Publications by authors named "Luis C Berrocal-Almanza"

12 Publications

  • Page 1 of 1

Defining the Role of Cellular Immune Signatures in Diagnostic Evaluation of Suspected Tuberculosis.

J Infect Dis 2021 Jul 31. Epub 2021 Jul 31.

TB Research Centre, National Heart and Lung Institute, Imperial College London, London, United Kingdom.

Background: Diagnosis of paucibacillary tuberculosis (TB) including extrapulmonary TB is a significant challenge, particularly in high-income, low-incidence settings. Measurement of Mycobacterium tuberculosis (Mtb)-specific cellular immune signatures by flow cytometry discriminates active TB from latent TB infection (LTBI) in case-control studies; however, their diagnostic accuracy and clinical utility in routine clinical practice is unknown.

Methods: Using a nested case-control study design within a prospective multicenter cohort of patients presenting with suspected TB in England, we assessed diagnostic accuracy of signatures in 134 patients who tested interferon-gamma release assay (IGRA)-positive and had final diagnoses of TB or non-TB diseases with coincident LTBI. Cellular signatures were measured using flow cytometry.

Results: All signatures performed less well than previously reported. Only signatures incorporating measurement of phenotypic markers on functional Mtb-specific CD4 T cells discriminated active TB from non-TB diseases with LTBI. The signatures measuring HLA-DR+IFNγ + CD4 T cells and CD45RA-CCR7-CD127- IFNγ -IL-2-TNFα + CD4 T cells performed best with 95% positive predictive value (95% confidence interval, 90-97) in the clinically challenging subpopulation of IGRA-positive but acid-fast bacillus (AFB) smear-negative TB suspects.

Conclusions: Two cellular immune signatures could improve and accelerate diagnosis in the challenging group of patients who are IGRA-positive, AFB smear-negative, and have paucibacillary TB.
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July 2021

Transcriptomic signatures for diagnosing tuberculosis in clinical practice: a prospective, multicentre cohort study.

Lancet Infect Dis 2021 03 25;21(3):366-375. Epub 2021 Jan 25.

Tuberculosis Research Centre, National Heart and Lung Institute, Imperial College London, London, UK. Electronic address:

Background: Blood transcriptomic signatures for diagnosis of tuberculosis have shown promise in case-control studies, but none have been prospectively designed or validated in adults presenting with the full clinical spectrum of suspected tuberculosis, including extrapulmonary tuberculosis and common differential diagnoses that clinically resemble tuberculosis. We aimed to evaluate the diagnostic accuracy of transcriptomic signatures in patients presenting with clinically suspected tuberculosis in routine practice.

Methods: The Validation of New Technologies for Diagnostic Evaluation of Tuberculosis (VANTDET) study was nested within a prospective, multicentre cohort study in secondary care in England (IDEA 11/H0722/8). Patients (aged ≥16 years) suspected of having tuberculosis in the routine clinical inpatient and outpatient setting were recruited at ten National Health Service hospitals in England for IDEA and were included in VANTDET if they provided consent for genomic analysis. Patients had whole blood taken for microarray analysis to measure abundance of transcripts and were followed up for 6-12 months to determine final diagnoses on the basis of predefined diagnostic criteria. The diagnostic accuracy of six signatures derived from the cohort and three previously published transcriptomic signatures with potentially high diagnostic performance were assessed by calculating area under the receiver-operating characteristic curves (AUC-ROCs), sensitivities, and specificities.

Findings: Between Nov 25, 2011, and Dec 31, 2013, 1162 participants were enrolled. 628 participants (aged ≥16 years) were included in the analysis, of whom 212 (34%) had culture-confirmed tuberculosis, 89 (14%) had highly probable tuberculosis, and 327 (52%) had tuberculosis excluded. The novel signature with highest performance for identifying all active tuberculosis gave an AUC-ROC of 0·87 (95% CI 0·81-0·92), sensitivity of 77% (66-87), and specificity of 84% (74-91). The best-performing published signature gave an AUC-ROC of 0·83 (0·80-0·86), sensitivity of 78% (73-83), and specificity of 76% (70-80). For detecting highly probable tuberculosis, the best novel signature yielded results of 0·86 (0·71-0·95), 77% (56-94%), and 77% (57-95%). None of the relevant cohort-derived or previously published signatures achieved the WHO-defined targets of paired sensitivity and specificity for a non-sputum-based diagnostic test.

Interpretation: In a clinically representative cohort in routine practice in a low-incidence setting, transcriptomic signatures did not have adequate accuracy for diagnosis of tuberculosis, including in patients with highly probable tuberculosis where the unmet need is greatest. These findings suggest that transcriptomic signatures have little clinical utility for diagnostic assessment of suspected tuberculosis.

Funding: National Institute for Health Research.
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March 2021

Effectiveness of pre-entry active tuberculosis and post-entry latent tuberculosis screening in new entrants to the UK: a retrospective, population-based cohort study.

Lancet Infect Dis 2019 11 27;19(11):1191-1201. Epub 2019 Aug 27.

National Institute for Health Research Health Protection Research Unit in Respiratory Infections, National Heart and Lung Institute, Imperial College London, London, UK; Tuberculosis Unit, TARGET, National Infection Service, Public Health England, London, UK.

Background: Evaluating interventions that might lead to a reduction in tuberculosis in high-income countries with a low incidence of the disease is key to accelerate progress towards its elimination. In such countries, migrants are known to contribute a large proportion of tuberculosis cases to the burden. We assessed the effectiveness of screening for active tuberculosis before entry to the UK and for latent tuberculosis infection (LTBI) post-entry for reduction of tuberculosis in new-entrant migrants to the UK. Additionally, we investigated the effect of access to primary care on tuberculosis incidence in this population.

Methods: We did a retrospective, population-based cohort study of migrants from 66 countries who were negative for active tuberculosis at pre-entry screening between Jan 1, 2011, and Dec 31, 2014, and eligible for LTBI screening. We used record linkage to track their first contact with primary care, uptake of LTBI screening, and development of active tuberculosis in England, Wales, and Northern Ireland. To assess the effectiveness of the pre-entry screening programme, we identified a control group of migrants who were not screened for active tuberculosis using the specific code for new entrants to the UK registering in primary care within the National Health Service patient registration data system. Our primary outcome was development of active tuberculosis notified to the National Enhanced Tuberculosis Surveillance System.

Findings: Our cohort comprised 224 234 migrants who were screened for active tuberculosis before entry to the UK and a control group of 118 738 migrants who were not. 103 990 (50%) migrants who were screened for active tuberculosis registered in primary care; all individuals in the control group were registered in primary care. 1828 tuberculosis cases were identified during the cohort time, of which 31 were prevalent. There were 26 incident active tuberculosis cases in migrants with no evidence of primary care registration, and 1771 cases in the entire cohort of migrants who registered in primary care (n=222 728), giving an incidence rate of 174 (95% CI 166-182) per 100 000 person-years. 672 (1%) of 103 990 migrants who were screened for active tuberculosis went on to develop tuberculosis compared with 1099 (1%) of 118 738 not screened for active tuberculosis (incidence rate ratio [IRR] 1·49, 95% CI 1·33-1·67; p<0·0001). 2451 (1%) of the 222 728 migrants registered in primary care were screened for LTBI, of whom 421 (17%) tested positive and 1961 (80%) tested negative; none developed active tuberculosis within the observed time period. Migrants settling in the least deprived areas had a decreased risk of developing tuberculosis (IRR 0·74, 95% CI 0·62-0·89; p=0·002), and time from UK arrival to primary care registration of 1 year or longer was associated with increased risk of active tuberculosis (2·96, 2·59-3·38; p<0·0001).

Interpretation: Pre-entry tuberculosis screening, early primary care registration, and LTBI screening are strongly and independently associated with a lower tuberculosis incidence in new-entrant migrants.

Funding: National Institute for Health Research (NIHR) Health Protection Research Unit in Respiratory Infections and NIHR Imperial Biomedical Research Centre.
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November 2019

Predicting progression to active tuberculosis: A rate-limiting step on the path to elimination.

PLoS Med 2019 05 24;16(5):e1002814. Epub 2019 May 24.

Tuberculosis Research Centre, National Heart and Lung Institute, St Mary's Campus, Imperial College London, London, United Kingdom.

In a Perspective, Ajit Lalvani and colleagues discuss new approaches to predicting progression to active tuberculosis.
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May 2019

CD4+ T cell cytokine responses to the DAR-901 booster vaccine in BCG-primed adults: A randomized, placebo-controlled trial.

PLoS One 2019 23;14(5):e0217091. Epub 2019 May 23.

Geisel School of Medicine, Hanover, NH, United States of America.

Background: DAR-901 is an inactivated whole cell tuberculosis booster vaccine, prepared using a new scalable, broth-grown method from the master cell bank of SRL172, a vaccine previously shown to prevent tuberculosis. This study examined whether DAR-901 (a) induces CD4+ T cell cytokine profiles previously proposed as correlates of protection and (b) has a specific vaccine-induced immunological signature compared to BCG or placebo.

Methods: We analysed CD4+ T cell cytokine immune responses from 10 DAR-901 recipients, 9 BCG recipients and 9 placebo recipients from the Phase I DAR-901 MDES trial. In that study, HIV-negative, IGRA-negative participants with prior BCG immunization were randomized (double-blind) to receive three intradermal injections of DAR-901 or saline placebo or two injections of saline placebo followed by an intradermal injection of BCG. Antigen-specific functional and phenotypic CD4+ T cell responses along with effector phenotype of responder cells were measured by intracellular cytokine staining.

Results: DAR-901 recipients exhibited increased DAR-901 antigen-specific polyfunctional or bifunctional T cell responses compared to baseline. Vaccine specific CD4+ IFNγ, IL2, TNFα and any cytokine responses peaked at 7 days post-dose 3. Th1 responses predominated, with most responder cells exhibiting a polyfunctional effector memory phenotype. BCG induced greater CD4+ T cell responses than placebo while the more modest DAR-901 responses did not differ from placebo. Neither DAR-901 nor BCG induced substantial or sustained Th17 /Th22 cytokine responses.

Conclusion: DAR-901, a TB booster vaccine grown from the master cell bank of SRL 172 which was shown to prevent TB, induced low magnitude polyfunctional effector memory CD4+ T cell responses. DAR-901 responses were lower than those induced by BCG, a vaccine that has been shown ineffective as a booster to prevent tuberculosis disease. These results suggest that induction of higher levels of CD4+ cytokine stimulation may not be a critical or pre-requisite characteristic for candidate TB vaccine boosters.

Trial Registration: NCT02063555.
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January 2020

S100A12 is up-regulated in pulmonary tuberculosis and predicts the extent of alveolar infiltration on chest radiography: an observational study.

Sci Rep 2016 08 19;6:31798. Epub 2016 Aug 19.

Septomics Research Center, Jena University Hospital, Jena, Germany.

Pulmonary tuberculosis (PTB) results in lung functional impairment and there are no surrogate markers to monitor the extent of lung involvement. We investigated the clinical significance of S100A12 and soluble receptor for advanced glycation end-products (sRAGE) for predicting the extent of lung involvement. We performed an observational study in India with 119 newly diagnosed, treatment naïve, sputum smear positive, HIV-negative PTB patients and 163 healthy controls. All patients were followed-up for six months. Sociodemographic variables and the serum levels of S100A12, sRAGE, esRAGE, HMGB-1, TNF-α, IFN-γ and CRP were measured. Lung involvement in PTB patients was assessed by chest radiography. Compared with healthy controls, PTB patients had increased serum concentrations of S100A12 while sRAGE was decreased. S100A12 was an independent predictor of disease occurrence (OR 1.873, 95%CI 1.212-2.891, p = 0.004). Under DOTS therapy, S100A12 decreased significantly after 4 months whereas CRP significantly decreased after 2 months (p < 0.0001). Importantly, although CRP was also an independent predictor of disease occurrence, only S100A12 was a significant predictor of lung alveolar infiltration (OR 2.60, 95%CI 1.35-5.00, p = 0.004). These results suggest that S100A12 has the potential to assess the extent of alveolar infiltration in PTB.
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August 2016

A Novel Reading Scheme for Assessing the Extent of Radiographic Abnormalities and Its Association with Disease Severity in Sputum Smear-Positive Tuberculosis: An Observational Study in Hyderabad/India.

PLoS One 2015 18;10(9):e0138070. Epub 2015 Sep 18.

Septomics Research Center, Jena University Hospital, Jena, Germany.

Background: Existing reading schemes for chest X-ray (CXR) used to grade the extent of disease severity at diagnosis in patients with pulmonary tuberculosis (PTB) are often based on numerical scores that summate specific radiographic features. However, since PTB is known to exhibit a wide heterogeneity in pathology, certain features might be differentially associated with clinical parameters of disease severity.

Objective: We aimed to grade disease severity in PTB patients at diagnosis and after completion of DOTS treatment by developing a reading scheme based on five different radiographic manifestations and analyze their association with the clinical parameters of systemic involvement and infectivity.

Methods: 141 HIV-negative adults with newly diagnosed sputum smear-positive PTB were enrolled in a prospective observational study in Hyderabad, India. The presence and extent on CXRs of five radiographic manifestations, i.e., lung involvement, alveolar infiltration, cavitation, lymphadenopathy and pleural effusion, were classified using the new reading scheme by using a four-quadrant approach. We evaluated the inter-reader reliability of each manifestation, and its association with BMI and sputum smear positivity at diagnosis. The presence and extent of these radiographic manifestations were further compared with CXRs on completion of DOTS treatment.

Results: At diagnosis, an average lung area of 51.7% +/- 23.3% was affected by radiographic abnormalities. 94% of the patients had alveolar infiltrates, with 89.4% located in the upper quadrants, suggesting post primary PTB and in 34.8% of patients cavities were found. We further showed that the extent of affected lung area was a negative predictor of BMI (β value -0.035, p 0.019). No significant association of BMI with any of the other CXR features was found. The extent of alveolar infiltrates, along with the presence of cavitation, were strongly associated with sputum smear positivity. The microbiological cure rate in our cohort after 6 months of DOTS treatment was 95%. The extent of the affected lung area in these patients decreased from 56.0% +/- 21.5% to 31.0 +/- 20% and a decrease was also observed in the extent of alveolar infiltrates from 98.4% to 25.8% in at least one quadrant, presence of cavities from 34.8% to 1.6%, lymphadenopathy from 46.8% to 16.1%, and pleural effusion from 19.4% to 6.5%.

Conclusions: We established a new assessment scheme for grading disease severity in PTB by specifically considering five radiographic manifestations which were differently associated with the BMI and sputum smear positivity, changed to a different extent after 6 months of treatment and exhibited an excellent agreement between radiologists. Our results suggest that this reading scheme might contribute to the estimation of disease severity with respect to differences in disease pathology. Further studies are needed to determine a correlation with short and long-term pulmonary function impairment and whether there would be any benefit in lengthening or modulating therapy based on this CXR severity assessment.
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June 2016

Erratum: Less functional variants of TLR-1/-6/-10 genes are associated with age.

Immun Ageing 2015 11;12:10. Epub 2015 Aug 11.

Institute of Microbiology and Hygiene, Charité University Medical Center Berlin, Charitéplatz 1, Berlin, 10117 Germany.

[This corrects the article DOI: 10.1186/s12979-015-0034-z.].
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August 2015

Less functional variants of TLR-1/-6/-10 genes are associated with age.

Immun Ageing 2015 8;12. Epub 2015 Jul 8.

Institute of Microbiology and Hygiene, Charité University Medical Center Berlin, Charitéplatz 1, 10117 Berlin, Germany.

Background: Determining the prerequisites for healthy aging is a major task in the modern world characterized by a longer lifespan of the individuals. Besides lifestyle and environmental influences genetic factors are involved as shown by several genome-wide association studies. Older individuals are known to have an impaired immune response, a condition recently termed "inflamm-aging". We hypothesize that the induction of this condition in the elderly is influenced by the sensitivity of the innate immune system. Therefore, we investigated genetic variants of the Toll-like receptor (TLR) family, one of the major family of innate immune receptors, for association with age in two cohorts of healthy, disease-free subjects.

Results: According to sex we found a positive association of loss-of-function variants of TLR-1 and -6 with healthy aging with odds ratios of 1.54 in males for TLR-6 (249 S/S), and 1.41, 1.66, and 1.64 in females for TLR-1 prom., TLR-1 (248 S/S), and TLR-1 (602 S/S), respectively. Thus, the presence of these variants increases the probability of achieving healthy old age and indicates that a reduced TLR activity may be beneficial in the elderly.

Conclusions: This is the first report showing an association of TLR variants with age. While a loss of function of an important immune receptor may be a risk factor for acute infections as has been shown previously, in the setting of healthy ageing it appears to be protective, which may relate to "inflamm-aging". These first results should be reproduced in larger trials to confirm this hypothesis.
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July 2015

Toll-like receptor 1 variations influence susceptibility and immune response to Mycobacterium tuberculosis.

Tuberculosis (Edinb) 2015 May 28;95(3):328-35. Epub 2015 Feb 28.

Institute for Microbiology and Hygiene, Charité-Universitätsmedizin, Berlin, Germany; Department of Psychosomatic Medicine, Charité-Universitätsmedizin, Berlin, Germany. Electronic address:

Background: Tuberculosis (TB), a disease caused by Mycobacterium tuberculosis (MTB) infection, is still a global public health problem. TB susceptibility varies greatly in infected individuals, and mycobacterial recognition by the innate immune system likely affects disease course and outcome. This research describes a single nucleotide polymorphism in the Toll-like receptor (TLR) 1 gene that functionally alters the innate immune response to MTB and is associated with TB susceptibility in India.

Methods: 206 TB patients and 239 healthy controls from Hyderabad, India were analyzed for SNPs in the TLR1 and TLR2 genes, which were subsequently correlated to TB susceptibility. To test individual responses to MTB lysates, we stimulated PBMCs from genotyped healthy German individuals, as well as HEK cells transfected with TLR1/2 variants. TNF production and NF-kB activation were assessed respectively.

Results: Cohort analysis associated the TLR1-248N SNP (RS4833095) with TB protection. TLR1-248N expressing PBMCs from healthy controls exhibited an increased TNF response to MTB lysates. In addition to this, functional studies using HEK cell lines transfected with TLR1-248N and stimulated with MTB showed an increased NF-kB activation.

Conclusion: SNP TLR1-248N is associated with TB protection in an Indian population and exhibits an increased immune response to MTB lysate in vitro.
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May 2015

The course of gastric cancer following surgery is associated with genetic variations of the interleukin-1 receptor antagonist and interleukin-1β.

Gastric Cancer 2015 Jan 21;18(1):77-83. Epub 2014 Feb 21.

Institute for Microbiology and Hygiene, Charité University Medicine Berlin, Hindenburgdamm 27, 12203, Berlin, Germany.

Background: Inflammation, especially the cytokine response of the IL-1 family, has been shown to influence susceptibility to gastric cancer. In addition, several other pro-inflammatory cytokines have been demonstrated to influence metastasis and resistance to chemotherapy. Therefore, genetic variations within these genes may not only affect susceptibility but also influence the outcome of gastric cancer patients. A limited number of studies showed indeed an association of IL-1β and IL-1RN variations with survival of gastric cancer patients. However, results are inconsistent, possibly because of different patient cohorts and different therapies.

Methods: In this retrospective cohort study we genotyped 154 patients with gastric cancer for IL-1β and IL-1RN variations. Patients had undergone pathologically proven R0 resection and had received no additional adjuvant treatment.

Results: We show here a protective association with disease-free survival for both heterozygous genotypes, IL-1β SNP C-511T (rs16944) and IL-1RN VNTR. The combination of both heterozygous genotypes is the strongest predictor independent of UICC stage.

Conclusion: Genetic variations in the IL-1β and IL-1RN genes influence disease progression in gastric cancer. Screening for these genetic variations might help to stratify therapies for gastric cancer patients in the future.
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January 2015