Scientific Program

Flavodoxins as drug targets. Novel Helicobacter pylori specific antimicrobials

Antimicrobial resistance constitutes a global health challenge that must be met by developing novel therapeutic tools. Flavodoxins are small bacterial proteins that participate in many key metabolic pathways and are promising targets for the development of novel antimicrobials against pathogenic bacteria. The main structural and biochemical features of flavodoxins will be described and the bacteria where they have been proved to be essential proteins or at least virulence factors will be highlighted. One such bacterium is Helicobacter pylori (Hp). This organism establishes long-life infections in about 50 % humans. Eradication of Hp infection, which is based on the combination of different antimicrobials, is being challenged by increasing antimicrobial resistance. To contribute to Hp-specific eradication, we have developed a novel family of Hp-specific antimicrobials. Their efficacy towards resistant clinical isolates and in the mouse model of Hp Infection has been evaluated. We have thus designed, synthesized and tested redox variants of two families of compounds (containing nitroethylene or 7-nitrobenzoxadiazole) that inhibit the activity of Hp-flavodoxin. Derivatives of nitroethylene show similar decreased cytotoxicity and antimicrobial activity and do not constitute a significant improvement from their parent compound. Derivatives of 7-nitrobenzoxadiazole display largely increased therapeutic indexes against several reference Hp strains. These inhibitors are effective against metronidazole-, clarithromycin- and rifampicin-resistant clinical isolates of Hp. Besides, their toxicity for mice after oral administration is greatly reduced compared to that of the lead compound. When given individually at single daily doses for 8 days in a mice model of Hp infection, they reduced significantly the Hp gastric colonization rates and were able to eradicate the infection in up to 60% of the mice.

Non-typhoidal Salmonella: Why Should We Care?

Non-typhoidal Salmonella (NTS) Dublin has adapted to cause invasive illness in humans. There is no vaccine against iNTS and disease management is further complicated by the emergence of multidrug resistant (MDR) strains. It is estimated that invasive non-typhoidal Salmonella (iNTS) serovars cause over 680,000 human deaths per year. We apply next generation sequencing (NGS) technologies and associated bioinformatics analyses tools to understand the genetic basis of iNTS Dublin.

Whole genome sequencing (WGS) of human S. Dublin strains revealed several virulence factors and mobile genetic elements (MGEs) that may contribute to bacterial virulence and enable the bacteria to cause invasive disease in humans including Gifsy-2 prophage, virulence plasmid and Salmonella pathogenicity islands; SPI-7 harbouring the Vi antigen, SPI-6 and SPI-19 harbouring two different T6SSs and the novel pathogenicity island ST313-GI. Understanding the genetic basis of virulence of iNTS Dublin will provide insights into developing novel therapeutics and effective vaccine against human infection.

Interestingly, NTS have developed MDR against current antibiotics including the last resort; colistin (polymexins). Bacteriophage therapy is therefore the hope for treatment of MDR bacterial infections however one of the key limitations to therapeutic use of phages, is the limited host range of many phages and the ease of development of bacterial resistance to phages. A solution is to develop one or a cocktail of engineered phage that overcome these limitations. An essential step towards this goal is understanding the complex dynamics of bacteria-phage interaction. We therefore use Anderson phage typing scheme as a valuable model system for study of phage-host interaction to characterize all bacterial antiviral systems (including clustered regularly interspaced short palindromic repeat (CRISPRs) loci and CRISPR-associated (Cas) proteins (CRISPR-Cas) immune systems, superinfection exclusion (Sie) and restriction-modification (R-M) systems) as well as phage evasion strategies (including anti-CRISPR). Understanding the dynamics of bacteria-phage interaction will provide new insights into phage biology and strategies for genetic modification of phages and designing effective broad spectrum engineered phages to overcome the limitations of bacteriophages as therapeutic agents.

Direct evidence of viral infection and mitochondrial alterations in the brain of fetuses at high risk for schizophrenia

There is increasing evidences that favor the prenatal beginning of schizophrenia. These evidences point toward intra-uterine environmental factors that act specifically during the second pregnancy trimester producing a direct damage of the brain of the fetus [1]. The current available technology doesn't allow observing what is happening at cellular level since the human brain is not exposed  to a direct analysis in that stage of the life in subjects at high risk of developing schizophrenia. Methods. In 1977 we began a direct electron microscopic research of the brain of fetuses at high risk from schizophrenic mothers in order to finding differences at cellular level in relation to controls. Results. In these studies we have observed within the nuclei of neurons the presence of complete and incomplete viral particles that reacted in positive form with antibodies to herpes simplex hominis type I [HSV1] virus, and mitochondria alterations [2]. Conclusion. The importance of these findings can have practical applications in the prevention of the illness keeping in mind its direct relation to the aetiology and physiopathology of schizophrenia. A study of the gametes or the amniotic fluid cells in women at risk of having a schizophrenic offspring is considered. Of being observed the same alterations that those observed previously in the cells of the brain of the studied foetuses, it would intend to these women in risk of having a schizophrenia descendant, previous information of the results, the voluntary medical interruption of the pregnancy or an early anti HSV1 viral treatment as preventive measure of the later development of the illness.

Combined Tuberculosis/HIV infection in Russia: Current status and perspectives

Objective: Epidemiology of combined TB/HIV infection in the Russian Federation over the past 10 years (2009-2018).

Materials and methods: retrospective analysis of statistical data received from the reporting forms of the ROSSTAT and MoH of Russia for the period (2009-2018). For the analysis, cartographic and correlation analyses were used.

Results: over the past 10 years, in Russia, against background of a steady decline in the incidence of tuberculosis (TB), there has been a radical change in the structure of TB patients due to increase in proportion of TB/HIV patients, which in 2018 was 23.1% among newly diagnosed patients and 20.7 % among TB contingents. The incidence of TB/HIV for the same period increased by 1,2 times and in 2018 amounted to 7,6 per 100000 of population, while the total rates of growth were 16,9%. The spread of TB/HIV was mainly observed in the age groups of 25-44 years, the proportion of which throughout the entire observation period was 76% among newly diagnosed patients and 77% among TB/HIV contingents. In the structure of TB/HIV patients there was a high proportion of patients with severe HIV infection with manifestations of multiple infections and diseases (ICD-10  B20.7, B22.7), accounting in 2018 for 39% among newly diagnosed patients and 40% among contingents, that evidenced of late diagnosis of HIV. The proportion of TB/HIV patients who were diagnosed Hepatitis (ICD-10 B20.7) was high and in 2018 accounted for 38% among newly diagnosed patients and 44% - among contingents, prevailing Hepatitis C in their structure, whose proportion was 93%.

The spread of TB/HIV in 85 constituent entities of Russia was characterized by apparent irregularity: if in 35 constituent entities of Russia (41% of their total number) the incidence of TB/HIV was high and exceeded that in Russia, then in 50 constituent entities of Russia (59% of their total number), on the contrary, it was low and did not reach that in Russia. The most severe group was MDR-TB/HIV patients, whose proportion in 2018 was 18% among newly diagnosed patients and 17% among contingents.   In 48 constituent entities of Russia, where the proportion of TB/HIV patients was >7% of newly diagnosed TB patients, a direct correlation was revealed (r = 0.33, p <0.05, t-statistics = 1.4) between the proportion of patients with MDR-TB and the proportion of TB/HIV patients as well as the proportion of TB/HIV patients who died during the first year of observation (r = 0.45, p <0.01, t-statistics = 3.5).

Among patients with TB/HIV, a consistently high level of immunosuppression was observed (CD4 lymphocyte count of <350 cells/μl), and, for example, in 2018 it was observed in 68% of newly diagnosed patients, which had a negative impact on the results of their treatment.

In Russia over the past 10 years, the effectiveness of TB/HIV patients’ treatment has been low and averaged 47%. Proportion of deaths, on the contrary, was high and in 2018 amounted to 22%. In 22 constituent entities of Russia, it was even higher and ranged from 25% to 88%. In structure of the deceased patients, 81% were people aged 25-44 years.

Conclusions: High prevalence of TB/HIV, currently registered in 35 out of 85 constituent entities of the Russian Federation, facilitates to slowing down the rates of decrease in the incidence of TB, which may not be enough to achieve the WHO targets in the program to eliminate tuberculosis in Russia.

Zoonotic Tuberculosis: Characterization of Antibody Responses in Various Host Species for Improved Serodiagnosis

Bovine tuberculosis (bTB) has a broad range of mammalian host species including various livestock and wildlife animals. Ante-mortem testing for bTB traditionally relies on cell-mediated immune responses, such as tuberculin skin test reactions or in vitro interferon-gamma release. However, serological methods using novel antigens and advanced assay formats have recently emerged to offer attractive options for rapid identification of infected animals.

Using Dual-Path Platform (DPP) technology, IgM and IgG antibody responses to two antigens, MPB70/MPB83 and CFP10/ESAT6 fusion proteins, were studied in cattle and domestic pigs infected with Mycobacterium bovis. Both test antigens elicited IgM responses in pigs, whereas cattle produced IgM antibodies only to MPB70/MPB83. DPP assay detected robust IgG responses to both antigens in the vast majority of infected animals, with MPB70/MPB83 recognized more frequently than CFP10/ESAT6 in these host species. Detecting IgM antibody did not improve seroreactivity rates based on IgG antibody detection in cattle and pigs. CFP10/ESAT6 antigen showed limited value in identifying infected animals by DPP assay using MPB70/MPB83 antigen to measure IgG antibody responses. The overall diagnostic accuracy of DPP assay was estimated to be higher in pigs (sensitivity 98%, specificity 100%) than in cattle (sensitivity 78%, specificity 97%).

Characterization of the humoral immune responses to M. bovis in cattle and pigs reveals distinct patterns of antigen recognition by IgM and IgG antibodies. The findings suggest that development of improved serologic tests for various hosts will require further research on comparative bTB immunobiology focusing on inter-species differences in antibody profiles.

Sepsis as a metabolic adaptation failure

Sepsis is a most deadly unmet medical need. It hits 30 million people yearly, leading to 8 million deaths, amongst which 3 million children. Usually, sepsis starts with a serious (bacterial) infection that spreads and triggers many systems of the host, including inflammation, immunity, coagulation, complement, but also thermoregulation, circadian rhythm and other systems are modulated. Of note, despite the enormous investments in sepsis, no major new therapies at the bedside have emerged, and sepsis is reputed for its high amounts of failed clinical trials. Patients are treated by antibiotics and organ support. The reasons behind these difficulties are hard to find, but we suggest that an over-emphasis on inflammation and inflammation-blockers is maybe one of the reasons. If the major probleam in sepsis, responsible for lethality, is not inflammation, then what is it?

To our opinion, the very well-known metabolic reprogramming in sepsis is the key. It has been known since many years, that sepsis patients undergo severe metabolic shifts, as they show hyperglycemia, followed by hypoglycemia, high lactate levels, and high blood concentrations of free fatty acids (FFAs) and glycerol. In a mouse model of sepsis, the cecal ligation and puncture (CLP) model, these typical metabolic changes are observed, and we interpret them as an accelerated and profound starvation response, based on lack of calorie intake and high calorie needs. The animals perform lipolysis as well as consume their glycogen stores and perform muscle degradation. A massive amount of energy-rich metabolites is released in the blood, but the major catabolic systems in the liver are failing to consume these metabolites. I will show that the receptor for Glucocorticoids (GR) quickly loses function in septic liver, and that PPARa, the major FFA sensor and driver of beta oxidations and ketogenesis is reducing in amounts in this organ. The mechanism underlying the GR problem is not known but based on a highly artificial model system in cell cultures using the cytokine TNF as a trigger, a mechanism of competition between transcription factors and GR for transcriptional co-regulators, such as p300, is one of the possibilities. The status of our work in relation to GR in sepsis will be shown, as well as the potential complications for the progression of sepsis. Equally so, I will show our findings on PPARa, and how this important transcription factor is modulated in sepsis.

Potential for spread of Leishmania tropica in southern Europe by local sand flies

Various Leishmania species constitute an important global health problem with millions of people affected. Leishmania tropica is the causative agent of cutaneous leishmaniases (CL) in the Old World, occurring in many countries of Asia and Africa. Currently, a great concern is posed by outbreaks of CL in the Middle East, connected with people displaced from their homes due to civil war. In addition, imported cases of CL caused by L. tropica are increasingly documented in Europe among the general and migrant populations. The only proven vectors of Leishmania parasites are phlebotomine sand flies (Diptera: Phlebotominae).  In most foci, L. tropica is transmitted by its specific vector Phlebotomus sergenti, a sand fly which is very rarely found in Europe. However, southern Europe is inhabited by other sand fly species and there is increasing evidence that they may also serve as vectors for L. tropica. We investigated the susceptibility of Phlebotomus perniciosus and Phlebotomus tobbi, two species widely distributed in the western and eastern parts of the Mediterranean basin, respectively, to infection with L. tropica. Laboratory-reared sand flies were infected experimentally with two L. tropica strains differing in geographical origin, epidemiology and surface molecules, particularly lipophosphoglycan epitopes. High infection rates, heavy parasite loads and fully developed late-stage infections, including colonization of the stomodeal valve, were observed in all parasite-vector combinations. In conclusion, we demonstrated that both sand fly species studied are susceptible to full development of various L. tropica  strains which suggests that they may play a role in circulation of this parasite in southern Europe, the Middle East and northern Africa.

Infections caused by antibiotic-resistant ESKAPE group in Mexico

Infections caused by antibiotic-resistant ESKAPE group microorganisms possess high risk to patients' lives, because  treatment is limited to few therapeutic options. The resistance mechanisms are: production of Extended Spectrum Lactamases (BLEE), Carbapenemes type MBL, enzymatic production of aminoglycoside resistance (HLRA), resistance to vancomycin and methicillin by modification of target site. In Mexico, epidemiological surveillance as well as control and prevention measures are regulated for the control and prevention of nosocomial infections. The American Society of Infectious Diseases (IDSA) defined a group of bacteria included in the term ESKAPE: Enterococcus faecium resistant to vancomycin (ERV), methicillin-resistant Staphylococcus aureus (MRSA), Klebsiella  pneumoniae  with expected -extended spectrum lactamic (BLEEs), Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter species, as high-priority pathogens, for representing relevant clinical or public health problems, as well as being very hard therapeutic alternatives.

In this talk I would like to present experiences and work of ours staff group and some other groups have done in Mexico, where the ESKAPE group is already being studied. The audience will be able to learn what methods we suggest to detect BLEEs, AmpC production by inhibition with fenil-boronic-acid and how to be confirmed by PCR assay. Metallo-β-lactamases-(MBL) phenotype production determined by disk diffusion with EDTA. Genes bla_KPC, bla_VIM, bla_IMP, bla_NDM and bla_OXA-24-like with PCR and efflux pumps using inhibitor of efflux pumps CCCP. Clonality analysis by PGE dissemination in all floors of hospitalization. Our knowledge help to understand the accuracy of results and provide information to assist and resolve similar problems and stablish prevention methods as hand washing.

Standardization of a pncA Gene Complementation Technique in Mycobacterium tuberculosis pncA-knockout: Tool for the Study of the Relationship Between Mutations in pncA and Phenotypic Parameters

Mutations in the pncA gene are the major cause of resistance to pyrazinamide (PZA), but there are alternative mechanisms that also provide resistance. The objective of the study was to standardize the pncA gene complementation technique in Mycobacterium tuberculosis H37Rv pncA-knockout to evaluate the effect of mutations in the pncA gene, in a scenario controlled by the genetic variability of clinical isolates.

Strains complemented with pncA wild type and D49N, H51R, G78C and F94L mutant genes were generated through the pNIT-1 expression system to evaluate pyrazinoic acid (POA) production by means of the quantitative Wayne test, pncA gene expression level and PZA susceptibility using gradual concentrations of PZA under non-induced and induced conditions.

The pNIT-pncA expression system restored the susceptible phenotype of the pncA-knockout strain. The basal expression levels of the pncA genes were constant and similar to that of the H37Rv strain. The pncA wild type complemented strain showed POA production and Minimum Inhibitory Concentration of PZA similar to H37Rv (0.6 uM and 100 ug/mL, respectively). Although the overexpression of pncA wild type in the complemented strain induced higher production of POA (3 uM), this did not interfere with susceptibility to PZA. However, the mutant pncA strains complemented were resistant in both conditions and the overexpression of the mutant pncA genes, nine times greater than that of the H37Rv strain (P<0.005), made it possible to verify the total or partial loss of the enzymatic function of the mutant pyrazinamidase.

The complementation of the pncA-knockout strain with the pncA wild type gene, by means of the pNIT expression system, allowed the restoration of the susceptible phenotype, and the overexpression of pncA with D49N, H51R, G78C and F94L mutations in the complemented strains really confer resistance to PZA.

Viral pathogens associated with acute lower respiratory tract infections in Bulgaria

Acute lower respiratory tract infections are a major cause of morbidity and hospital admissions. This study aimed to determine the viral etiology of these infections among the patients of different age groups in Bulgaria during the period October 2016-September 2019. Nasopharyngeal specimens were collected from patients with bronchiolitis, pneumonia, and exacerbation of chronical respiratory diseases (asthma, COPD). The viral etiology was determined by individual Real-Time PCR assays against 12 respiratory viruses. Of the 644 patients examined, 476 (73.9%) were positive for at least one virus. Co-infections with two and three viruses were found in 81 (17%) of the infected patients. Among the 532 children younger than 5 years, respiratory-syncytial virus (RSV) was the predominant pathogen (37.4%), followed by rhinoviruses (14.1%), bocaviruses (8.8%), human metapneumovirus (7%), adenoviruses (6.8%), influenza A(H3N2) (4.7%) and influenza A(H1N1)pdm09 (3.8%) viruses. RSV-B were more prevalent than RSV-A throughout the study period. At least one respiratory virus was identified in 82.9% and 67.2% of the children with bronchiolitis and pneumonia, respectively. Among the patients >5 years, influenza A(H1N1)pdm09 virus was the most frequently detected (16.5%), followed by RSV (8.8%) and influenza A(H3N2) (7.8%). Diagnostic testing for respiratory viruses using molecular methods may lead to the reduced use of antibiotics and may assist in measures to control infection.

Study of Patterns and Markers of Human Immune Deficiency Virus -1 (HIV-1) Progression and Unemployment Rate among Patients from Alexandria, Egypt

The Middle East and North Africa (MENA) new HIV cases show the highest increase among all regions in the world. Even though Egypt has a low prevalence among the general population (< 0.02%), a national HIV epidemic occurs in certain population risk groups. The current study was conducted to asses clinical and immunological disease progression; following up viral load (VL) and detecting delta-32 CCR5 genotype polymorphism in selected cases, determining the unemployment rate and identify predictors of employment for HIV-cases. A cross-sectional design was adopted. HIV infected cases attending Alexandria Fever Hospital (AFH) for one year. Interview questionnaire and four CD+4 counts were done for all patients, HIV VL and delta-32 CCR5 polymorphism were done for selected cases. Sexual transmission and drug abuse are the most important risk factors. Infectious comorbidity increases the rate of HIV progression. CD4+ count at the end of the study; CD+4 (4), the count was significantly higher than all other CD4+ readings among the whole cohort and among the treated group. Also, VL at the end of the study; VL(2), was significantly higher than VL(1) among the untreated group. The unemployment rate was 40%. Male gender and obtaining vocational training were significant predictors of employment. It can be concluded that having a family member living with HIV and drug abusers are high-risk groups for HIV acquisition. Factors responsible for the progression of HIV should be further investigated. Antiretroviral therapy is very effective in checking the HIV replication rate, delaying the progression of HIV, reconstituting the immune response and should be available for all cases detected.

Diagnostic utility of bronchoalveolar lavage and computerized tomography in diagnosis of recurrence among sputum scarce and sputum negative treated pulmonary tuberculosis suspects

Objective: There are a large number of patients who present with chest symptoms of short duration at secondary and tertiary care centers with a background history of anti-tubercular therapy in the past. The literature regarding the evaluation and management of such patients is scarce. Therefore a study was planned with the aim of to assess the sensitivity, specificity and diagnostic accuracy of various CT chest findings, BAL GeneXpert, AFB and Culture (MGIT- 960) in diagnosing recurrence of pulmonary tuberculosis.

Methods: A prospective observational study was conducted in a tertiary care hospital of North India wherein 62 suspects with past history of treatment for pulmonary tuberculosis, who presented with symptoms suggestive of recurrence, were included. Patients underwent BAL and CT chest and other relevant investigations. They were followed up till treatment completion or clinical cure.

Results: A total of 62 patients were included in the final analysis. Recurrence of TB was present in 29 %( 18) of patients. The mean age of the patients with recurrent PTB was 27.5 years, 55.5% (10) were males and 44.5% (8) were females. Median duration between two episodes of PTB was 1.5 years. Cough was the universal symptom in these patients with recurrent symptoms (> 90%). Hemoptysis was predominant symptom among patients with Aspergillosis (66.6%). Mean CRP was higher among recurrent PTB groups compared to patients in another group (83.03 & 11.73 respectively) which was statistically significant. BAL Xpert® MTB/RIF and MGIT had good sensitivity (83.33% & 84.62% respectively), specificity (100% for both) and excellent diagnostic
accuracy (95.16% & 96.36%) for diagnosing recurrence in sputum negative and sputum scarce patient, (p<0.001). Among CT findings, necrotic mediastinal lymph nodes had a good diagnostic accuracy of 88.71% with AUC of 0.806, (p < 0.001).

Conclusion: BAL Xpert® MTB/RIF and MGIT, have good sensitivity (83.33% & 84.62%), specificity and excellent diagnostic accuracy (95.16% & 96.36%) for diagnosing recurrence in sputum negative and sputum scarce patient. The presence of mediastinal necrotic lymph node is the single most CT finding that can truly differentiate recurrent TB from post TB sequelae. Bronchoscopy & BAL should be considered for microbiological confirmation and sensitivity testing before embarking on unwarranted ATT initiation, in cases of suspected recurrence.