The role of the efflux mechanisms in multidrug resistance in Mycobacterium tuberculosis

Abstract The emergence of multi and extensively drug resistant tuberculosis (MDRTB and XDRTB) has increased the concern of public health authorities around the world. The World Health Organization has defined MDRTB as tuberculosis (TB) caused by organisms resistant to at least isoniazid and rifampicin, the main first-line drugs used in TB therapy, whereas XDRTB refers to TB resistant not only to isoniazid and rifampicin, but also to a fluoroquinolone and to at least one of the three injectable second-line drugs, kanamycin, amikacin and capreomycin. Resistance in Mycobacterium tuberculosis is mainly due to the occurrence of spontaneous mutations and followed by selection of mutants by subsequent treatment. However, some resistant clinical isolates do not present mutations in any genes associated with resistance to a given antibiotic, which suggests that other mechanism(s) are involved in the development of drug resistance, namely the presence of efflux pump systems that extrude the drug to the exterior of the cell, preventing access to its target. Increased efflux activity can occur in response to prolonged exposure to subinhibitory concentrations of anti-TB drugs, a situation that may result from inadequate TB therapy. The inhibition of efflux activity with a non-antibiotic inhibitor may restore activity of an antibiotic subject to efflux and thus provide a way to enhance the activity of current anti-TB drugs. The work described in this thesis foccus on the study of efflux mechanisms in the development of multidrug resistance in M. tuberculosis and how phenotypic resistance, mediated by efflux pumps, correlates with genetic resistance. In order to accomplish this goal, several experimental protocols were developed using biological models such as Escherichia coli, the fast growing mycobacteria Mycobacterium smegmatis, and Mycobacterium avium, before their application to M. tuberculosis. This approach allowed the study of the mechanisms that result in the physiological adaptation of E. coli to subinhibitory concentrations of tetracycline (Chapter II), the development of a fluorometric method that allows the detection and quantification of efflux of ethidium bromide (Chapter III), the characterization of the ethidium bromide transport in M. smegmatis (Chapter IV) and the contribution of efflux activity to macrolide resistance in Mycobacterium avium complex (Chapter V). Finally, the methods developed allowed the study of the role of efflux pumps in M. tuberculosis strains induced to isoniazid resistance (Chapter VI). By this manner, in Chapter II it was possible to observe that the physiological adaptation of E. coli to tetracycline results from an interplay between events at the genetic level and protein folding that decrease permeability of the cell envelope and increase efflux pump activity. Furthermore, Chapter III describes the development of a semi-automated fluorometric method that allowed the correlation of this efflux activity with the transport kinetics of ethidium bromide (a known efflux pump substrate) in E. coli and the identification of efflux inhibitors. Concerning M. smegmatis, we have compared the wild-type M. smegmatis mc2155 with knockout mutants for LfrA and MspA for their ability to transport ethidium bromide. The results presented in Chapter IV showed that MspA, the major porin in M. smegmatis, plays an important role in the entrance of ethidium bromide and antibiotics into the cell and that efflux via the LfrA pump is involved in low-level resistance to these compounds in M. smegmatis. Chapter V describes the study of the contribution of efflux pumps to macrolide resistance in clinical M. avium complex isolates. It was demonstrated that resistance to clarithromycin was significantly reduced in the presence of efflux inhibitors such as thioridazine, chlorpromazine and verapamil. These same inhibitors decreased efflux of ethidium bromide and increased the retention of [14C]-erythromycin in these isolates. Finaly, the methods developed with the experimental models mentioned above allowed the study of the role of efflux pumps on M. tuberculosis strains induced to isoniazid resistance. This is described in Chapter VI of this Thesis, where it is demonstrated that induced resistance to isoniazid does not involve mutations in any of the genes known to be associated with isoniazid resistance, but an efflux system that is sensitive to efflux inhibitors. These inhibitors decreased the efflux of ethidium bromide and also reduced the minimum inhibitory concentration of isoniazid in these strains. Moreover, expression analysis showed overexpression of genes that code for efflux pumps in the induced strains relatively to the non-induced parental strains. In conclusion, the work described in this thesis demonstrates that efflux pumps play an important role in the development of drug resistance, namely in mycobacteria. A strategy to overcome efflux-mediated resistance may consist on the use of compounds that inhibit efflux activity, restoring the activity of antimicrobials that are efflux pump substrates, a useful approach particularly in TB where the most effective treatment regimens are becoming uneffective due to the increase of MDRTB/XDRTB.

Characterization of the non-apparent clinical form in the initial phase of schistosomiasis mansoni

In this paper the history of 115 recruits that had bathed simultaneously in streams contaminated with Schistosoma mansoni, during military maneuvers, is reported. Thirty four of the infected patients presented the initial phase of the infection diagnosed through epidemiologic, clinical and laboratorial parameters. Three out of the 34 patients did not reveal the clinical picture of the infection, thus being considered representatives of the non-apparent form of the disease. Differences between the intensity of blood eosinophilia, the area of immediate cutaneous reaction and the number of Schistosoma eggs eliminated in the stools proved not to be statistically significant (p>0.05) when the non-apparent and acute cases of schistosomiasis were compared. These cases actually may be considered evidences of the non-apparent form hitherto merely taken for granted in the literature.

Functional studies on magnetic resonance

Dissertation submitted in Faculdade de Ciências e Tecnologia of Universidade Nova de Lisboa for the degree of Master in Biomedical Engineering

Acute schistosomiasis: clinical, diagnostic and therapeutic features

Three distinct syndromes caused by schistosomiasis have been described: cercarial dermatitis or swimmer's itch, acute schistosomiasis or Katayama fever, and chronic schistosomiasis. Complications of acute schistosomiasis have also been reported. The absence of a serological marker for the acute stage has hindered early diagnosis and treatment. Recently, an ELISA test using KLH (keyhole limpet haemocyanin) as antigen, has proved useful in differentiating acute from chronic schistosomiasis mansoni. Clinical and experimental evidence indicate that steroids act synergistically with schistosomicides in the treatment of Katayama syndrome. In this paper, clinical, diagnostic and therapeutic features of acute schistosomiasis are updated.

Laboratory acquired infection by the virus SP H 114202 (Arenavirus: Arenaviridae): clinical and laboratory findings

Here in is described the clinical and laboratorial findings of a laboratory-acquired infection caused by the virus SP H 114202 (Arenavirus, family Arenaviridae) a recently discovered agent responsible for a viral hemorrhagic fever. The patient was sick for 13 days. The disease had an abrupt onset characterized by high fever (39ºC.), headache, chills and myalgias for 8 days. In addition, on the 3rd day, the patient developed nauseas and vomiting, and in the 10th, epigastralgia, diarrheia and gengivorrhagia. Leucopenia was seen within the 1 st week of onset, with counts as low as 2,500 white cells per mm³. Counts performed after the 23th day of the onset were within normal limits. With the exception of moderate lymphocitosis, no changes were observed in differential counts. An increase in the liter of antibodies by complement fixation, neutralization and ELISA (IgM) was detected. Suckling mice and baby hamsters were inoculated intracerebrally with 0.02 ml of blood samples collected in the 2nd and 7th days of disease. Attempts to isolate the virus were also made in Vero cells. No virus was isolated. This virus was isolated before in a single occasion in São Paulo State, in 1990, from the blood of a patient with hemorrhagic fever with a fatal outcome. The manipulation of the virus under study, must be done carefully, since the transmission can occur through aerosols.

Clinical and laboratorial features of visceral toxocariasis in infancy

Forty children with a diagnosis of Visceral Toxocariasis were evaluated prospectively from February 1982 to June 1989. Diagnosis was established by clinical, laboratorial and serological (ELISA - ES Toxocara canis antigen) evaluations. A great clinical polymorphism was found in our patients, ranging from unspecific or absent manifestations to an exhuberant symptomatology. The laboratorial findings were: leukocytosis,eosinophilia and elevation of serum gammaglobulin and isohemagglutinin levels. No significant relationship between clinical findings and laboratorial parameters was found. Serology (ELISA) was a method of great diagnostic support but did not show a correlation with clinical and laboratorial findings in this study. There was a significant relationship between pulmonary manifestations and the presence of signs and/or symptoms, when the patients were sent to us. Our findings, especially the high incidence of pulmonary manifestations, suggest that Visceral Toxocariasis has to be included in the differential diagnostic of children with pulmonary manifestations, characteristic epidemiological data and associated eosinophilia.

Severe scorpion envenomation in Brazil: clinical, laboratory and anatomopathological aspects

Scorpion stings in Brazil are important not only because of their incidence but also for their potential ability to induce severe, and often fatal, clinical situations, especially among children. In this report we present the clinical and laboratory data of 4 patients victims of scorpion stings by T. serrulatus, who developed heart failure and pulmonary edema, with 3 of them dying within 24 hours of the sting. Anatomopathologic study of these patients revealed diffuse areas of myocardiocytolysis in addition to pulmonary edema. The surviving child presented enzymatic, electrocardiographic and echocardiographic changes compatible with severe cardiac involvement, which were reversed within 5 days. These findings reinforce the need for continuous monitoring of patients with severe scorpion envenoming during the hours immediately following the sting.

Off-the-person electrocardiography: performance assessment and clinical correlation

Previous work by our group introduced a novel concept and sensor design for “off-the-person” ECG, for which evidence on how it compares against standard clinical-grade equipment has been largely missing. Our objectives with this work are to characterise the off-the-person approach in light of the current ECG systems landscape, and assess how the signals acquired using this simplified setup compare with clinical-grade recordings. Empirical tests have been performed with real-world data collected from a population of 38 control subjects, to analyze the correlation between both approaches. Results show off-the-person data to be correlated with clinical-grade data, demonstrating the viability of this approach to potentially extend preventive medicine practices by enabling the integration of ECG monitoring into multiple dimensions of people’s everyday lives. © 2015, IUPESM and Springer-Verlag Berlin Heidelberg.

Schistosoma mansoni circulating polysaccharide and protein antigens recognized by sheep antisera in patients with different clinical forms of schistosomiasis before and after treatment

Two sheep antisera, one of which raised against polysaccharide (Po) and other against protein (Pt) components of Schistosoma mansoni adult worms, were assessed by ELISA for their ability to detect circulating parasite antigens in patients with different clinical forms of chronic schistosomiasis mansoni. The former antiserum detected parasite antigens in liver granulomata and the latter in renal glomeruli from schistosomiasis patients and mice experimentally infected with S. mansoni. In general, the levels and/or positivity rate of circulating antigens and specific IgG antibodies were significantly higher in patients with hepatointestinal (HI) and hepatosplenic (HS) forms than in mild intestinal (I) forms. An association between Po antigens and clinical features of the disease was observed, as the level of these antigens was low (137 ng/ml) as well as the positivity rate (7.9%) in patients with I forms; values that were intermediate (593 ng/ml and 33.3%) in those with HI forms, and high (1.563 ng/ml and 50.0%) in more severe HS forms. The Pt antigens were detected in the studied clinical forms not differing statistically but, the positivity rate was significantly higher in HS forms comparatively to I forms. The antisera studied revealed distinct circulating antigen profiles, and the prognostic value of Po and Pt antigens was suggested.

Downstream processing development of enveloped viruses for clinical applications: innovative tools for rational process optimization

Dissertation presented to obtain a Ph.D. degree in Engineering and Technology Sciences, Biotechnology at the Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa

Clinical-epidemiologic study of schistosomiasis mansoni in Ponte do Pasmado, a village in the municipality of Itinga, State of Minas Gerais, Brazil, 1992

A clinical-epidemiologic study of schistosomiasis mansoni was conducted in the population of Ponte do Pasmado, a village in the municipality of Itinga, state of Minas Gerais, Brazil. Faecal Parasitology by the Kato-Katz method and clinical examination were performed in 93.8% and 82.8% of the local population, respectively. A socioeconomic survey was also made and the signs and symptoms presented by the patients were recorded, as well as their contacts with natural waters. The rate of Schistosoma mansoni infection was 50.3%; the peak of infection occurred during the second decade of life; there was a predominance of low egg counts in faeces (85.89% of positive patients eliminated less than 500 eggs per gram of faeces); the splenomegaly rate was 1.23%. When the risk factors for S. mansoni infection were studied, significant risks were detected in activities such as fetching water, washing dishes, bathing, and crossing streams.

Seroepidemiological and clinical study of Chagas' disease in Nicaragua

With the aim of determining the prevalence, immunological profile, and knowing the electrocardiographic alterations, a clinical and Seroepidemiological study of Chagas' disease was performed in three rural settlements located at the North, East and West of Nicaragua. Anti T. cruzi antibodies were searched by indirect immunofluorescence (IFI) and hemagglutination (IHA) in a total of 803 subjects. Seropositives and the same number of seronegatives, matched by age and sex, were included in a case-control design for the electrocardiographic assessment. Antibody prevalence was 13.1, 4.3 and 3.2% in the respective settlements. In the first two the immunological profile corresponds to that of an endemic zone of long standing, were transmission has decreased, and in the third the pattern is of a zone under control. Electrocardiographic changes compatible with Chagas' disease were found in seropositive individuals, but difference with control group was not statistically significant. It is concluded that the disease is endemic in the three settlements and the clinical aspect requires further evaluation, including additional cardiologic techniques.

Jungle yellow fever: clinical and laboratorial sudies emphasizing viremia on a human case

The authors report the clinical, laboratorial and epidemiological aspects of a human case of jungle yellow fever. The patient suffered from fever, chills, sweating, headaches, backaches, myalgia, epigastric pains, nausea, vomiting, diarrhea and prostration. He was unvaccinated and had been working in areas where cases of jungle yellow fever had been confirmed. Investigations concerning the yellow fever virus were performed. Blood samples were collected on several days in the course of the illness. Three of these samples (those obtained on days 5,7 and 10) were inoculated into suckling mice in attempt to isolate virus and to titrate the viremia level. Serological surveys were carried out by using the IgM Antibodies Capture Enzyme Linked Immunosorbent Assay (MAC-ELISA), Complement Fixation (CF), Hemagglulinalion Inhibition (HI) and Neutralization (N) tests. The yellow fever virus, recovered from the two first samples and the virus titration, showed high level of viremia. After that, specific antibodies appeared in all samples. The interval between the end of the viremia and the appearance of the antibodies was associated with the worsening of clinical symptoms, including bleeding of the mucous membrane. One must be aware of the risk of having a urban epidemics in areas where Aedes aegypti is found in high infestation indexes.

Clinical and endoscopic findings in the mucosae of the upper respiratory and digestive tracts in post-treatment follow-up of paracoccidioidomycosis patients

Systematic examination of the upper respiratory and digestive tracts (URDT) was performed in a group of 80 paracoccidioidomycosis (PCM) patients submitted to post-treatment follow-up ranging from 8 months to 17 years. Mucosae of the URDT had been involved prior to specific treatment in 74 patients, distributed as follows: oropharynx, 50 (41 alone, 7 in association with the larynx, and 2 with the nasal mucosa); larynx, 30 (23 alone and 7 in association); and nasal mucosa, 3(1 alone and 2 in association). Inactive lesions were observed in all the 50 patients with lesions of the oropharynx, 3 of whom with deforming scars (1 with retraction of the tongue and 2 with narrowing of the oral orifice). One case presented a destructive lesion, with perfuration of the palate. Of the other 46 cases, examination showed nacreous white striated scars which were nearly imperceptible in some cases and in others displayed partial retraction of anatomical structures without any alteration of their features. Patients presented a high rate of missing teeth. In 3 patients with involvement of the nasal mucosa, none of whom presented active PCM lesions, 2 still had nasal voices. In 30 patients with lesions of the larynx, 1 suffered a relapse of PCM and 2 developed epidermoid carcinoma. Of the other 27 cases, none of whom had active PCM lesions, 15 presented dysphonia, 3 were tracheotomized, and 9 were asymptomatic.

Intestinal microsporidiosis in HIV-positive patients with chronic unexplained diarrhea in Rio de Janeiro, Brazil: diagnosis, clinical presentation and follow-up

After the diagnosis of two cases of microsporidial intestinal infection in 1992, in Rio de Janeiro, we have started looking for this parasite in HIV-infected patients with chronic unexplained diarrhea. We have studied 13 patients from Hospital Evandro Chagas, IOC-FIOCRUZ. Fecal specimens from these patients were examined for the presence of Cryptosporidia and Microsporidia, in addition to routine examination. Spores of Microsporidia were found in the stools of 6 (46.1%) of the 13 patients studied, with 2 histological jejunal confirmations. The Microsporidia-infected patients presented chronic diarrhea with about 6 loose to watery bowel movements a day. Five infected patients were treated with Metronidazole (1.5 g/day). They initially showed a good clinical response, but they never stopped eliminating spores. After about the 4th week of therapy, their diarrhea returned. Two patients utilized Albendazole (400 mg/day-4 weeks) with a similar initial improvement and recurrence of the diarrhea. Intestinal Microsporidiosis seems to be a marker of advanced stages of AIDS, since 5 of our 6 infected patients were dead after a 6 month period of follow-up. The present study indicates that intestinal microsporidiosis may be a burgeoning problem in HIV-infected patients with chronic diarrhea in Brazil, which deserves further investigation.