Guadalupe García-Elorriaga

Display and release of the Plasmodium falciparum circumsporozoite protein using the autotransporter MisL of Salmonella enterica

The Salmonella enterica MisL (protein of membrane insertion and secretion) is an autotransporter with high homology to AIDA-I (adhesin involved in diffuse adherence) of enteropathogenic Escherichia coli. Considering that it has been reported that the MisL beta translocator domain is able to display heterologous passenger peptides to the bacterial surface, we developed a system to display proteins and release them to the external environment by means of proteolytic cleavage. Plasmids were constructed encoding 8 or 53 repeats of the NANP (Asp-Ala-Asp-Pro) tetrapeptide, which is the main B cell epitope of the Plasmodium falciparum circumsporozoitic protein (CSP), fused to the the MisL beta-domain and including the recognition cleavage sequence from the E. coli OmpT surface protease. E. coli XL-10Gold and BL21(DE3) (OmpT positive and negative, respectively) and Salmonella enterica serovar Typhimurium SL3261 (Aro A(-)) were transformed with the plasmids and, both expression and localization of the fusion proteins were assessed by Western blot, indirect immunofluorescence, and flow cytometry, using a monoclonal antibody against (NANP)(3). Higher expression of the (NANP)(8) and (NANP)(53) fusion proteins was demonstrated on the bacterial surface of the OmpT negative E. coli strains and the (NANP)(53) in the culture supernatant of E. coli XL-10Gold indicating a protease mediated cleavage. The flow cytometry analysis suggested 71 and 98% cleavage efficiency for the (NANP)(8) and (NANP)(53), respectively, in E. coli XL-10Gold. Similar results were obtained in S. enterica serovar Typhimurium SL3261, suggesting the involvement of other proteases related to OmpT. These results demonstrate that MisL may be used for the autodisplay and release of passenger proteins in attenuated Salmonella or E. coli strains, which may have several applications in vaccine design.

−592 and −1082 interleukin-10 polymorphisms in pulmonary tuberculosis with type 2 diabetes

OBJECTIVE To determine the polymorphisms of Interleukin-10 (IL-10) (-592, -1082) in pulmonary tuberculosis (PTB) with and without type 2 diabetes (T2D). METHODS We studied a Mexican mestizo population of 37 patients with TB in remission (TBr) and 40 with active pulmonary TB (PTB), 21 patients with TB + T2D, 47 blood donors accepted, and 13 healthy health-care workers with tuberculin skin test positive. Determination of IL-10 polymorphisms was performed by real-time Polymerase chain reaction. RESULTS IL-10-592C/A presented in a greater proportion in healthy individuals than in patients with type 2 diabetes and TB in a not quite significant statistically manner. IL-10-1082A/A presented more frequently in the group of patients with both diseases, not being statistically significant in comparison with the group of healthy subjects. CONCLUSIONS This study describes two important new findings. First, it reveals that the IL-10 (-592 A/A and -592 C/C) polymorphisms were found in a greater proportion in a group of patients with T2D and TB than in healthy subjects. Second, the study provides evidence that the (-1082 G/G) polymorphism presented with greater frequency in healthy individuals than in patients with both of these diseases.

Polymorphisms in Tumor Necrosis Factor and Lymphotoxin A in Tuberculosis without and with Response to Treatment

This study compared the frequency of the genetic polymorphisms of tumor necrosis factor (TNF) in pulmonary tuberculosis without and with response to treatment. We carried out an observational, prospective, comparative study. Three groups were studied: healthy subjects, responders, and non-responders to directly observed treatment short-course. We took a peripheral blood sample for identification of polymorphic genotypes TNF −308G/A and lymphotoxin A (LTA) +252G/A by polymerase chain reaction, and their later digestion with the Nco1 restriction enzyme. We studied a total of 138 subjects: 42 (non-responders) and 48 in each of the remaining groups. Healthy subjects had significantly high frequency of the LTA +252A allele compared to groups of patients and could be related with protection from the disease. Patients had higher frequency of the non-polymorphic allele LTA +252G than healthy subjects. With regard to LTA +252G/A genotype, we did find a significant difference with a greater frequency in the group of patients. The LTA +252G/A genotype was associated with impaired response to treatment.

Molecular Analysis of Mycobacteria Isolated in Mexican Patients with Different Immunodeficiencies in a Tertiary Care Hospital

BACKGROUND AND AIMS The prevalence of infections with Mycobacterium tuberculosis (MTB) and non-tuberculous mycobacteria (NTM) species in patients with immunodeficiencies in Mexico is unknown. The aim of this study was to identify, at the molecular level, the mycobacterial species most frequently affecting patients with immunodeficiencies and evaluate the genotypic diversity of MTB complex strains. METHODS We conducted a retrospective study of 97 strains in patients with the diagnosis of pulmonary (all isolates were of pathological significance) or extrapulmonary tuberculosis. PCR analysis was performed to determine whether they belonged to the MTB complex (MTC) or the Mycobacterium avium complex (MAC). Noncharacterized NTM were sequenced and, finally, MTC were genotyped by MIRUs-VNTR and spoligotyping. RESULTS Of the 97 mycobacterial strains isolated, 53% were M. tuberculosis, 10% M. bovis, 24% M. avium, 9% M. simiae, 2% M. kansasii and 2% M. gordonae. A great genetic diversity was found by MIRU-VNTR with the greatest polymorphism in MIRU 10, 16, 23 and 27. By spoligotyping, the predominant family was T1. Combining both methods, the association of 13 strains in four different groups was found. CONCLUSIONS This is the first molecular analysis of mycobacteria isolated from patients with immunodeficiencies in Mexico, describing the prevalence of different mycobacterial species in this population. A great genetic diversity of MTB strains was identified. This is also the first report in Mexico describing clinically important isolates of M. simiae.

Clinical usefulness of the nested polymerase chain reaction in the diagnosis of extrapulmonary tuberculosis.

OBJECTIVE To evaluate the effectiveness of nested polymerase chain reaction (PCR) for diagnosis of extrapulmonary tuberculosis (ETB), as well as the impact of PCR results on clinical management. MATERIALS AND METHODS We conducted a study of nested PCR tests in 45 patients and a review of patient hospital files, calculating sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV). RESULTS PCR was positive in 51% of cases; PCR sensitivity for diagnosing TB was 86%, specificity was 79%, PPV was 76%, and NPV was 88%. When solely analyzing urine samples, sensitivity and NPV increased to 100%. PCR exerted an influence on management in 27% of patients. CONCLUSIONS PCR for rapid diagnosis of extrapulmonary TB has an adequate effect, which improves when performed on urine. The results of PCR exerted an acceptable impact on the clinical management of these patients.

Clinical, radiological and molecular diagnosis correlation in serum samples from patients with osteoarticular tuberculosis

OBJECTIVE To assess the role of polymerase chain reaction (PCR) in serum samples, in the diagnosis of osteoarticular tuberculosis (OTB) in a setting where only clinical and imaging diagnoses determine the treatment. METHODS A total of 44 consecutive serum specimens were collected from clinically suspected OTB patients, based on clinical and radiological [X-ray or magnetic resonance imaging/computed tomography] features. They were screened by in-house nested PCR. In addition, a few specimens were examined by Gram stain, acid-fast bacilli stain, histopathology and routine bacterial culture. A total of 39 specimens were collected from patients suffering from other bone diseases of nontuberculous origin and included as negative controls. RESULTS Of the 44 clinically suspected OTB patients, in-house nested PCR was positive in 40 (91%) cases; PCR was negative in 38 (97%) negative controls. Sensitivity and specificity of our in-house nested PCR was 90.9% and 97.4%, respectively. The PCR report was available within 48 h. It was possible to standardize serum PCR technique and in positive cases, a good correlation was observed in terms of an adequate treatment response. CONCLUSIONS Nested PCR in serum samples is a rapid, highly sensitive and specific modality for OTB detection. PCR should be performed in addition to clinical evaluation, imaging studies, acid-fast bacilli staining, culture and histopathology diagnosis, if possible.

Anti-Chlamydophila pneumoniae Antibodies as Associated Factor for Carotid Atherosclerosis in Patients with AIDS

Atherosclerosis is a multifactor disease. Lately, infectious factors such as C. pneumoniae have been found to be involved. To determine whether the infection by C. pneumoniae is a risk factor for atherosclerosis in patients with AIDS. Case-control study on 43 patients with AIDS under HAART (16 cases and 27 controls). To document atherosclerosis, a carotid and transcranial Doppler ultrasound was performed. Anti-C pneumoniae antibodies were searched using a microimmunofluorescence test for IgM and IgG levels. To study the associations with risk of atherosclerosis, Odds Ratios were calculated for each IgG anti-C. pneumoniae antibody titre. A titre of 1:64 significantly increased the risk of atherosclerosis. These results suggest that hypertriglyceridemia and C. pneumoniae infection coexistence significantly increases the risk of atherosclerosis. The inverse geometric average of the antibodies titre against C. pneumoniae in individuals with atheromatous plaque fell to 64, two titres above the controls. This difference turned out to be statistically significant. Exposure to C. pneumoniae with antibodies (IgG) should be considered in any HIV diagnosed patient as a risk factor for atherosclerosis, having found that the inverse geometric averages of antibodies titre are significantly different comparing cases and controls, especially in patients with dyslipidemia, hypertriglyceridemia or in patients whose treatments could cause these conditions. In patients with concomitant hypertriglyceridemia, the association increases up to three times. It is advisable that AIDS patients take a serological test to determine exposure to C. pneumoniae, and to assess treatment options.

Interferon γ in patients with HIV/AIDS and suspicion or latent tuberculosis infection

OBJECTIVE To assess the usefulness of IGRA test (QuantiFERON(®)-Cell mediated immune) compared with the tuberculin skin test. METHODS A cross-sectional study was carried out in Mexico, 25 infected patients with HIV-AIDS and the suspicion or with latent tuberculous infection (LTBI) who were >18 years of age and without treatment for tuberculosis (TB), were enrolled in the study. RESULTS Median cluster of differentiation (CD4) count was 364 cells/μ L and median HIV viral load was 50 copies/mL. Overall, 20 patients (80%) had at least one positive diagnostic test for LTBI: four (16%) had a positive tuberculin skin test and 19 (76%), a positive QuantiFERON(®)-tuberculosis. CONCLUSIONS No agreement is found between the two diagnostic tests: k = -0.004, 95% confidence interval (-0.2219, 0.2210). Additional longitudinal studies among HIV-infected populations with high prevalence of TB are needed to further assess the usefulness of IGRAs in this patient population.

Asociación entre enfermedad cardiovascular y anticuerpos contra Chlamydia pneumoniae

Objetivo. Precisar si existe asociacion entre enfermedad cardiovascular (ECV) y anticuerpos contra Chlamydia en poblacion mexicana. Material y metodos. Estudio transversal, realizado en la Unidad de Investigacion en Inmunologia e Infectologia -Hospital de Infectologia del Centro Medico Nacional La Raza (CMNR)- y en el Servicio de Cirugia Cardiovascular y Asistencia Circulatoria, del Hospital General del CMNR, Instituto Mexicano del Seguro Social (IMSS), de agosto de 1998 a abril de 2000. Se determinaron anticuerpos IgG e IgM contra C. psittaci, C. trachomatis y C. pneumoniae mediante microinmunofluorescencia, en suero de 70 pacientes con ECV hospitalizados en el CMNR, mayores de 30 anos, de uno u otro sexo, y se compararon con 140 sanos, pareados por edad y sexo. Se utilizaron muestras aleatorias simples, con un tamano poblacional de 110, una prevalencia de 50% y un nivel de confianza de 99%. Para establecer la diferencia entre las proporciones de los titulos se utilizo ji cuadrada y se calculo la razon de momios. Resultados. El 94.3% (66/70) de los pacientes presento IgG en contra de C. pneumoniae vs 37% (52/140) de los individuos sanos (p<0.001). Conclusiones. Existe una fuerte asociacion entre anticuerpos IgG hacia C. pneumoniae y ECV.

Microbiología de lavado broncoalveolar en lactantes con neumonía bacteriana adquirida en la comunidad de mala evolución

BACKGROUND Community-acquired pneumonia (CAP) is one of the most common infectious causes of morbidity and mortality in children <5 years of age. The aim of the study was to clarify the bacterial etiologic diagnosis in infants with CAP. METHODS A prospective, cross-sectional and descriptive study in patients 6 months to 2 years 11 months of age with CAP with poor outcome was conducted. Patients were admitted to the Pediatric Pneumology Service and underwent bronchoscopy with bronchoalveolar lavage (BAL), taking appropriate measures during the procedure to limit the risk of contamination. RESULTS Aerobic bacteria isolated were Moraxella sp. 23%, Streptococcus mitis 23%, Streptococcus pneumoniae 18%, Haemophilus influenzae 12%, Streptococcus oralis 12%, and Streptococcus salivarius 12%. CONCLUSIONS In contrast to other reports, we found Moraxella sp. to be a major bacterial pathogen, possibly because of improved detection with bronchoscopy plus BAL.