chemokine and cytokine secretion by DCs differentiated by M. tb antigens such as CFP-10 play major roles in regulating protective immune responses at sites of infection.     

Reduced transmissibility of East African Indian strains of Mycobacterium tuberculosis

Background

Mycobacterium tuberculosis (MTB) has been classified into 4 main lineages. Some reports have associated certain lineages with particular clinical phenotypes, but there is still insufficient information regarding the clinical and epidemiologic implications of MTB lineage variation.

Methods

Using large sequence polymorphisms we classified MTB isolates from a population-based study in Montreal, Canada into the 4 major lineages, and identified the associated clinical and epidemiologic features. In addition, IS6110-RFLP and spoligotyping were used as indicators of recent TB transmission. The study population was divided into a derivation cohort, diagnosed between 2001 and 2007, and a separate validation cohort, diagnosed between 1996 and 2000.

Results

In the derivation cohort, when compared to the other MTB lineages, the East African-Indian (EAI) lineage was associated with lower rates of TB transmission, as measured by: positive TST among close contacts of pulmonary TB cases (adjusted odds ratio 0.6: [95% confidence interval 0.4–0.9]), and clustered TB cases (0.3: [<0.001–0.6]). Severe forms of TB were also less likely among the EAI group (0.4: [<0.001–0.8]). There were no significant differences when comparing patients with the other MTB lineages. In the validation cohort, the EAI lineage was associated with lower rates of positive TST among contacts (0.5: [0.3–0.9]) and a trend towards less clustered TB cases (0.5: [0.1–1.8]) when compared to the other lineages. Disease severity among the different groups was not significantly different in the validation cohort.

Conclusions

We conclude that in Montreal, EAI strains were associated with reduced transmission compared to other MTB lineages.     

Can Interferon-Gamma or Interferon-Gamma-Induced-Protein-10 Differentiate Tuberculosis Infection and Disease in Children of High Endemic Areas?

Background

Diagnosis of childhood tuberculosis (TB) is difficult in high TB burden settings. Interferon-gamma-induced protein 10 (IP10) has been suggested as a marker of TB infection and disease, but its ability to differentiate the two conditions remains uncertain.

Objectives

To describe Interferon-gamma (INFγ) and IP10 expression in children with TB infection and disease and controls to assess their potential to differentiate latent and active TB.

Methods

This was a cross sectional study of 322 1–15 years old children with symptoms of TB (28 confirmed, 136 probable and 131 unlikely TB), 335 children in contact with adults with pulmonary TB and 156 community controls in Southern Ethiopia. The Tuberculin Skin Test (TST) and Quantiferon-In-Tube (QFT-IT) were performed. INFγ and IP10 were measured in plasma supernatants.

Results and Interpretation

Children with confirmed and probable TB and contacts were more likely to have TST+ (78.6%, 59.3% and 54.1%, respectively) than children with unlikely TB (28.7%) and controls (12.8%) (p<0.001). Children with confirmed TB (59.3%) and contacts (44.7%) were more likely to have INFγ+ than children with probable (37.6%) or unlikely TB (28.1%) and controls (13.1%) (p<0.001). IP10 concentrations were higher in INFγ+ children independently of TST (p<0.001). There was no difference between IP10 concentrations of children with confirmed TB and contacts (p = 0.8) and children with and without HIV (p>0.1).INFγ and IP10 can identify children with TB infection and disease, but cannot differentiate between the two conditions. HIV status did not affect the expression of IP10.     

Mycobacterium tuberculosis complex mycobacteria as amoeba-resistant organisms

Background

Most environmental non-tuberculous mycobacteria have been demonstrated to invade amoebal trophozoites and cysts, but such relationships are largely unknown for members of the Mycobacterium tuberculosis complex. An environmental source has been proposed for the animal Mycobacterium bovis and the human Mycobacterium canettii.

Methodology/Principal Findings

Using optic and electron microscopy and co-culture methods, we observed that 89±0.6% of M. canettii, 12.4±0.3% of M. tuberculosis, 11.7±2% of M. bovis and 11.2±0.5% of Mycobacterium avium control organisms were phagocytized by Acanthamoeba polyphaga, a ratio significantly higher for M. canettii (P = 0.03), correlating with the significantly larger size of M. canetti organisms (P = 0.035). The percentage of intraamoebal mycobacteria surviving into cytoplasmic vacuoles was 32±2% for M. canettii, 26±1% for M. tuberculosis, 28±2% for M. bovis and 36±2% for M. avium (P = 0.57). M. tuberculosis, M. bovis and M. avium mycobacteria were further entrapped within the double wall of <1% amoebal cysts, but no M. canettii organisms were observed in amoebal cysts. The number of intracystic mycobacteria was significantly (P = 10⁻⁶) higher for M. avium than for the M. tuberculosis complex, and sub-culturing intracystic mycobacteria yielded significantly more (P = 0.02) M. avium organisms (34×10⁴ CFU/mL) than M. tuberculosis (42×10¹ CFU/mL) and M. bovis (35×10¹ CFU/mL) in the presence of a washing fluid free of mycobacteria. Mycobacteria survived in the cysts for up to 18 days and cysts protected M. tuberculosis organisms against mycobactericidal 5 mg/mL streptomycin and 2.5% glutaraldehyde.

Conclusions/Significance

These data indicate that M. tuberculosis complex organisms are amoeba-resistant organisms, as previously demonstrated for non-tuberculous, environmental mycobacteria. Intercystic survival of tuberculous mycobacteria, except for M. canettii, protect them against biocides and could play a role in their life cycle.     

Novel M tuberculosis antigen-specific T-cells are early markers of infection and disease progression

Background

Mycobacterium tuberculosis Region-of-Difference-1 gene products present opportunities for specific diagnosis of M. tuberculosis infection, yet immune responses to only two gene-products, Early Secretory Antigenic Target-6 (ESAT-6) and Culture Filtrate Protein-10 (CFP-10), have been comprehensively investigated.

Methods

T-cell responses to Rv3873, Rv3878 and Rv3879c were quantified by IFN-γ-enzyme-linked-immunospot (ELISpot) in 846 children with recent household tuberculosis exposure and correlated with kinetics of tuberculin skin test (TST) and ESAT-6/CFP-10-ELISpot conversion over six months and clinical outcome over two years.

Results

Responses to Rv3873, Rv3878, and Rv3879c were present in 20–25% of contacts at enrolment. Rv3873 and Rv3879c responses were associated with and preceded TST conversion (P = 0.02 and P = 0.04 respectively), identifying these antigens as early targets of cell-mediated immunity following M. tuberculosis exposure. Responses to Rv3873 were additionally associated with subsequent ESAT-6/CFP-10-ELISpot conversion (P = 0.04). Responses to Rv3873 and Rv3878 predicted progression to active disease (adjusted incidence rate ratio [95% CI] 3.06 [1.05,8.95; P = 0.04], and 3.32 [1.14,9.71; P = 0.03], respectively). Presence of a BCG-vaccination scar was associated with a 67% (P = 0.03) relative risk reduction for progression to active tuberculosis.

Conclusions

These RD1-derived antigens are early targets of cellular immunity following tuberculosis exposure and T-cells specific for these antigens predict progression to active tuberculosis suggesting diagnostic and prognostic utility.     

Levels and determinants of inflammatory biomarkers in a Swiss population-based sample (CoLaus study)

Objective

to assess the levels and determinants of interleukin (IL)-1β, IL-6, tumour necrosis factor (TNF)-α and C-reactive protein (CRP) in a healthy Caucasian population.

Methods

population sample of 2884 men and 3201 women aged 35 to 75. IL-1β, IL-6 and TNF-α were assessed by a multiplexed particle-based flow cytometric assay and CRP by an immunometric assay.

Results

Spearman rank correlations between duplicate cytokine measurements (N = 80) ranged between 0.89 and 0.96; intra-class correlation coefficients ranged between 0.94 and 0.97, indicating good reproducibility. Among the 6085 participants, 2289 (37.6%), 451 (7.4%) and 43 (0.7%) had IL-1β, IL-6 and TNF-α levels below detection limits, respectively. Median (interquartile range) for participants with detectable values were 1.17 (0.48–3.90) pg/ml for IL-1β; 1.47 (0.71–3.53) pg/ml for IL-6; 2.89 (1.82–4.53) pg/ml for TNF-α and 1.3 (0.6–2.7) ng/ml for CRP. On multivariate analysis, greater age was the only factor inversely associated with IL-1β levels. Male sex, increased BMI and smoking were associated with greater IL-6 levels, while no relationship was found for age and leisure-time PA. Male sex, greater age, increased BMI and current smoking were associated with greater TNF-α levels, while no relationship was found with leisure-time PA. CRP levels were positively related to age, BMI and smoking, and inversely to male sex and physical activity.

Conclusion

Population-based levels of several cytokines were established. Increased age and BMI, and to a lesser degree sex and smoking, significantly and differentially impact cytokine levels, while leisure-time physical activity has little effect.     

Deep sequencing analyses of low density microbial communities: working at the boundary of accurate microbiota detection

Introduction

Accurate analyses of microbiota composition of low-density communities (10³–10⁴ bacteria/sample) can be challenging. Background DNA from chemicals and consumables, extraction biases as well as differences in PCR efficiency can significantly interfere with microbiota assessment. This study was aiming to establish protocols for accurate microbiota analysis at low microbial density.

Methods

To examine possible effects of bacterial density on microbiota analyses we compared microbiota profiles of serial diluted saliva and low (nares, nasopharynx) and high-density (oropharynx) upper airway communities in four healthy individuals. DNA was extracted with four different extraction methods (Epicentre Masterpure, Qiagen DNeasy, Mobio Powersoil and a phenol bead-beating protocol combined with Agowa-Mag-mini). Bacterial DNA recovery was analysed by 16S qPCR and microbiota profiles through GS-FLX-Titanium-Sequencing of 16S rRNA gene amplicons spanning the V5–V7 regions.

Results

Lower template concentrations significantly impacted microbiota profiling results. With higher dilutions, low abundant species were overrepresented. In samples of <10⁵ bacteria per ml, e.g. DNA <1 pg/µl, microbiota profiling deviated from the original sample and other dilutions showing a significant increase in the taxa Proteobacteria and decrease in Bacteroidetes. In similar low density samples, DNA extraction method determined if DNA levels were below or above 1 pg/µl and, together with lysis preferences per method, had profound impact on microbiota analyses in both relative abundance as well as representation of species.

Conclusion

This study aimed to interpret microbiota analyses of low-density communities. Bacterial density seemed to interfere with microbiota analyses at < than 10⁶ bacteria per ml or DNA <1 pg/µl. We therefore recommend this threshold for working with low density materials. This study underlines that bias reduction is crucial for adequate profiling of especially low-density bacterial communities.     

Detection of Microbial Translocation in HIV and SIV Infection Using the Limulus Amebocyte Lysate Assay is Masked by Serum and Plasma

Objective

Microbial translocation (MT) is thought to be a major contributor to the pathogenesis of HIV-related immune activation, and circulating lipopolysaccharide (LPS) from Gram-negative bacteria is the principle measurement of this process. However, related research has been impeded by inconsistent LPS test results.

Methods

Specimens were obtained from HIV-infected adults enrolled in the PEARLS study (ACTG A5175) and HIV-HCV co-infected participants enrolled in a study of liver disease staging using MRI elastography. Pig-tailed macaque specimens were obtained from SIV-infected and –uninfected animals. Samples were tested for LPS using the LAL assay with diazo-coupling modifications to improve sensitive detection.

Results

When exogenous LPS was added to macaque plasma, >25% inhibition of LPS detection was found in 10/10 (100%) samples at 20% plasma concentration compared to control; in contrast 5/10 (50%) samples at 2% plasma concentration (p = 0.07) and 0/10 (0%) at 0.1% plasma concentration (p = 0.004) showed >25% inhibition of LPS detection. Similarly, when LPS was added to human serum, >25% inhibition of LPS detection was found in 5/12 (42%) of samples at 2% serum concentration compared to control, while 0/12 (0%) of samples in 0.1% serum showed >25% inhibition of LPS detection (p = 0.07). Likewise, LPS detection in human sera without exogenous LPS was improved by dilution: LPS was detected in 2/12 (17%) human samples in 2% serum, ranging from 3,436–4,736 pg/mL, compared to 9/12 (75%) samples in 0.1% serum, ranging from 123 pg/mL –60,131 pg/mL (p = 0.016). In a separate validation cohort of HIV-HCV co-infected participants sampled at two different times on the same day, LPS measured in 0.2% plasma and with diazo-coupling was closely correlated between the first and second samples (R = 0.66, p<0.05).

Conclusions

Undiluted serum and plasma mask LPS detection. The extent of MT may be substantially underestimated.     

Qualitative immune modulation by interleukin-2 (IL-2) adjuvant therapy in immunological non responder HIV-infected patients

Background

Treatment of HIV-infected patients with interleukin-2 (IL-2) produces significant increases in CD4 T cell counts; however an associated qualitative improvement in cells function has yet to be conclusively demonstrated. By measuring mycobacterial killing activity, we evaluated IL-2-mediated functional immune enhancement ex vivo in immunological non-responders (INRs).

Methods and Findings

PBMC from 12 immunological non-responders (INRs) (CD4+<200/µl, HIV-RNA<50 cp/ml) on combination antiretroviral treatment (cART) were collected at baseline, and after 3 IL-2 cycles. Eight INRs receiving only cART were studied as controls. After 21 days of PBMC incubation with a virulent M. avium suspension, counts of residual colony forming units (CFUs) and concentrations of TNF-α, IL-10 and IFN-γ were determined. In IL-2 treated patients, a significant reduction in mean residual CFUs of PBMC cultures was observed (p<0.01). Moreover, following IL-2 treatment, significant increases in PBMC''s IFNγ production (p = 0.02) and substantial reductions in IL-10 levels were observed.

Conclusions

IL-2 therapy restores the ability of the lympho-monocyte system in eliciting an effective response against mycobacterial infections. Our data indicate the possibility of a clinical role held by IL-2 in enhancing the immune function of subjects unable to achieve immune competence through cART alone.