Molnupiravir increases SARS-CoV-2 genome diversity and complexity: A case-control cohort study.

Molnupiravir, an oral direct-acting antiviral effective in vitro against SARS-CoV-2, has been largely employed during the COVID-19 pandemic, since December 2021. After marketing and widespread usage, a progressive increase in SARS-CoV-2 lineages characterized by a higher transition/transversion ratio, a characteristic signature of molnupiravir action, appeared in the Global Initiative on Sharing All Influenza Data (GISAID) and International Nucleotide Sequence Database Collaboration (INSDC) databases. Here, we assessed the drug effects by SARS-CoV-2 whole-genome sequencing on 38 molnupiravir-treated persistently positive COVID-19 outpatients tested before and after treatment. Seventeen tixagevimab/cilgavimab-treated outpatients served as controls. Mutational analyses confirmed that SARS-CoV-2 exhibits an increased transition/transversion ratio seven days after initiation of molnupiravir. Moreover we observed an increased G->A ratio compared to controls, which was not related to apolipoprotein B mRNAediting enzyme, catalytic polypeptide-like (APOBEC) activity. In addition, we demonstrated for the first time an increased diversity and complexity of the viral quasispecies.

Temporal intra-host variability of mpox virus genomes in multiple body tissues.

Whole-genome sequencing (WGS) has been widely used for the genomic characterization and the phylogenesis of mpox virus (MPXV) 2022 multi-country outbreak. To date, no evidence has been reported on intra-host evolution within samples collected over time from a single patient with long-term infection. Fifty-one samples were collected from five patients at different time points post-symptom onset. All samples were confirmed as MPXV DNA positive, amplified by a multiplexed PCR amplicon, and sequenced by WGS. Complete MPXV genomes were assembled by reference mapping and then aligned to perform phylogenetic and hierarchical clustering analysis. Large intra-host variability was observed among the MPXV genomes sequenced from samples of two immunocompromised with advanced HIV-1 infection patients with prolonged MPXV shedding. Overall, 20 nucleotide mutations were identified in the 32 genomes from HIV patients, differently distributed in samples collected from different tissues and at different time points. No sequence compartmentalization nor variation was observed in the three patients with rapid viral clearance. MPXV exhibits adaptation to changing environments within the infected host and consequently demonstrates tissue compartmentalization. Further studies are needed to elucidate the role of this adaptation in forming a pool of genetic variability and contributing to viral persistence and its clinical implications.

Genomic surveillance of SARS-CoV-2 positive passengers on flights from China to Italy, December 2022.

With numbers of COVID-19 cases having substantially increased at the end of 2022 in China, some countries have started or expanded testing and genomic surveillance of travellers. We report screening results in Italy in late December 2022 of 556 flight passengers in provenance from two Chinese provinces. Among these passengers, 126 (22.7%) tested SARS-CoV-2 positive. Whole genome sequencing of 61 passengers' positive samples revealed Omicron variants, notably sub-lineages BA.5.2.48, BF.7.14 and BQ.1.1, in line with data released from China.

Genetic Diversity of Mycobacterium tuberculosis Isolates in the Metropolitan Area of Rome.

BACKGROUND: The presence in a geographic area of Mycobacterium tuberculosis (Mtb) strains belonging to different phylogeographic lineages and showing different drug susceptibility patterns may suggest recent transmission, with implications in terms of patient clinical management and disease control. The aim of this study was to carry out a preliminary epidemiological investigation of tuberculosis (TB) cases in Rome. METHODS: A total of 232 Mtb isolates, collected from new or previously treated patients, admitted between 2008 and 2014 at 2 hospital settings in Rome with a diagnosis of TB, were analyzed by spoligotyping and analyzing 24 variable-number tandem repeats (VNTR) mycobacterial interspersed repetitive-unit (MIRU) loci. The SITVIT2 database and the MIRU-VNTRplus web applications were used to identify the strain genotypes and to generate phylogenetic trees. RESULTS: Based on the position on the phylogenetic tree, 97.4% of the strains were associated with 1 of the 7 main lineages. The Euro-American lineage was the most commonly represented (81.9%) within both Italian and foreign-born populations, although all main lineages were present. The highest frequency of drug-resistant strains was found among the East-Asian lineage (Beijing genotype) isolated from foreign-born patients. CONCLUSIONS: Dynamics of TB transmission in Rome indicate recent spread of Mtb strains belonging to phylogeographic lineages and clades usually found in countries and geographic areas with a high incidence of TB, similarly to what is observed in most metropolitan areas in Western Europe. Knowledge from molecular and classical epidemiology provides an important tool for disease control.

Screening of Klebsiella pneumoniae subsp. pneumoniae Strains with Multi-Drug Resistance and Virulence Profiles Isolated from an Italian Hospital between 2020 and 2023.

Klebsiella pneumoniae strains that are resistant to multiple drugs (KPMDRs), which are often acquired in hospital settings and lead to healthcare-associated infections, pose a serious public health threat, as does hypervirulent K. pneumoniae (hvKp), which can also cause serious infections in otherwise healthy individuals. The widespread and often unnecessary use of antibiotics seen during the recent COVID-19 pandemic has exacerbated the challenges posed by antibiotic resistance in clinical settings. There is growing concern that hypervirulent (hvKp) strains may acquire genes that confer antimicrobial resistance, thus combining an MDR profile with their increased ability to spread to multiple body sites, causing difficult-to-treat infections. This study aimed to compare resistance and virulence profiles in KPC-3-producing K. pneumoniae isolates collected over four years (2020-2023). A genome-based surveillance of all MDR CRE-K. pneumoniae was used to identify genetic differences and to characterize the virulence and resistance profiles. Our results provide a picture of the evolution of resistance and virulence genes and contribute to avoiding the possible spread of isolates with characteristics of multi-drug resistance and increased virulence, which are thought to be one of the main global challenges to public health, within our hospital.

Implementation of Whole Genome Sequencing of Tuberculosis Isolates in a Referral Center in Rome: Six Years' Experience in Characterizing Drug-Resistant TB and Disease Transmission.

Over the past years, Tuberculosis (TB) control strategies have been effective in reducing drug-resistant (DR) TB globally; however, a wider implementation of new diagnostic strategies, such as Whole genome sequencing (WGS), would be critical for further improvement. The aim of this study, based on WGS of Mycobacterium tuberculosis (MTB) strains isolated in a TB referral center over 6 years, was to evaluate the efficacy of this methodology in improving therapy guidance for clinicians and in improving the understanding of the epidemiology of TB transmission. WGS was performed in addition to pDST on 1001 strains consecutively isolated between January 2016 and December 2021; the results allowed us to improve the quality of data on resistance and to identify possible clusters of transmission. Prediction of rifampicin-resistant (RR) or multi-drug-resistant TB strains (MDR-TB, defined as resistance to at least rifampicin and isoniazid) was obtained for 50 strains (5%). Mutations predictive of an MDR isolate were further characterized, and Ser450Leu and Ser315Thr were found to be the most frequent mutations in rpoB and katG genes, respectively. Discordances between WGS and phenotypic drug susceptibility testing (pDST) were found in few strains, and their impact on clinical decisions and outcome was addressed. The introduction of WGS in our Institute improved our diagnostic routine, allowing accurate patient management, and was a valid instrument for epidemiological investigations and infection control.

Specific T cells restore the autophagic flux inhibited by Mycobacterium tuberculosis in human primary macrophages.

BACKGROUND: Autophagy inhibits survival of intracellular Mycobacterium tuberculosis when induced by rapamycin or interferon γ (IFN-γ), but it remains unclear whether M. tuberculosis itself can induce autophagy and whether T cells play a role in M. tuberculosis-mediated autophagy. The aim of this study was to evaluate the impact of M. tuberculosis on autophagy in human primary macrophages and the role of specific T cells in this process. METHODS: M. tuberculosis (H37Rv)-infected macrophages were incubated with naive or M. tuberculosis-specific T cells. Autophagy was evaluated at 4 hours and 8 hours after infection by analyzing the levels of LC3-II (a hallmark of autophagy) and p62 (a protein degraded by autophagy). M. tuberculosis survival was evaluated by counting the colony-forming units. RESULTS: M. tuberculosis infection of macrophages inhibited the autophagic process at 8 hours after infection. Naive T cells could not rescue this block, whereas M. tuberculosis-specific T cells restored autophagy degradation, accompanied by enhanced bacterial killing. Notably, the effect of M. tuberculosis-specific T cells was not affected by neutralization of endogenous IFN-γ and tumor necrosis factor α and was blocked by preventing contact between macrophages and T cells, suggesting that cell-cell interaction is crucial. CONCLUSIONS: M. tuberculosis inhibits autophagy in human primary macrophages, and specific T cells can restore functional autophagic flux through cell-cell contact.

Genomic and Epidemiologic Surveillance of SARS-CoV-2 in the Pandemic Period: Sequencing Network of the Lazio Region, Italy.

Since the beginning of the COVID-19 pandemic, large-scale genomic sequencing has immediately pointed out that SARS-CoV-2 has rapidly mutated during the course of the pandemic, resulting in the emergence of variants with a public health impact. In this context, strictly monitoring the circulating strains via NGS has proven to be crucial for the early identification of new emerging variants and the study of the genomic evolution and transmission of SARS-CoV-2. Following national and international guidelines, the Lazio region has created a sequencing laboratory network (WGSnet-Lazio) that works in synergy with the reference center for epidemiological surveillance (SERESMI) to monitor the circulation of SARS-CoV-2. Sequencing was carried out with the aims of characterizing outbreak transmission dynamics, performing the genomic analysis of viruses infecting specific categories of patients (i.e., immune-depressed, travelers, and people with severe symptoms) and randomly monitoring variant circulation. Here we report data emerging from sequencing activities carried out by WGSnet-Lazio (from February 2020 to October 2022) linked with epidemiological data to correlate the circulation of variants with the clinical and demographic characteristics of patients. The model of the sequencing network developed in the Lazio region proved to be a useful tool for SARS-CoV-2 surveillance and to support public health measures for epidemic containment.

Epidemiological and Molecular Investigation of the Heater-Cooler Unit (HCU)-Related Outbreak of Invasive Mycobacterium chimaera Infection Occurred in Italy.

BACKGROUND: From 2013 onwards, a large outbreak of Mycobacterium chimaera (MC) invasive infection, which was correlated with the use of contaminated heater-cooler units (HCUs) during open chest surgery, was reported from all over the world. Here, we report the results of the epidemiological and molecular investigations conducted in Italy after the alarm raised about this epidemic event. METHODS: MC strains isolated from patients or from HCU devices were characterized by genomic sequencing and molecular epidemiological analysis. RESULTS: Through retrospective epidemiological analysis conducted between January 2010 and December 2022, 40 possible cases of patients infected with MC were identified. Thirty-six strains isolated from these patients were analysed by whole genome sequencing (WGS) and were found to belong to the genotypes 1.1 or 1.8, which are the genotypes correlated with the outbreak. Most of the cases presented with prosthetic valve endocarditis, vascular graft infection or disseminated infection. Among the cases found, there were 21 deaths. The same analysis was carried out on HCU devices. A total of 251 HCUs were found to be contaminated by MC; genotypes 1.1 or 1.8 were identified in 28 of those HCUs. CONCLUSIONS: To ensure patients' safety and adequate follow-up, clinicians and general practitioners were made aware of the results and public health measures, and recommendations were issued to prevent further cases in the healthcare settings. The Italian Society of Cardiac Surgery performed a national survey to assess the incidence of HCU-related MC prosthetic infections in cardiac surgery. No cases were reported after HCU replacement or structural modification and disinfection and possibly safe allocation outside surgical rooms.

Treatment-Emergent Cilgavimab Resistance Was Uncommon in Vaccinated Omicron BA.4/5 Outpatients.

Mutations in the SARS-CoV-2 Spike glycoprotein can affect monoclonal antibody efficacy. Recent findings report the occurrence of resistant mutations in immunocompromised patients after tixagevimab/cilgavimab treatment. More recently, the Food and Drug Agency revoked the authorization for tixagevimab/cilgavimab, while this monoclonal antibody cocktail is currently recommended by the European Medical Agency. We retrospectively reviewed 22 immunocompetent patients at high risk for disease progression who received intramuscular tixagevimab/cilgavimab as early COVID-19 treatment and presented a prolonged high viral load. Complete SARS-CoV-2 genome sequences were obtained for a deep investigation of mutation frequencies in Spike protein before and during treatment. At seven days, only one patient showed evidence of treatment-emergent cilgavimab resistance. Quasispecies analysis revealed two different deletions on the Spike protein (S:del138-144 or S:del141-145) in combination with the resistance S:K444N mutation. The structural and dynamic impact of the two quasispecies was characterized by using molecular dynamics simulations, showing the conservation of the principal functional movements in the mutated systems and their capabilities to alter the structure and dynamics of the RBD, responsible for the interaction with the ACE2 human receptor. Our study underlines the importance of prompting an early virological investigation to prevent drug resistance or clinical failures in immunocompetent patients.

Molecular Characterization of Whole-Genome SARS-CoV-2 from the First Suspected Cases of the XE Variant in the Lazio Region, Italy.

We report two cases of SARS-CoV-2 recombinant variant XE detected in nasopharyngeal swabs (NPS) of hospitalized patients with no evident epidemiological link in Lazio, Central Italy. Whole-Genome Sequencing (WGS) performed on an Ion Torrent GSS5 platform according to Italian flash surveys showed genomes corresponding to the PANGOLIN unclassified lineage and the Nextclade XE clade. Further analyses were then carried out to investigate more deeply the genetic characteristics of these XE-like sequences. When phylogenetic trees, by using IQ-TREE, were built splitting the genome into two regions according to the putative XE recombination site, the upstream and downstream regions were seen to be clustered near BA.1 and BA.2 sequences, respectively. However, our XE-like sequences clustered separately, with a significant bootstrap, from the classified European and Italian XE strains, although the recombination site between BA.1 and BA.2 was identified at the nucleotide site 11556 by RDP4 software, consistent with the putative XE breakpoint. These findings show the risk of the introduction of novel recombinant variants of SARS-CoV-2 and the existence of XE-like strains, phylogenetically separated, that could make their exact taxonomy difficult. It follows the need for continued SARS-CoV-2 surveillance by WGS.

Importance of Surveillance of New Delhi Metallo-Beta-Lactamase Klebsiella pneumoniae: Molecular Characterization and Clonality of Strains Isolated in the Lazio Region, Italy.

INTRODUCTION: New Delhi metallo-ß-lactamase producing Klebsiella pneumoniae (NDM-Kpn) strains have been causing healthcare-associated infections worldwide. The aim of this study was to describe the molecular mechanisms of antimicrobial resistance and to analyze the clonality of NDM-Kpn isolates collected between January 2019 and June 2020 from patients admitted to hospitals from the Lazio region, Italy. METHODS: We performed a retrospective cohort study. Whole-genome sequencing (WGS) was performed on all NDM-Kpn strains; clonality and genetic relationships were further investigated. RESULTS: During the surveillance period, 17 NDM-Kpn isolates were obtained from 17 patients admitted to seven different hospitals. Eight different sequence types (STs) were detected: ST147 (n = 4), ST383 (n = 4), ST15 (n = 3), ST11 (n = 2), ST17 (n = 1), ST29 (n = 1), ST307 (n = 1) and the newly identified ST4853 (n = 1). Genetic relationships were further investigated by the WGS-based core genome MLST (cgMLST) scheme, and 5 cluster types (CTs) were identified. Whereas a substantial overall heterogeneity among isolates was detected (8 different STs were identified out of 17 isolates), the strains within each cluster showed a very high level of genome similarity. DISCUSSION: Our study highlights the key role of surveillance, which allowed taking a picture of a part of the NDM-Kpn strains circulating in Italy, adding further insight into their molecular features.

Molecular analysis of clinical isolates of ceftazidime-avibactam-resistant Klebsiella pneumoniae.

OBJECTIVES: To analyse the strains collected during a 1-year survey of ceftazidime-avibactam-resistant KPC-producing Klebsiella pneumoniae, in order to investigate the molecular mechanisms potentially responsible for their resistant phenotype. METHODS: Clinical KPC-producing K. pneumoniae isolates were collected from 31 patients in six different hospitals in Rome. For eight of the patients, an additional strain grown before the start of treatment was also available, bringing the total of isolates studied to 39. Antimicrobial susceptibility was determined by automated system, broth microdiluition and E-test as appropriate. In silico analysis of acquired resistance genes was achieved by whole-genome sequencing, while multilocus sequence typing and core genome multilocus sequence typing were employed for molecular typing. Mutations associated with ceftazidime-avibactam resistance were identified by Sanger sequencing of the blaKPC gene. Possible mutations in OmpK35 and OmpK36 outer membrane proteins were also investigated. RESULTS: Molecular analyses highlighted the circulation of the ST512, 101 and 307 high-risk clones; 26 of the 31 patients carried a mutated KPC variant, five had a wild-type KPC-3. Among the KPC variants detected, 11 were different mutations within the blaKPC-3 gene, four of which were novel mutational changes. CONCLUSIONS: Different mutations including single amino-acid substitutions, insertions or deletions within the blaKPC gene were found in 26/31 ceftazidime-avibactam-resistant KPC-producing K. pneumoniae strains belonging to high-risk clones circulating in Italy. Of note, in 14/31 cases the isolates displayed resistance to both ceftazidime-avibactam and carbapenems, raising concerns for the possible selection of a multidrug-resistant phenotype.

Multicenter analysis of sputum microbiota in tuberculosis patients.

The impact of tuberculosis and of anti-tuberculosis therapy on composition and modification of human lung microbiota has been the object of several investigations. However, no clear outcome has been presented so far and the relationship between M. tuberculosis pulmonary infection and the resident lung microbiota remains vague. In this work we describe the results obtained from a multicenter study of the microbiota of sputum samples from patients with tuberculosis or unrelated lung diseases and healthy donors recruited in Switzerland, Italy and Bangladesh, with the ultimate goal of discovering a microbiota-based biomarker associated with tuberculosis. Bacterial 16S rDNA amplification, high-throughput sequencing and extensive bioinformatic analyses revealed patient-specific flora and high variability in taxon abundance. No common signature could be identified among the individuals enrolled except for minor differences which were not consistent among the different geographical settings. Moreover, anti-tuberculosis therapy did not cause any important variation in microbiota diversity, thus precluding its exploitation as a biomarker for the follow up of tuberculosis patients undergoing treatment.

New tools for detecting latent tuberculosis infection: evaluation of RD1-specific long-term response.

BACKGROUND: Interferon-gamma (IFN-gamma) release assays (IGRAs) were designed to detect latent tuberculosis infection (LTBI). However, discrepancies were found between the tuberculin skin test (TST) and IGRAs results that cannot be attributed to prior Bacille Calmètte Guerin vaccinations. The aim of this study was to evaluate tools for improving LTBI diagnosis by analyzing the IFN-gamma response to RD1 proteins in prolonged (long-term response) whole blood tests in those subjects resulting negative to assays such as QuantiFERON-TB Gold In tube (QFT-IT). METHODS: The study population included 106 healthy TST+ individuals with suspected LTBI (recent contact of smear-positive TB and homeless) consecutively enrolled. As controls, 13 healthy subjects unexposed to M. tuberculosis (TST-, QFT-IT-) and 29 subjects with cured pulmonary TB were enrolled. IFN-gamma whole blood response to RD1 proteins and QFT-IT were evaluated at day 1 post-culture. A prolonged test evaluating long-term IFN-gamma response (7-day) to RD1 proteins in diluted whole blood was performed. RESULTS: Among the enrolled TST+ subjects with suspected LTBI, 70/106 (66.0%) responded to QFT-IT and 64/106 (60.3%) to RD1 proteins at day 1. To evaluate whether a prolonged test could improve the detection of LTBI, we set up the test using cured TB patients (with a microbiologically diagnosed past pulmonary disease) who resulted QFT-IT-negative and healthy controls as comparator groups. Using this assay, a statistically significant difference was found between IFN-gamma levels in cured TB patients compared to healthy controls (p < 0.006). Based on these data, we constructed a receiver operating characteristic (ROC) curve and we calculated a cut-off. Based on the cut-off value, we found that among the 36 enrolled TST+ subjects with suspected LTBI not responding to QFT-IT, a long term response to RD1 proteins was detected in 11 subjects (30.6%). CONCLUSION: These results indicate that IFN-gamma long-term response to M. tuberculosis RD1 antigens may be used to detect past infection with M. tuberculosis and may help to identify additional individuals with LTBI who resulted negative in the short-term tests. These data may provide useful information for improving immunodiagnostic tests for tuberculosis infection, especially in individuals at high risk for active TB.

Isoniazid prophylaxis differently modulates T-cell responses to RD1-epitopes in contacts recently exposed to Mycobacterium tuberculosis: a pilot study.

RATIONALE: Existing data on the effect of treatment of latent tuberculosis infection (LTBI) on T-cell responses to Mycobacterium tuberculosis (MTB)-specific antigens are contradictory. Differences in technical aspects of the assays used to detect this response and populations studied might explain some of these discrepancies. In an attempt to find surrogate markers of the effect of LTBI treatment, it would be important to determine whether, among contacts of patients with contagious tuberculosis, therapy for LTBI could cause changes in MTB-specific immune responses to a variety of RD1-antigens. METHODS AND RESULTS: In a longitudinal study, 44 tuberculin skin test+ recent contacts were followed over a 6-month period and divided according to previous exposure to MTB and LTBI treatment. The following tests which evaluate IFN-gamma responses to RD1 antigens were performed: QuantiFERON TB Gold, RD1 intact protein- and selected peptide-based assays. Among the 24 contacts without previous exposure that completed therapy, we showed a significant decrease of IFN-gamma response in all tests employed. The response to RD1 selected peptides was found to be more markedly decreased compared to that to other RD1 antigens. Conversely, no significant changes in the response to RD1 reagents were found in 9 treated subjects with a known previous exposure to MTB and in 11 untreated controls. CONCLUSION: These data suggest that the effect of INH prophylaxis on RD1-specific T-cell responses may be different based on the population of subjects enrolled (recent infection versus re-infection) and, to a minor extent, on the reagents used.

Thymidine kinase and deoxycytidine kinase activity in mononuclear cells from antiretroviral-naive HIV-infected patients.

OBJECTIVE: To evaluate whether an inter-individual variability in the activity of thymidine kinase (TK) and deoxycytidine kinase (dCK), which are involved in the first step of phosphorylation of some nucleoside analogues, exists in antiretroviral-naive, HIV-seropositive patients. DESIGN: Forty-five randomly selected antiretroviral-naive HIV-infected patients were recruited, together with 26 healthy volunteers with no concurrent infection and under no pharmacological treatment. METHODS: Peripheral blood mononuclear cells (PBMC) were isolated from venous blood and their TK and dCK activities evaluated. CD4 T cells and HIV-RNA were measured in HIV-infected patients, too. RESULTS: There was a broad range of variability in TK activity in HIV-infected individuals. Furthermore, the activity in PBMC was significantly higher in HIV-infected individuals than in healthy volunteers. dCK activity in seropositive patients was significantly lower than in healthy volunteers. A marked inter-individual variability in dCK levels was observed in the HIV-infected group. No correlations were found between TK or dCK activities and plasma viral load, CD4 cell count, sex or age of patients. CONCLUSIONS: A marked range of inter-individual variability of TK and dCK activities in PBMC exists in HIV-infected individuals but not in healthy volunteers, indicating that the activity of enzymes with key roles in drug activation could vary greatly from one patient to another. Furthermore, TK expression is greater in HIV-infected individuals than in healthy volunteers. Better understanding of the viral or cellular factors that contribute to this variability, as well as their effect on responses to antiretroviral treatment, may aid optimization of the management of HIV-infected patients.

A multicentre evaluation of the accuracy and performance of IP-10 for the diagnosis of infection with M. tuberculosis.

IP-10 has potential as a diagnostic marker for infection with Mycobacterium tuberculosis, with comparable accuracy to QuantiFERON-TB Gold In-Tube test (QFT-IT). The aims were to assess the sensitivity and specificity of IP-10, and to evaluate the impact of co-morbidity on IP-10 and QFT-IT. 168 cases with active TB, 101 healthy controls and 175 non-TB patients were included. IP-10 and IFN-γ were measured in plasma of QFT-IT stimulated whole blood and analyzed using previously determined algorithms. A subgroup of 48 patients and 70 healthy controls was tested in parallel with T-SPOT.TB IP-10 and QFT-IT had comparable accuracy. Sensitivity was 81% and 84% with a specificity of 97% and 100%, respectively. Combining IP-10 and QFT-IT improved sensitivity to 87% (p < 0.0005), with a specificity of 97%. T-SPOT.TB was more sensitive than QFT-IT, but not IP-10. Among non-TB patients IP-10 had a higher rate of positive responders (35% vs 27%, p < 0.02) and for both tests a positive response was associated with relevant risk factors. IFN-γ but not IP-10 responses to mitogen stimulation were reduced in patients with TB and non-TB infection. This study confirms and validates previous findings and adds substance to IP-10 as a novel diagnostic marker for infection with M. tuberculosis. IP-10 appeared less influenced by infections other than TB; further studies are needed to test the clinical impact of these findings.

Methylated HBHA produced in M. smegmatis discriminates between active and non-active tuberculosis disease among RD1-responders.

BACKGROUND: A challenge in tuberculosis (TB) research is to develop a new immunological test that can help distinguish, among subjects responsive to QuantiFERON TB Gold In tube (QFT-IT), those who are able to control Mtb replication (remote LTBI, recent infection and past TB) from those who cannot (active TB disease). IFN-γ response to the Heparin-binding-hemagglutinin (HBHA) of Mtb has been associated with LTBI, but the cumbersome procedures of purifying the methylated and immunological active form of the protein from Mtb or M. bovis Bacillus Calmette et Guerin (BCG) have prevented its implementation in a diagnostic test. Therefore, the aim of the present study was to evaluate the IFN-γ response to methylated HBHA of Mtb produced in M. smegmatis (rHBHAms) in individuals at different stages of TB who scored positive to QFT-IT. METHODOLOGY/PRINCIPAL FINDINGS: 87 individuals at different stages of TB who scored positive to QFT-IT were selected. IFN-γ response to in vitro whole blood stimulation with rHBHAms was evaluated by short-term and long-term tests and detected by ELISA or flow cytometry. We demonstrated that the IFN-γ response to rHBHAms is mediated by CD4(+) T-cells with an effector-memory phenotype. This response, evaluated by short-term-tests, is significantly lower in active TB than in remote LTBI (p = 0.0010) and past TB (p = 0.0152). These results were confirmed by long-term tests. The qualitative data confirmed that IFN-γ responses higher than the cut-off point identified by ROC analysis are associated with the status of non-active disease. CONCLUSIONS: In this study we show that the T-cell response to a recombinant and methylated HBHA of Mtb produced in M. smegmatis is useful to discriminate between active and non-active TB disease among those responsive to QFT-IT in a whole blood system. Further studies are needed to improve the accuracy of the assay.