Genetic Polymorphisms in Cytokine Genes in Colombian Patients with Ocular Toxoplasmosis.

Toxoplasmosis is caused by infection with the protozoan parasite Toxoplasma gondii, which has the capacity to infect all warm-blooded animals worldwide. Toxoplasmosis is a major cause of visual defects in the Colombian population; however, the association between genetic polymorphisms in cytokine genes and susceptibility to ocular toxoplasmosis has not been studied in this population. This work evaluates the associations between polymorphisms in genes coding for the cytokines tumor necrosis factor alpha (TNF-α) (rs1799964, rs1800629, rs1799724, rs1800630, and rs361525), interleukin 1ß (IL-1ß) (rs16944, rs1143634, and rs1143627), IL-1α (rs1800587), gamma interferon (IFN-γ) (rs2430561), and IL-10 (rs1800896 and rs1800871) and the presence of ocular toxoplasmosis (OT) in a sample of a Colombian population (61 patients with OT and 116 healthy controls). Genotyping was performed with the "dideoxynucleotide (ddNTP) primer extension" technique. Functional-effect predictions of single nucleotide polymorphisms (SNPs) were done by using FuncPred. A polymorphism in the IL-10 gene promoter (-1082G/A) was significantly more prevalent in OT patients than in controls (P = 1.93e-08; odds ratio [OR] = 5.27e+03; 95% confidence interval [CI] = 3.18 to 8.739; Bonferroni correction [BONF] = 3.48e-07). In contrast, haplotype "AG" of the IL-10 gene promoter polymorphisms (rs1800896 and rs1800871) was present at a lower frequency in OT patients (P = 7e-04; OR = 0.10; 95% CI = 0.03 to 0.35). The +874A/T polymorphism of IFN-γ was associated with OT (P = 3.37e-05; OR = 4.2; 95% CI = 2.478 to 7.12; BONF = 6.07e-04). Haplotype "GAG" of the IL-1ß gene promoter polymorphisms (rs1143634, rs1143627, and rs16944) appeared to be significantly associated with OT (P = 0.0494). The IL-10, IFN-γ, and IL-1ß polymorphisms influence the development of OT in the Colombian population.

Prevalence of Toxoplasma gondii in cats from Colombia, South America and genetic characterization of T. gondii isolates.

Cats are important in the epidemiology of Toxoplasma gondii infection because they are the only hosts that can excrete the environmentally-resistant oocysts. In the present study, prevalence of T. gondii was determined in serum, feces, and tissues of 170 unwanted cats from Colombia, South America. Antibodies to T. gondii were assayed by the modified agglutination test and found in 77 of 170 (45.2%) cats with titers of <1:5 in 93, 1:5 in eight, 1:10 in 17, 1:20 in 10, 1:40 in seven, 1:80 in four, 1:160 in eight, 1:320 in six, and 1:640 or higher in 17 cats. T. gondii oocysts were not found in feces of any cat as ascertained by bioassay in mice. Tissues (brain, heart, tongue) of 116 cats were bioassayed in mice or cats. T. gondii was isolated from tissues of 15 of the 42 cats with titers of 1:40 or higher and not from any of the 90 cats titers of 1:20 or lower. Of the 29 cats whose tissues were bioassayed individually, T. gondii was isolated from the tongues of nine, hearts of eight, and brains of five. Mice inoculated with tissues of 12 of 15 infected cats died of toxoplasmosis; with nine T. gondii isolates all infected mice died. Overall, 65 of 92 (70%) of T. gondii-infected mice died of toxoplasmosis. Genotyping of these 15 isolates using polymorphisms at the SAG1, SAG2, SAG3, BTUB, and GRA6 loci revealed that three isolates (TgCtCo1, 2, and 7) had Type I alleles and one isolate (TgCtCo8) had Type II allele at all five loci. Eleven isolates contained the combination of Type I and III alleles and were divided into three genotypes, with TgCtCo3,5,6,9,12,13 and 15 had alleles I, I, III, I and III, TgCtCo4,10,11 had alleles I, III, III, I and I, and TgCtCo14 had alleles I, III, III, III, and III, at loci SAG1, SAG2, SAG3, BTUB and GRA6, respectively. All infected mice from each group had identical genotype except one mouse infected with TgCtCo5 had a Type III allele at locus BTUB and a unique allele (u-1) at locus SAG1 indicating mixed infection for TgCtCo5, whereas the rest seven mice had a Type I alleles at both loci.

A maternal screening program for congenital toxoplasmosis in Quindio, Colombia and application of mathematical models to estimate incidences using age-stratified data.

We studied 937 pregnant women from Quindio, Colombia for the presence of specific anti-Toxoplasma gondii IgG antibodies using the indirect immunofluorescence antibody technique (IFAT-IgG). Specific anti-T. gondii IgM antibodies detected using the immunosorbent agglutination assay (ISAgA-IgM) were investigated in patients with high titers in the IFAT-IgG (dilutions > or = 1:1,024). We used mathematical models based on the age prevalence results of the IFAT-IgG to estimate the number of seroconversions and these were compared with the results predicted by the IgM based-incidence results. We found 15 positive cases by ISAgA-IgM and we were able to follow the children of six mothers from this group in which we found one case of congenital toxoplasmosis with the development of a retinal scar despite prenatal and postnatal treatment. The estimation of new cases for the annual total of pregnancies (approximately 8,000) in the Quindio region was 30-120 according to the ISAgA-IgM results and 57-85 using mathematical models. Thus, mathematical models based on age prevalence can give useful estimations of the magnitude of the problem.

Frequency of tuberculous and non-tuberculous mycobacteria in HIV infected patients from Bogota, Colombia.

BACKGROUND: The prevalence of infections by Mycobacterium tuberculosis and non-tuberculous Mycobacterium species in the HIV-infected patient population in Colombia was uncertain despite some pilot studies. We determined the frequency of isolation of Mycobacterium tuberculosis and of non-tuberculous Mycobacterium species in diverse body fluids of HIV-infected patients in Bogota, Colombia. METHODS: Patients who attended the three major HIV/AIDS health care centres in Bogota were prospectively studied over a six month period. A total of 286 patients were enrolled, 20% of them were hospitalized at some point during the study. Sixty four percent (64%) were classified as stage C, 25% as stage B, and 11% as stage A (CDC staging system, 1993). A total of 1,622 clinical samples (mostly paired samples of blood, sputum, stool, and urine) were processed for acid-fast bacilli (AFB) stain and culture. RESULTS: Overall 43 of 1,622 cultures (2.6%) were positive for mycobacteria. Twenty-two sputum samples were positive. Four patients were diagnosed with M. tuberculosis (1.4%). All isolates of M. tuberculosis were sensitive to common anti-tuberculous drugs. M. avium was isolated in thirteen patients (4.5%), but only in three of them the cultures originated from blood. The other isolates were obtained from stool, urine or sputum samples. In three cases, direct AFB smears of blood were positive. Two patients presented simultaneously with M. tuberculosis and M. avium. CONCLUSIONS: Non-tuberculous Mycobacterium infections are frequent in HIV infected patients in Bogota. The diagnostic sensitivity for infection with tuberculous and non-tuberculous mycobacteria can be increased when diverse body fluids are processed from each patient.

Does human toxoplasmosis involve an imbalance in T1/T2 cytokines?

The T1 (interferon-gamma, interleukin-12, interleukin-2) and T2 (interleukin-4, interleukin-10, interleukin-6) cytokine groups constitute two polar responses of the immune system. The T1 group is a predominantly cellular response, while the T2 group response is mainly humoral. The hypothesis forwarded here links these subgroups of induced cytokines to the various clinical forms of human toxoplasmosis. Ocular toxoplasmosis in immunocompetent patients could be attributed to a T1 hyper-response, whereas congenital toxoplasmosis, toxoplasmic encephalitis (in immunodeficient patients) and active chronic toxoplasmosis (with persistent lymphadenophathy) would be characterized by a predominantly T2 response. Confirmation that this kind of immunological imbalance effectively underlies the various clinical forms of toxoplasmosis would open the way for a new range of treatments based on immunomodulation.

Incidence and clinical characteristics in a Colombian cohort of ocular toxoplasmosis.

PURPOSE: To evaluate the incidence and clinical features of patients with ocular toxoplasmosis in a Colombian cohort. METHODS: We collected prospectively the clinical features of patients with ocular toxoplasmosis seen at the 'Universidad del Quindio' health centre between September 2005 and September 2007 (24 months). RESULTS: Seventy patients were included in the analysis (95 affected eyes). The median age at the first episode was 21 years (range: 1-54 years). Thirty-two patients had active lesions (45.7%), one of them had active lesions in both eyes. The acquisition of infection was determined in 14 patients as congenital (20%), in seven as an acquired postnatal infection (10%), and in 49 as undetermined (70%). Bilateral involvement was found in 25 cases (35.7%). Unilateral legal blindness (<20/200) was found in 14 of 37 inactive cases (37.8%). The most frequent complication was strabismus (n: 12; 13.4%). CONCLUSIONS: There is a high incidence of cases of ocular toxoplasmosis in Quindio region (three new episodes by 100,000 inhabitants by year). Clinical features of ocular toxoplasmosis in Colombia are similar to those reported in other regions but there are no comparative data about the size and number of lesions.

Frequency and factors associated with recurrences of ocular toxoplasmosis in a referral centre in Colombia.

AIM: To determine the frequency and clinically related factors for recurrences in toxoplasmic retinochoroiditis in Colombian patients. METHODS: Cross-sectional analysis based on clinical charts of patients examined during the period of September 2005 to July 2008 at the University medical centre in Quindio (Colombia). Patients with retinochoroidal lesions consistent with Toxoplasma infection were included. Comparisons were made with an index of recurrences adjusted for months of follow-up or of the available data of periods with and without recurrences RESULTS: The clinical charts of 56 patients were analysed. In total, 25 patients (44%) were seen during an active episode, and 31 patients during inactive periods. There were 25 patients (44%) without episodes of recurrence. The total number of recurrences was 80 episodes. The mean number of recurrences was of two recurrences each 11 years. Adjusted recurrences index indicated that the most important factors associated with recurrence were previous therapy with steroids without antibiotics and previous subconjunctival injection of steroids. CONCLUSIONS: The use of systemic steroids without antibiotics and subconjunctival injection of steroids were identified as the main factors related to recurrence in this group of patients.

Detection of specific immunoglobulin E during maternal, fetal, and congenital toxoplasmosis.

Toxoplasma immunoglobulin E (IgE) antibodies in 664 serum samples were evaluated by using an immunocapture method with a suspension of tachyzoites prepared in the laboratory in order to evaluate its usefulness in the diagnosis of acute Toxoplasma gondii infection during pregnancy, congenital infection, and progressive toxoplasmosis. IgE antibodies were never detected in sera from seronegative women, from patients with chronic toxoplasma infection, or from infants without congenital toxoplasmosis. In contrast, they were detected in 86.6% of patients with toxoplasmic seroconversion, and compared with IgA and IgM, the short kinetics of IgE was useful to date the infection precisely. For the diagnosis of congenital toxoplasmosis, specific IgE detected was less frequently than IgM or IgA (25 versus 67.3%), but its detection during follow-up of children may be interesting, reflecting an immunological rebound. Finally, IgE was detected early and persisted longer in progressive toxoplasmosis with cervical adenopathies, so it was also a good marker of the evolution of toxoplasma infection.

Frequency of specific anti-Toxoplasma gondii IgM, IgA and IgE in colombian patients with acute and chronic ocular toxoplasmosis.

We studied the frequency of specific anti-Toxoplasma IgM, IgA and IgE antibodies in serum of 28 immunocompetent Colombian patients, selected by ophthalmologists and with lesions that were compatible with ocular toxoplasmosis. Patients were classified in three groups: (i) group 1 consisted of ten patients with a first episode; (ii) group 2, with seven patients with a recurrence and (iii) group 3, consisted of eleven patients with chronic chorioretinal lesion without uveitis. We found that 10/28 (35%) of Colombian patients with ocular toxoplasmosis possessed at least one serological marker for Toxoplasma infection different from IgG. In group 1 (first episode), we found simultaneous presence of specific IgM plus IgA plus IgE in 1/10 (10%). In group 2 (recurrences) in 1/7 (14%) we found IgM and IgA test positives and in 1/7 (14%) we found IgM and IgE tests positives. In group 3 (toxoplasmic chorioretinal scar) the IgA serological test was positive in 2/11 (18%). These results show that serum IgM or IgA or IgE can be present during recurrences.

Interferon-gamma signal transduction during parasite infection: modulation of MAP kinases in the infection of human monocyte cells (THP1) by Toxoplasma gondii.

We assayed mitogen-activated protein (MAP) kinase phosphorylation in a human monocyte cell line (THP1) during their infection by Toxoplasma gondii. In addition, we tested the effect of specific MAP kinase inhibitors (PD098059 and SB203580) on parasite invasion. MAP kinase phosphorylation was increased in the cytosol and membrane fractions of THP1 infected with T. gondii. The MAP kinase phosphorylation of uninfected THP1 cells was not significantly modified by incubation for 20 h with 1000 U/ml of IFN-gamma. However, IFN-gamma treatment of infected cells significantly reduces the increase in phosphorylation caused by parasite infection. There was also MAP kinase activity in the cytosol and membrane fractions of extracellular T. gondii tachyzoites. IFN-gamma altered the distribution of activity in subcellular fractions of extracellular T. gondii tachyzoites. This indicates that IFN-gamma directly affects parasite MAP kinase activity. The results provide evidence that MAP kinase pathways participate in the infection by T. gondii and that the decrease in MAP kinase activity in infected cells caused by IFN-gamma may be involved in mediating their protective signals.

Detection and localization of a Ca2+-ATPase activity in Toxoplasma gondii.

Toxoplasma gondii, the agent causing toxoplasmosis, is an obligate intracellular protozoan parasite. A calcium signal appears to be essential for intracellular transduction during the active process of host cell invasion. We have looked for a Ca2+-transport ATPase in tachyzoites and found Ca2+-ATPase activity (11-22 nmol Pi liberated/mg protein/min) in the tachyzoite membrane fraction. This ATP-dependent activity was stimulated by Ca2+ and Mg2+ ions and by calmodulin, and was inhibited by pump inhibitors (sodium orthovanadate or thapsigargin). We used cytochemistry and X-ray microanalysis of cerium phosphate precipitates and immunolabelling to find the Ca2+, Mg2+-ATPase. It was located mainly in the membrane complex, the conoid, nucleus, secretory organelles (rhoptries, dense granules) and in vesicles with a high calcium concentration. Thus, Toxoplasma gondii possesses Ca2+-pump ATPase (Ca2+, Mg2+-ATPase) as do eukaryotic cells.

Restriction fragment length polymorphism (RFLP) analysis and tuberculosis epidemiology.

In order to study polymorphisms of the DNA insertion sequence 6110 (IS6110) in Mycobacterium tuberculosis strains isolated from Colombian patients, together with resistance to antituberculous medications in the Department of Quindío, Colombia, a prospective study was conducted using a consecutive sample of 59 patients with symptomatic pulmonary tuberculosis whose cases had been confirmed by bacilloscopy, both with and without a history of treatment. The patients, who were participating in the Tuberculosis Control Program of the Regional Health Institute of Quindío in Armenia, included all individuals attending local health centers and hospitals between March and July 1993 who were referred to the regional institute. Sputum specimens from each patient were cultured and subjected to drug sensitivity tests. Subsequently, restriction fragment length polymorphisms (RFLP) of IS6110 from 27 patients were analyzed. The patients' treatment histories were used to classify their cases according to WHO criteria. Forty-five cultures were found positive, 44 for M. tuberculosis and 1 for M. africanum. Initial drug resistance was observed in 4 of 42 new cases, or 9.5% (95% CI: 0.6, 18), 2 showing resistance to isoniazid (INH) and 2 to isoniazid plus streptomycin (INH-SM). Acquired resistance was observed in 2 of the 3 chronic cases and relapses, the bacteria being resistant to isoniazid, rifampicin, and streptomycin (INH-RM-SM) in one case and to isoniazid, ethambutol, rifampicin, and streptomycin (INH-EMB-RM-SM) in the other. In those 27 strains subjected to RFLP analysis, the number of copies of IS6110 ranged from 6 to 17. Similarity coefficients revealed five distinct groups of strains. Overall, the RFLP analysis permitted most of the strains to be distinguished from one another, implying that the polymorphisms involved are sufficient to permit effective employment of this technique, which appears to have considerable potential for use in epidemiologic studies and in work designed to provide a basis for tuberculosis control program decision-making.

[Analysis of restriction fragment length polymorphism (RFLP) in epidemiology of tuberculosis]. / Análisis de polimorfismos de los fragmentos de restricción (RFLP) y epidemiología de la tuberculosis.

The purpose of this study was to determine the polymorphism of insertion segment 6110 (IS6110) in strains of Mycobacterium tuberculosis isolated from Colombian patients as well as the current status of resistance to antituberculosis drugs in the department of Quindío, Colombia. To this end, a prospective study was performed with a consecutive sample of 59 patients who sought care at local health centers and hospitals in rural and urban areas of Quindío from March to July 1993. The patients in the sample had symptomatic pulmonary tuberculosis confirmed by bacteriologic inspection of sputum, with and without a history of treatment, and were participants in the Tuberculosis Control Program of the Sectional Health Institute of Quindío in Armenia, Colombia. Sputum cultures and drug sensitivity tests were done. Later, restriction fragment length polymorphisms (RFLP) of IS6110 were analyzed in accordance with the protocols of van Soolingen et al. (1992). Cases were classified by treatment history, applying the criteria of WHO (1991). The results showed 44 cultures positive for M. tuberculosis and one positive for M. africanum. Primary drug resistance was found in 4 of 42 cultures, or 9.5% (CI 95%: 0.6 to 18); 4.8% were resistant to isoniazid (INH) and 4.8% to isoniazid and streptomycin (INH-SM). Acquired resistance was found in two of three cultures, or 66% (to isoniazid, rifampicin, and streptomycin [INH-RM-SM] and to isoniazid, ethambutol, rifampicin, and streptomycin [INH-EMB-RM-SM]). In 27 strains submitted to RFLP analysis, the number of copies of IS6110 varied from 6 to 17. Similarity coefficients revealed five distinct groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Role of interferon-gamma against invasion by Toxoplasma gondii in a human monocytic cell line (THP1): involvement of the parasite's secretory phospholipase A2.

We examined the role of IFN gamma in protection against Toxoplasma gondii in the monocytoid cell line THP1. The addition of IFN gamma to cultured infected THP1 cells reduced the number of parasitized cells without altering intracellular multiplication during the first 24 hr. This reduction was potentiated by bacterial lipopolysaccharide (LPS). We also examined the role of an enzyme important for T. gondii cellular invasion, secretory phospholipase-A2 (sPLA2) and its relation with IFN gamma-induced protection. Treatment of cells or parasites with a specific inhibitor of sPLA2 significantly reduced the number of infected cells at 6 hr. The addition of exogenous sPLA2 from Naja naja venom did not interfere with the protective effect of IFN gamma and conferred protection when used alone. PLA2 activity was measured in supernatants of parasites maintained in the presence of IFN gamma, and the results suggested that IFN gamma opposes cell invasion by T. gondii by suppressing parasite production of PLA2.

Involvement of tumor necrosis factor-alpha during infection of human monocytic cells by Toxoplasma gondii.

Although the involvement of tumor necrosis factor-alpha (TNF-alpha) during Toxoplasma gondii infection has largely been described in mouse models, only a few studies in human models have been reported. We demonstrated the role of TNF-alpha during the process of invasion by T. gondii in human monocytic cells. Cotreatment of cells with interferon-gamma (IFN-gamma) and lipolysaccharide (LPS) during T. gondii infection induced TNF-alpha production and decreased the number of parasitized cells (invasion index). Neutralization of the production of TNF-alpha using a specific monoclonal antibody or pentoxifylline enhanced the invasion index. The relationship between TNF-alpha production and protection of monocytic cells against T. gondii invasion was confirmed by treatment of infected cultures with exogenous TNF-alpha. Thus, in contrast to the results obtained in murine models but in accordance with those observed in other human models, the present study shows for the first time in human monocytic cells that T. gondii does not induce any TNF-alpha secretion and inhibits TNF-alpha production induced by IFN-gamma/LPS.

Fosfolipasas A2 y su importancia clínica / Phospholipases A2 and clinical importance

Se revisa el estado actual del conocimiento con respecto al papel fisiológico de las fosfolipasas A2, su clasificación funcional y molecular. Las PLA2 son enzimas que hidrolizan los ácidos grasos de membrana y llevan a la producción de derivados del ácido araquidónico entre otros. Se describen una serie de procesos patológicos en los cuales se encuentran involucradas. De otra parte se presentan los inhibidores específicos para cada una de las familias de PLA2 y su posible utilización terapéutica. Las PLA2 son enzimas implicadas en procesos inflamatorios, en acciones enzimáticas que deterioran varios sistemas y en el proceso de transducción de señal intracelular. El potencial de intervención con fines de tratamiento es bastante amplio y los inhibidores diseñados en los últimos años han sido muy específicos ya que han tenido en cuenta las particularidades estructurales de cada familia de PLA2, las cuales hacen que tengan funciones muy diferentes entre cada una de ellas. Se pueden augurar la aparición de nuevos tratamientos para enfermedades de tipo metabólico como la diabetes, inflamatorios como la sepsis, enfermedades autoinmunes e infecciones por la inhibición de procesos de invasión de organismos intracelulares obligatorios