A gain-of-function mutation in zinc cluster transcription factor Rob1 drives Candida albicans adaptive growth in the cystic fibrosis lung environment.

Candida albicans chronically colonizes the respiratory tract of patients with Cystic Fibrosis (CF). It competes with CF-associated pathogens (e.g. Pseudomonas aeruginosa) and contributes to disease severity. We hypothesize that C. albicans undergoes specific adaptation mechanisms that explain its persistence in the CF lung environment. To identify the underlying genetic and phenotypic determinants, we serially recovered 146 C. albicans clinical isolates over a period of 30 months from the sputum of 25 antifungal-naive CF patients. Multilocus sequence typing analyses revealed that most patients were individually colonized with genetically close strains, facilitating comparative analyses between serial isolates. We strikingly observed differential ability to filament and form monospecies and dual-species biofilms with P. aeruginosa among 18 serial isolates sharing the same diploid sequence type, recovered within one year from a pediatric patient. Whole genome sequencing revealed that their genomes were highly heterozygous and similar to each other, displaying a highly clonal subpopulation structure. Data mining identified 34 non-synonymous heterozygous SNPs in 19 open reading frames differentiating the hyperfilamentous and strong biofilm-former strains from the remaining isolates. Among these, we detected a glycine-to-glutamate substitution at position 299 (G299E) in the deduced amino acid sequence of the zinc cluster transcription factor ROB1 (ROB1G299E), encoding a major regulator of filamentous growth and biofilm formation. Introduction of the G299E heterozygous mutation in a co-isolated weak biofilm-former CF strain was sufficient to confer hyperfilamentous growth, increased expression of hyphal-specific genes, increased monospecies biofilm formation and increased survival in dual-species biofilms formed with P. aeruginosa, indicating that ROB1G299E is a gain-of-function mutation. Disruption of ROB1 in a hyperfilamentous isolate carrying the ROB1G299E allele abolished hyperfilamentation and biofilm formation. Our study links a single heterozygous mutation to the ability of C. albicans to better survive during the interaction with other CF-associated microbes and illuminates how adaptive traits emerge in microbial pathogens to persistently colonize and/or infect the CF-patient airways.

On the onset and dispersal of a major MDR TB clone among HIV-negative patients, Tunisia.

BACKGROUND: To carry out a whole genome sequencing (WGS)-based investigation on the emergence and spread of the largest multidrug-resistant tuberculosis (MDR TB) outbreak that has been thriving among HIV-negative patients, Tunisia, since the early 2000s. METHODS: We performed phylogeographic analyses and molecular dating based on a WGS dataset representing 68 unique Mycobacterium tuberculosis isolates, covering almost the entire MDR TB outbreak for the time period 2001-2016. RESULTS: The data indicate that the ancestor of the MDR TB outbreak emerged in the region of Bizerte, as early as 1974 (95% CI 1951-1985), from where it spread to other regions by 1992 (95% CI 1980-1996). Analysis of a minimum spanning tree based on core genome Multilocus Sequence Typing (cgMLST) uncovered the early spill-over of the fitness-compensated MDR TB strain from the prison into the general population. Indeed, cases with history of incarceration were found to be directly or indirectly linked to up to 22 new outbreak cases (32.35%) among the non-imprisoned population. By around 2008, the MDR TB outbreak strain had acquired additional resistance, leading to an XDR phenotype. CONCLUSIONS: WGS allowed refining our understanding of the emergence and evolution of the largest MDR TB outbreak in Tunisia, whose causative strain has been circulating silently for almost 26 years before. Our study lends further support to the critical role of prisons-related cases in the early spread of the outbreak among the general population. The shift to an XDR phenotype of such an epidemic clone prompts an urgent need to undertake drastic control measures.

HBHA-IGRA and cytotoxic mediators release assays for the diagnosis of cervical tuberculous lymphadenitis.

IMPORTANCE: Cervical tuberculous lymphadenitis (CTL), the most frequent extrapulmonary form of tuberculosis, is currently a major health problem in Tunisia and in several regions around the world. CTL diagnosis is challenging mainly due to the paucibacillary nature of the disease and the potential misdiagnosis as cervical non-tuberculous lymphadenitis. This study demonstrates the added value of the heparin-binding hemagglutinin-interferon-gamma release assay as an immunoassay in the context of CTL.

PHINDaccess Hackathons for COVID-19 and Host-Pathogen Interaction: Lessons Learned and Recommendations for Low- and Middle-Income Countries.

Hackathons are collaborative events that bring together diverse groups to solve predefined challenges. The COVID-19 pandemic caused by SARS-CoV-2 has emphasized the need for portable and reproducible genomics analysis pipelines to study the genetic susceptibility of the human host and investigate human-SARS-CoV-2 protein interactions. To build and strengthen institutional capacities in OMICS data analysis applied to host-pathogen interaction (HPI), the PHINDaccess project organized two hackathons in 2020 and 2021. These hackathons are aimed at developing bioinformatics pipelines related to the SARS-CoV-2 viral genome, its phylodynamic transmission, and the identification of human genome host variants, with a focus on addressing global health challenges, particularly in low- and middle-income countries (LMIC). This paper outlines the preparation, proceedings, and lessons learned from these hackathons, including the challenges faced by participants and our recommendations based on our experience for organizing hackathons in LMIC and beyond.

Escherichia coli Nissle 1917 Antagonizes Candida albicans Growth and Protects Intestinal Cells from C. albicans-Mediated Damage.

Candida albicans is a pathobiont of the gastrointestinal tract. It can contribute to the diversity of the gut microbiome without causing harmful effects. When the immune system is compromised, C. albicans can damage intestinal cells and cause invasive disease. We hypothesize that a therapeutic approach against C. albicans infections can rely on the antimicrobial properties of probiotic bacteria. We investigated the impact of the probiotic strain Escherichia coli Nissle 1917 (EcN) on C. albicans growth and its ability to cause damage to intestinal cells. In co-culture kinetic assays, C. albicans abundance gradually decreased over time compared with C. albicans abundance in the absence of EcN. Quantification of C. albicans survival suggests that EcN exerts a fungicidal activity. Cell-free supernatants (CFS) collected from C. albicans-EcN co-culture mildly altered C. albicans growth, suggesting the involvement of an EcN-released compound. Using a model of co-culture in the presence of human intestinal epithelial cells, we further show that EcN prevents C. albicans from damaging enterocytes both distantly and through direct contact. Consistently, both C. albicans's filamentous growth and microcolony formation were altered by EcN. Taken together, our study proposes that probiotic-strain EcN can be exploited for future therapeutic approaches against C. albicans infections.

Genomic changes underpinning the emergence of a successful Mycobacterium tuberculosis Latin American and Mediterranean clonal complex.

Introduction: The Latin American and Mediterranean sublineage (L4.3/LAM) is the most common generalist sublineage of Mycobacterium tuberculosis lineage 4 (L4), yet certain L4.3/LAM genotypes appear to be confined to particular geographic regions. This is typically the case of a L4.3/LAM clonal complex (CC), TUN4.3_CC1, which is the most preponderant in Tunisia (61.5% of L4.3/LAM). Methods: Here, we used whole-genome sequencing data of 346 globally distributed L4 clinical strains, including 278 L4.3/LAM isolates, to reconstruct the evolutionary history of TUN4.3_CC1 and delineate critical genomic changes underpinning its success. Results and Discussion: Phylogenomic coupled to phylogeographic analyses indicated that TUN4.3_CC1 has evolved locally, being confined mainly to North Africa. Maximum likelihood analyses using the site and branch-site models of the PAML package disclosed strong evidence of positive selection in the gene category "cell wall and cell processes" of TUN4.3_CC1. Collectively, the data indicate that TUN4.3_CC1 has inherited several mutations, which could have potentially contributed to its evolutionary success. Of particular interest are amino acid replacements at the esxK and eccC2 genes of the ESX/Type VII secretion system, which were found to be specific to TUN4.3_CC1, being common to almost all isolates. Because of its homoplastic nature, the esxK mutation could potentially have endowed TUN4.3_CC1 with a selective advantage. Moreover, we noticed the occurrence of additional, previously described homoplasic nonsense mutations in ponA1 and Rv0197. The mutation in the latter gene, a putative oxido-reductase, has previously been shown to be correlated with enhanced transmissibility in vivo. In sum, our findings unveiled several features underpinning the success of a locally evolved L4.3/LAM clonal complex, lending further support to the critical role of genes encoded by the ESX/type VII secretion system.

Evolutionary history and spread of the Mycobacterium tuberculosis Latin American and Mediterranean (L4.3/LAM) sublineage, Tunisia.

BACKGROUND: To infer the origin and spread of the Mycobacterium tuberculosis Latin American and Mediterranean (L4.3/LAM) sublineage in a Mediterranean country, Tunisia, where it predominates. METHODS: We combined Bayesian (STRUCTURE) and maximum likelihood (MIGRAINE) estimation approaches based on a global 24-loci mycobacterial interspersed repetitive units-variable numbers of tandem repeats (MIRU-VNTR24) genotyping dataset consisting of 1573 L4.3/LAM clinical strains from four continents, including 252 isolates originating from Tunisia. RESULTS: Phylogenetic analyses coupled with Bayesian estimations suggested that the most predominant L4.3/LAM subpopulation in Tunisia (65.07%), which is dominated by a single clonal complex, TUN4.3_CC1 (94.51%), has evolved from an ancestral pool that is restricted to Europe and Africa, contrasting with the remaining L4.3/LAM subpopulations whose ancestry was traced all over the word. Maximum likelihood analysis revealed that TUN4.3_CC1 has been undergoing a demographic expansion since 131 years ago (CI95%: 90.7-205), thus explaining its preponderance relative to the second most predominant CC, TUN4.3_CC2, whose population was found under contraction. CONCLUSIONS: The preponderance of L4.3/LAM in Tunisia stems from a 130-year expansion process of a locally evolved clone.

Phenotypic and genomic hallmarks of a novel, potentially pathogenic rapidly growing Mycobacterium species related to the Mycobacterium fortuitum complex.

Previously, we have identified a putative novel rapidly growing Mycobacterium species, referred to as TNTM28, recovered from the sputum of an apparently immunocompetent young man with an underlying pulmonary disease. Here we provide a thorough characterization of TNTM28 genome sequence, which consists of one chromosome of 5,526,191 bp with a 67.3% G + C content, and a total of 5193 predicted coding sequences. Phylogenomic analyses revealed a deep-rooting relationship to the Mycobacterium fortuitum complex, thus suggesting a new taxonomic entity. TNTM28 was predicted to be a human pathogen with a probability of 0.804, reflecting the identification of several virulence factors, including export systems (Sec, Tat, and ESX), a nearly complete set of Mce proteins, toxin-antitoxins systems, and an extended range of other genes involved in intramacrophage replication and persistence (hspX, ahpC, sodA, sodC, katG, mgtC, ClpR, virS, etc.), some of which had likely been acquired through horizontal gene transfer. Such an arsenal of potential virulence factors, along with an almost intact ESX-1 locus, might have significantly contributed to TNTM28 pathogenicity, as witnessed by its ability to replicate efficiently in macrophages. Overall, the identification of this new species as a potential human pathogen will help to broaden our understanding of mycobacterial pathogenesis.

High Frequency of Enterocytozoon bieneusi Genotype WL12 Occurrence among Immunocompromised Patients with Intestinal Microsporidiosis.

Microsporidiosis is an emerging opportunistic infection causing severe digestive disorders in immunocompromised patients. The aim of this study was to investigate the prevalence of intestinal microsporidia carriage among immunocompromised patients hospitalized at a major hospital complex in the Tunis capital area, Tunisia (North Africa), and perform molecular epidemiology and population structure analyses of Enterocytozoon bieneusi, which is an emerging fungal pathogen. We screened 250 stool samples for the presence of intestinal microsporidia from 171 patients, including 81 organ transplant recipients, 73 Human Immunodeficiency Virus (HIV)-positive patients, and 17 patients with unspecified immunodeficiency. Using a nested PCR-based diagnostic approach for the detection of E. bieneusi and Encephalitozoon spp., we identified 18 microsporidia-positive patients out of 171 (10.5%), among which 17 were infected with E. bieneusi. Microsporidia-positive cases displayed chronic diarrhea (17 out of 18), which was associated more with HIV rather than with immunosuppression other than HIV (12 out of 73 versus 6 out of 98, respectively, p = 0.02) and correlated with extended hospital stays compared to microsporidia-negative cases (60 versus 19 days on average, respectively; p = 0.001). Strikingly, internal transcribed spacer (ITS)-based genotyping of E. bieneusi strains revealed high-frequency occurrence of ITS sequences that were identical (n = 10) or similar (with one single polymorphic site, n = 3) to rare genotype WL12. Minimum-spanning tree analyses segregated the 17 E. bieneusi infection cases into four distinct genotypic clusters and confirmed the high prevalence of genotype WL12 in our patient population. Phylogenetic analyses allowed the mapping of all 17 E. bieneusi strains to zoonotic group 1 (subgroups 1a and 1b/1c), indicating loose host specificity and raising public health concern. Our study suggests a probable common source of E. bieneusi genotype WL12 transmission and prompts the implementation of a wider epidemiological investigation.

Successful expansion of Mycobacterium tuberculosis Latin American and Mediterranean sublineage (L4.3/LAM) in Tunisia mainly driven by a single, long-established clonal complex.

OBJECTIVES: To explore the evolutionary history of Mycobacterium tuberculosis Latin American and Mediterranean (L4.3/LAM) sublineage in Tunisia, where it predominates. METHODS: High-resolution genotyping of 252 L4.3/LAM clinical strains was undertaken, and whole-genome sequencing was performed on 31 representative isolates. RESULTS: Genotyping data coupled with Bayesian analyses split the Tunisian L4.3/LAM strain collection into two divergent entities (65.07% vs 34.92%): a major subpopulation, dominated by a single clonal complex (CC), TUN4.3_CC1 (94.51%); and a minor subpopulation, dominated by TUN4.3_CC2 (42.04%). TUN4.3_CC1 is clearly thriving in Tunisia, accounting for 61.5% of the L4.3/LAM sublineage. TUN4.3_CC1 displayed higher mean allelic richness compared with TUN4.3_CC2 and predominated throughout the entire region, indicating a long-established history. The very low proportion of drug resistance among TUN4.3_CC1 isolates is indicative of their intrinsic ability to spread successfully in the host population. Genomic analyses further confirmed the clear genetic separation between the two main CCs (pairwise fixation index 0.56), and suggested the relatively ancient origin of TUN4.3_CC1. Consistent with its successful expansion, TUN4.3_CC1 showed reduced mean pairwise genetic distance between genomes. CONCLUSIONS: These findings link the successful expansion of L4.3/LAM in Tunisia to a single long-established clone.

Performance of GeneXpert ultra in the diagnosis of Tuberculous Cervical lymphadenitis in formalin fixed paraffin embedded tissues.

The diagnosis of Tuberculous Cervical lymphadenitis (TCL) is challenging. The present study aimed to assess the performance of GeneXpert ultra (GXu) in the diagnosis of TCL on Formalin Fixed, Paraffin Embedded Tissues (FFPET). This study included 35 TCL cases confirmed by positive microbiology and/or positive GXu on Fresh Tissues (FT). The diagnostic performance parameters of GXu on FFPET were determined with reference to microbiology (positive Ziehl Neelsen and/or positive culture) and with reference to positive microbiology and/or positive GXu on FT. The GXu on FFPET was positive in 26/35 (74%) cases. With reference to positive ZN and or culture, the sensitivity, specificity, positive predictive value, and negative predictive value of GXu on FFPET were 63%, 100%, 100% and 71% respectively. With reference to positive microbiology and/or positive GXu on FT, these rates were 74%, 100%, 100% and 40% respectively. GXu on FFPET is a reliable tool for the detection of Mycobacterium tuberculosis complex particularly for cases where microbiological investigations have not been performed.

Clonal Spread of Tetracycline Resistance Among Mycoplasma hominis Clinical Strains, Tunisia.

Antimicrobial resistance in a number of bacterial pathogens has been shown to spread clonally. To our knowledge, data about the phylodistribution of drug resistance in Mycoplasma hominis are very scarce. The aims of this study were to assess the antimicrobial susceptibility of Mycoplasma hominis clinical strains in Tunisia, to identify the molecular basis of antibiotic resistance, and to investigate the phylogenetic relationships of resistant strains. This study included 65 molecularly typed Mycoplasma hominis clinical strains recovered from Tunisian patients over 18 years (2000-2018). The antimicrobial susceptibility was tested against nine antibacterial agents using the broth microdilution method. Minimum spanning tree was constructed to establish the phylogenetic relationships among resistant isolates. Fluoroquinolones, doxycycline, and josamycine were found to be the most effective antibacterial agents. However, 22 strains belonging to 11 expanded multilocus sequence types (eSTs) proved resistant to tetracycline. The majority of these eSTs were genetically related, indicative of clonal expansion of tetracycline resistance. The present study provides relevant information on the antibiotic susceptibility of Tunisian M. hominis clinical strains, lending support to a clonal transmission of tetracycline resistance. This is likely to have an important implication in monitoring the spread of drug resistance among M. hominis.

Spread of multidrug resistance among Ureaplasma serovars, Tunisia.

Background: Ureaplasma spp. have been implicated in a variety of clinical conditions and certain serovars are likely to be disease-associated. Hence, the ascending trend of Ureaplasma spp. resistance to antimicrobials should deserve more attention. Here we assessed the extent of antimicrobial resistance of Ureaplasma serovars in Tunisia, and investigated the underlying molecular basis. Methods: This study included 101 molecularly typed Ureaplasma spp. clinical strains isolated over a 12-year time period (2005-2017). The antimicrobial susceptibility was tested against nine antibacterial agents using the broth microdilution method. Neighbor-joining tree was constructed to establish the phylogenetic relationships among isolates. Results: We found that all ureaplasma isolates were resistant to ciprofloxacin and erythromycin, intermediately resistant to azithromycin, and susceptible to doxycycline, moxifloxacin and josamycin. Ofloxacin and levofloxacin resistance was found in 73.27 and 17.82%, respectively, while 37.62% of isolates proved resistant to tetracycline. Consequently, we detected an elevated multidrug resistance rate among ureaplasma isolates (37.62%), particularly among serovars 2, 5, 8, and 9 (77.77% overall), as well as serovars 4, 10, 12, and 13 (52.63% overall). In most cases, drug resistance was found to be associated with known molecular mechanisms, yet we have identified two novel mutations in the L22 protein, which might be associated with macrolide-resistance. Conclusion: To our knowledge, this is the first study that reports the widespread expansion of multidrug resistance among Ureaplasma serovars, a finding of importance in terms of both surveillance and antimicrobial usage.

Nontuberculous mycobacteria isolated from specimens of pulmonary tuberculosis suspects, Northern Tunisia: 2002-2016.

BACKGROUND: Reports on the worldwide ascending trend of pulmonary nontuberculous mycobacteria (NTM) isolation rates and their effective role in respiratory tract infections are compelling. However, as yet, there are no such data relating to Tunisia. METHODS: Here we carried out a retrospective review of mycobacterial cultures originating from Northern Tunisia, which have been processed in the laboratory of mycobacteria of the Institut Pasteur de Tunis, during the time period 2002-2016. All pulmonary NTM (PNTM) isolates available for culture were characterized phenotypically and their taxonomic status was further established based on polymorphisms in rpoB, 16S rRNA, hsp65, and sodA DNA gene sequences. RESULTS: Of the 10,466 specimens collected from HIV-negative Tunisian patients with presumptive clinical pulmonary TB, 60 (0.6%) yielded PNTM isolates. An overall annual PNTM isolation prevalence of 0.2/100,000 was estimated. As far as could be ascertained, this isolation rate accounts amongst the lowest reported hitherto throughout the world. Among the 30 NTM isolates that were available for culture, 27 (90.0%) have been identified to the species level. The most commonly encountered species was Mycobacterium kansasii (23.3%) subtype 1. Strikingly, all M. kansasii cases were male patients originating from Bizerte, an industrialized region particularly known for iron industry. The remaining NTM species were M. fortuitum (16.6%), M. novocastrense (16.6%), M. chelonae (10.0%), M. gordonae (6.6%), M. gadium (6.6%), M. peregrinum (3.3%), M. porcinum (3.3%), and M. flavescens (3.3%). There were no bacteria of the M. avium complex, the most frequently isolated NTM globally, and the main driver of the rise of NTM-lung diseases. CONCLUSIONS: This study uncovered an exceptional low prevalence of PNTM isolation among HIV-negative TB suspects in Northern Tunisia, suggesting a very low burden of NTM pulmonary disease. However, the frequent isolation of M. kansasii subtype 1, the most pathogenic subtype, particularly from the industrialized region of Bizerte, strongly suggests its effective involvement in a typical pulmonary disease.

Phylogenetics of Mycoplasma hominis clinical strains associated with gynecological infections or infertility as disclosed by an expanded multilocus sequence typing scheme.

To our knowledge, the phylodistribution of M. hominis clinical strains associated with various pathological conditions of the urogenital tract has not been explored hitherto. Here we analyzed the genetic diversity and phylogenetic relationships among 59 M. hominis Tunisian clinical isolates, categorized as gynecological infections- or infertility-associated pathotypes. For this purpose, we developed an expanded multilocus sequence typing (eMLST) scheme, combining the previously reported multilocus sequence typing (MLST) loci (gyrB, tuf, ftsY, uvrA, gap) with a new selected set of putative virulence genes (p120', vaa, lmp1, lmp3, p60), referred herein to as multi-virulence-locus sequence typing (MVLST) loci. In doing so, M. hominis population was segregated into two distinct genetic lineages, which were differentially associated with each pathotype. Such a clear dichotomy was supported by several phylogenetic and population genetic analysis tools. Recombination was found to take place, but not sufficient enough to break down the overall clonal population structure of M. hominis, most likely as a result of purifying selection, which accommodated the most fit clones. In sum, and owing to the eMLST scheme described herein, we provide insightful data on the phylogenetics of M. hominis, arguing for the existence of genetically differentiable urogenital pathotypes.

Automated IS6110-based fingerprinting of Mycobacterium tuberculosis: Reaching unprecedented discriminatory power and versatility.

BACKGROUND: Several technical hurdles and limitations have restricted the use of IS6110 restriction fragment length polymorphism (IS6110 RFLP), the most effective typing method for detecting recent tuberculosis (TB) transmission events. This has prompted us to conceive an alternative modality, IS6110-5'3'FP, a plasmid-based cloning approach coupled to a single PCR amplification of differentially labeled 5' and 3' IS6110 polymorphic ends and their automated fractionation on a capillary sequencer. The potential of IS6110-5'3'FP to be used as an alternative to IS6110 RFLP has been previously demonstrated, yet further technical improvements are still required for optimal discriminatory power and versatility. OBJECTIVES: Here we introduced critical amendments to the original IS6110-5'3'FP protocol and compared its performance to that of 24-loci multiple interspersed repetitive unit-variable number tandem repeats (MIRU-VNTR), the current standard method for TB transmission analyses. METHODS: IS6110-5'3'FP protocol modifications involved: (i) the generation of smaller-sized polymorphic fragments for efficient cloning and PCR amplification, (ii) omission of the plasmid amplification step in E. coli for shorter turnaround times, (iii) the use of more stable fluorophores for increased sensitivity, (iv) automated subtraction of background fluorescent signals, and (v) the automated conversion of fluorescent peaks into binary data. RESULTS: In doing so, the overall turnaround time of IS6110-5'3'FP was reduced to 4 hours. The new protocol allowed detecting almost all 5' and 3' IS6110 polymorphic fragments of any given strain, including IS6110 high-copy number Beijing strains. IS6110-5'3'FP proved much more discriminative than 24-loci MIRU-VNTR, particularly with strains of the M. tuberculosis lineage 4. CONCLUSIONS: The IS6110-5'3'FP protocol described herein reached the optimal discriminatory potential of IS6110 fingerprinting and proved more accurate than 24-loci MIRU-VNTR in estimating recent TB transmission. The method, which is highly cost-effective, was rendered versatile enough to prompt its evaluation as an automatized solution for a TB integrated molecular surveillance.

Efficacy of Fine-Needle Aspiration Cytology in the Diagnosis of Tuberculous Cervical Lymphadenitis.

OBJECTIVE: This study assesses the role of fine-needle aspiration cytology (FNAC) in the diagnosis of tuberculous lymphadenitis (TL) in comparison with histology and bacteriology findings. STUDY DESIGN: We undertook a descriptive retrospective study of 937 FNAC specimens from 851 patients with cervical lymph nodes. The FNAC findings were then compared to histopathology and bacteriology. RESULTS: Of the 937 aspirates, the cytopathological diagnoses consisted of 426 (55.9%) TL, 185 (24.3%) reactive lymphoid hyperplasia, 18 (2.3%) suppurative inflammation, 78 (10.2%) malignant metastatic tumor, and 54 (7%) lymphoma. Of the 426 TL cases, 171 were diagnosed by FNAC combined with bacteriological examination. In this group, 22 cases were found to be positive on Ziehl-Neelsen stain and 16 by culture. A histopathology report was available for 62 cases. Compared to histopathology, the overall diagnostic sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of FNAC in the diagnosis of cervical TL were, respectively, 96.77, 100, 100, and 96.67%. When comparing bacteriology to histopathology, these values were 97.44, 100, 100, and 91.67%. CONCLUSION: Our study shows that FNAC is a sensitive and specific tool for the diagnosis of cervical TL.

Mycobacterium tuberculosis lineage 4 comprises globally distributed and geographically restricted sublineages.

Generalist and specialist species differ in the breadth of their ecological niches. Little is known about the niche width of obligate human pathogens. Here we analyzed a global collection of Mycobacterium tuberculosis lineage 4 clinical isolates, the most geographically widespread cause of human tuberculosis. We show that lineage 4 comprises globally distributed and geographically restricted sublineages, suggesting a distinction between generalists and specialists. Population genomic analyses showed that, whereas the majority of human T cell epitopes were conserved in all sublineages, the proportion of variable epitopes was higher in generalists. Our data further support a European origin for the most common generalist sublineage. Hence, the global success of lineage 4 reflects distinct strategies adopted by different sublineages and the influence of human migration.

MDR-TB Outbreak among HIV-Negative Tunisian Patients followed during 11 Years.

BACKGROUND: Multidrug-resistant tuberculosis (MDR-TB) outbreaks that evolve, from the outset, in a context strictly negative for HIV infection deserve special consideration since they reflect the true intrinsic epidemic potential of the causative strain. To our knowledge, the long-term evolution of such exceptional outbreaks and the treatment outcomes for the involved patients has never been reported hitherto. Here we provide a thorough description, over an 11-year period, of an MDR-TB outbreak that emerged and expanded in an HIV-negative context, Northern Tunisia. METHODOLOGY/PRINCIPAL FINDINGS: From October 2001 to June 2011, the MDR-TB outbreak involved 48 HIV-negative individuals that are mainly young (mean age 31.09 yrs; 89.6% male) and noninstitutionalized. Drug susceptibility testing coupled to mutational analysis revealed that initial transmission involved an isolate that was simultaneously resistant to isoniazid, rifampicin, ethambutol, and streptomycin. The causative Haarlem3-ST50 outbreak strain expanded mainly as an 11-banded IS6110 RFLP profile (77.1%), from which a 12-banded subclone evolved. After undergoing a 2-year treatment with second-line drugs, 22 (45.8%) patients were cured and 3 (6.2%) completed treatment, thus yielding an overall treatment success rate of 52.1%. Among the patients that experienced unfavorable treatment outcomes, 10 (20.8%) failed treatment, 3 (6.2%) were lost to follow-up, 5 (10.4%) died, and 5 (10.4%) could not be evaluated. Poor adherence to treatment was found to be the main independent predictor of unfavorable outcomes (HR: 9.15; 95% CI 1.72-48.73; P = 0.014). Intriguingly, the evolved 12-banded subclone proved significantly associated with unfavorable outcomes (HR: 4.90; 95% CI 1.04-23.04, P = 0.044). High rate of fatality and relapse was further demonstrated at the long-term, since 70% of those whose treatment failed have died, and 24% among those deemed successfully treated have relapsed. CONCLUSIONS/SIGNIFICANCE: Taken together, the data obtained in this study indicate that MDR-TB clinical isolates could become fit enough to cause large and severe outbreaks in an HIV-negative context. Such MDR-TB outbreaks are characterized by low treatment success rates and could evolve towards increased severity, thus calling for early detection of cases and the necessity to raise the bar of surveillance throughout and beyond the treatment period.

Evidence for the critical role of a secondary site rpoB mutation in the compensatory evolution and successful transmission of an MDR tuberculosis outbreak strain.

BACKGROUND: MDR Mycobacterium tuberculosis clinical strains that cause large outbreaks, particularly among HIV-negative patients, are likely to have undergone the most successful compensatory evolution. Hence, mutations secondary to the acquisition of drug resistance are worthy of consideration in these highly transmissible strains. Here, we assessed the role of a mutation within rpoB, rpoB V615M, secondary to the rifampicin resistance-conferring mutation rpoB S531L, which is associated with a major MDR tuberculosis outbreak strain that evolved in an HIV-negative context in northern Tunisia. METHODS: Using BCG as a model organism, we engineered strains harbouring either the rpoB S531L mutation alone or the double mutation rpoB S531L, V615M. Individual and competitive in vitro growth assays were performed in order to assess the relative fitness of each BCG mutant. RESULTS: The rpoB V615M mutation was found to be invariably associated with rpoB S531L. Structural analysis mapped rpoB V615M to the same bridge helix region as rpoB compensatory mutations previously described in Salmonella. Compared with the rpoB single-mutant BCG, the double mutant displayed improved growth characteristics and fitness rates equivalent to WT BCG. Strikingly, the rpoB double mutation conferred high-level resistance to rifampicin. CONCLUSIONS: Here, we demonstrated the fitness compensatory role of a mutation within rpoB, secondary to the rifampicin resistance mutation rpoB S531L, which is characteristic of an MDR M. tuberculosis major outbreak strain. The finding that this secondary mutation concomitantly increased the resistance level to rifampicin argues for its significant contribution to the successful transmission of the MDR-TB strain.