The molecular mechanisms determining transmissibility and prevalence of drug-resistant tuberculosis in a population were investigated through whole genome sequencing of 1,000 prospectively-obtained patient isolates from Russia. Two-thirds belonged to the Beijing lineage, which was dominated by two homogeneous clades. MDR genotypes were found in 48% of isolates overall and 87% of the major clades. The most common rifampicin-resistance rpoB mutation was associated with fitness-compensatory mutations in rpoA or rpoC, and a novel intragenic compensatory substitution was identified. The proportion of MDR cases with XDR-tuberculosis was 16% overall with 65% of MDR isolates harboring eis mutations, selected by kanamycin therapy, which may drive the expansion of strains with enhanced virulence. The combination of drug resistance and compensatory mutations displayed by the major clades confer clinical resistance without compromising fitness and transmissibility, revealing a biological contribution to the tuberculosis program weaknesses driving the persistence and spread of M/XDR-tuberculosis in Russia and beyond.

In October 2018 a large number of international experts with complementary expertise came together in Taormina to participate in a workshop on occult hepatitis B virus infection (OBI). The objectives of the workshop were to review the existing knowledge on OBI, to identify issues that require further investigation, to highlight both existing controversies and newly emerging perspectives, and ultimately to update the statements previously agreed in 2008. This paper represents the output from the workshop.

An important prognostic factor in any form of infection seems to be glucose control in patients with type 2 diabetes. There is no information about the effects of tight glycemic control on coronavirus disease 2019 (COVID-19) outcomes in patients with hyperglycemia. Therefore, we examined the effects of optimal glycemic control in patients with hyperglycemia affected by COVID-19. RESEARCH DESIGN AND METHODS Fifty-nine patients with COVID-19 hospitalized with moderate disease were evaluated. On the basis of admission glycemia >7.77 mmol/L, patients were divided into hyperglycemic and normoglycemic groups. Interleukin 6 (IL-6) and D-dimer levels were evaluated at admission and weekly during hospitalization. The composite end point was severe disease, admission to an intensive care unit, use of mechanical ventilation, or death. RESULTS Thirty-four (57.6%) patients were normoglycemic and 25 (42.4%) were hyperglycemic. In the hyperglycemic group, 7 (28%) and 18 (72%) patients were diagnosed with diabetes already before admission, and 10 (40%) and 15 (60%) were treated without and with insulin infusion, respectively. The mean of glycemia during hospitalization was 10.65 6 0.84 mmol/L in the no insulin infusion group and 7.69 6 1.85 mmol/L in the insulin infusion group. At baseline, IL-6 and D-dimer levels were significantly higher in the hyperglycemic group than in the normoglycemic group (P < 0.001). Even though all patients were on standard treatment for COVID-19 infection, IL-6 and D-dimer levels persisted higher in patients with hyperglycemia during hospitalization. In a risk-adjusted Cox regression analysis, both patients with hyperglycemia and patients with diabetes had a higher risk of severe disease than those without diabetes and with normoglycemia. Cox regression analysis evidenced that patients with hyperglycemia treated with insulin infusion had a lower risk of severe disease than patients without insulin infusion. CONCLUSIONS Insulin infusion may be an effective method for achieving glycemic targets and improving outcomes in patients with COVID-19.

Background: The genome of Mycobacterium tuberculosis harbors four copies of a cluster of genes termed mce operons. Despite extensive research that has demonstrated the importance of these operons on infection outcome, their physiological function remains obscure. Expanding databases of complete microbial genome sequences facilitate a comparative genomic approach that can provide valuable insight into the role of uncharacterized proteins.

Two-component regulatory signal transduction systems are widely distributed among bacteria and enable the organisms to make coordinated changes in gene expression in response to a variety of environmental stimuli. The genome sequence of Mycobacterium tuberculosis contains 11 complete two-component systems, four isolated homologous regulators, and three isolated homologous sensors. We have constructed defined mutations in six of these genes and measured virulence in a SCID mouse model. Mice infected with four of the mutants (deletions of devR, tcrXY, trcS, and kdpDE) died more rapidly than those infected with wild-type bacteria. The other two mutants (narL and Rv3220c) showed no change compared to the wild-type H37Rv strain. The most hypervirulent mutant (devR⌬) also grew more rapidly in the acute stage of infection in immunocompetent mice and in gamma interferon-activated macrophages. These results define a novel class of genes in this pathogen whose presence slows down its multiplication in vivo or increases its susceptibility to host killing mechanisms. Thus, M. tuberculosis actively maintains a balance between its own survival and that of the host.Mycobacterium tuberculosis is an extraordinarily successful pathogen that currently infects approximately one-third of the global population and causes 8 million new cases of tuberculosis annually (41). The tubercle bacillus functions within several hostile environments in order to survive within the human host and cause disease. For instance, the bacteria must be able to gain entry into macrophages, multiply intracellularly, survive within the lung granuloma for years, and disperse to a new host via aerosols (9). It is therefore likely that the expression of different sets of genes by M. tuberculosis at various stages of infection is crucial to its survival. Two-component regulatory systems (2CRs) are widely distributed among bacteria and plants and enable the organisms to respond to many different external stimuli (20,37,38). These systems form a large family of related proteins that consist of a membrane-bound sensor protein that activates an effector protein, generally a transcriptional regulator, by phosphorylation. 2CRs have been shown to play a crucial role in the controlled expression of virulence genes in other bacteria (11). For example, in Salmonella enterica serovar Typhimurium, the membrane-bound sensor protein PhoQ is activated by changing levels of magnesium, and this activates the PhoP response regulator. PhoP is a DNA-binding protein that binds in a sequence-specific manner to selected promoters, thereby inducing or repressing their transcription, and S. enterica serovar Typhimurium phoP mutants are highly attenuated (14, 17).The genome of M. tuberculosis contains 11 paired 2CRs, four isolated regulators, and three isolated sensors (6). Mutants in two of these, phoP and mprA are highly attenuated in a murine model (31, 43), while a prrA mutant grows more slowly in macrophages (12), indicating that these regulators control genes which are important f...

An estimated one-third of the world's population is latently infected with Mycobacterium tuberculosis, the etiologic agent of tuberculosis. Here, we demonstrate that, unlike wild-type M. tuberculosis, a strain of M. tuberculosis disrupted in the mce1 operon was unable to enter a stable persistent state of infection in mouse lungs. Instead, the mutant continued to replicate and killed the mice more rapidly than did the wild-type strain. Histological examination of mouse lungs infected with the mutant strain revealed diffusely organized granulomas with aberrant inflammatory cell migration. Murine macrophages infected ex vivo with the mutant strain were reduced in their ability to produce tumor necrosis factor ␣, IL-6, monocyte chemoattractant protein 1, and nitric oxide (NO), but not IL-4. The mce1 mutant strain complemented with the mce1 genes stimulated tumor necrosis factor ␣ and NO production by murine macrophages at levels stimulated by the wild-type strain. These observations indicate that the mce1 operon mutant is unable to stimulate T helper 1-type immunity in mice. The hypervirulence of the mutant strain may have resulted from its inability to stimulate a proinflammatory response that would otherwise induce organized granuloma formation and control the infection without killing the organism. The mce1 operon of M. tuberculosis may be involved in modulating the host inflammatory response in such a way that the bacterium can enter a persistent state without being eliminated or causing disease in the host. Approximately 60% of people who become infected with Mycobacterium tuberculosis develop asymptomatic latent infection (1). This reservoir of latently infected individuals has a 2-23% lifetime risk of developing active disease, referred to as reactivation tuberculosis (1). How M. tuberculosis establishes and maintains latent infection in an animal host is poorly understood. Several candidate M. tuberculosis genes have been recently reported to be important for persistence in the mouse model of tuberculosis. They include the isocitrate lyase gene (icl), mycolic acid cyclopropane synthase gene (pca), and a two-component response regulator called mprA (2)(3)(4). In each case, the disruption of the gene led to attenuation of the mutant strains in the mouse model of infection, whereas their in vitro growth kinetics remained similar to that of the respective wild-type strain (2-4).We reported previously the identification of a M. tuberculosis gene mce1A (Rv0169, Sanger Centre genome sequence designation) that conferred on a nonpathogenic Escherichia coli strain an ability to enter nonphagocytic cells (5). The encoded product facilitated uptake of synthetic microspheres into nonphagocytic cells, and an active domain of the protein was recently shown to cause cytoskeletal rearrangement in HeLa cells that was both microfilament-and microtubule-dependent (6, 7). The gene mce1A is located in a putative operon called mce1 containing 12 genes in the M. tuberculosis H37Rv genome (8) (Fig. 1A). The genome of M. tuberculosi...

Hepatitis B virus (HBV), hepatitis D virus (HDV), and hepatitis C virus (HCV) are responsible for the majority of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma (HCC) cases worldwide. 1-4 Multiple HBV and HDV infection has been extensively investigated in past years and found to be associated with a fulminant course of acute hepatitis, with the more severe forms of chronic liver disease, and with a rapid progression to liver cirrhosis. 5-8 An inhibitory effect exerted by HDV on HBV replication has also been shown. 9 The prevalence of patients with HCV and HBV coinfection has been described as high in geographic areas where a high endemic level of both infections is reported, such as Western Asia and the Mediterranean Basin. 10-14 The interaction between HCV and HBV has so far been poorly investigated. Little is known on the clinical presentation, the natural history, and the response to antiviral treatment of liver diseases associated with HBV and HCV coinfection. However, small pilot studies have prompted the hypothesis that HCV may have an inhibitory effect on HBV replication [15][16][17][18][19] and that multiple HBV and HCV infection may be associated with a more severe clinical presentation. 10,[20][21][22][23][24][25] The interaction between HDV and HCV in hepatitis B surface antigen (HBsAg) chronic carriers has not as yet been investigated.In this article we report the data from an Italian multicenter case-control study with incident cases, performed on a high number of patients with chronic hepatitis from a multiple hepatitis virus infection who were compared with patients with chronic hepatitis caused by a single virus. We investigated the interference between viruses, the clinical impact of a multiple virus infection compared with a single HBV or HCV infection, and tested the hypothesis that anti-HCV-positive/ anti-hepatitis B core antigen (HBc)-positive patients lacking both HBsAg and anti-hepatitis B surface antigen (HBs) might be a subgroup of patients with a multiple HBV and HCV infection. MATERIALS AND METHODSPatients. Seven liver units in different geographic areas of Italy, 1 in the north (Padua), 1 in the center (Rome), 2 in the south (Naples and Bari), 1 in Sardinia (Sassari), and 2 in Sicily (Palermo and Messina), participated in the study. These liver units had participated in numerous multicenter studies on chronic hepatitis in the past 2 decades and had been using similar criteria for the clinical approach and the histologic diagnosis. The investigation was planned as a cross-sectional case-control study with incident cases during a preliminary consensus meeting of the senior investigators from the participating centers.Chronic hepatitis was diagnosed on the basis of high serum transaminase values for at least 6 months.

Extensively drug-resistant (XDR) tuberculosis (TB), which is resistant to both first- and second-line antibiotics, is an escalating problem, particularly in the Russian Federation. Molecular fingerprinting of 2348 Mycobacterium tuberculosis isolates collected in Samara Oblast, Russia, revealed that 72% belonged to the Beijing lineage, a genotype associated with enhanced acquisition of drug resistance and increased virulence. Whole-genome sequencing of 34 Samaran isolates, plus 25 isolates representing global M. tuberculosis complex diversity, revealed that Beijing isolates originating in Eastern Europe formed a monophyletic group. Homoplasic polymorphisms within this clade were almost invariably associated with antibiotic resistance, indicating that the evolution of this population is primarily driven by drug therapy. Resistance genotypes showed a strong correlation with drug susceptibility phenotypes. A novel homoplasic mutation in rpoC, found only in isolates carrying a common rpoB rifampicin-resistance mutation, may play a role in fitness compensation. Most multidrug-resistant (MDR) isolates also had mutations in the promoter of a virulence gene, eis, which increase its expression and confer kanamycin resistance. Kanamycin therapy may thus select for mutants with increased virulence, helping preserve bacterial fitness and promoting transmission of drug-resistant TB strains. The East European clade was dominated by two MDR clusters, each disseminated across Samara. Polymorphisms conferring fluoroquinolone resistance were independently acquired multiple times within each cluster, indicating that XDR TB is currently not widely transmitted.