Epitope of dengue virus E protein detect human antibodies associated with mild disease: a potential peptide for vaccine development

ABSTRACT The antigenic potential of seven immunogenic peptides of the dengue virus was evaluated in the sera of patients with dengue confirmed by IgM/IgG serology. Antibodies IgM and IgG against dengue virus peptides were analyzed by ELISA in 31 dengue sero-positive and 20 sero-negative patients. The P5 peptide showed significant IgG immunoreactivity mostly in the sera of patients with dengue without warning signs in comparison with patients with dengue with warning signs, correlating with mild disease. This finding suggests that the low antibody response against P5 epitope could be a risk factor for higher susceptibility to dengue virus infection with warning signs, and that P5 could be a potential antigen for vaccine development.

Epitope of dengue virus E protein detect human antibodies associated with mild disease: a potential peptide for vaccine development.

The antigenic potential of seven immunogenic peptides of the dengue virus was evaluated in the sera of patients with dengue confirmed by IgM/IgG serology. Antibodies IgM and IgG against dengue virus peptides were analyzed by ELISA in 31 dengue sero-positive and 20 sero-negative patients. The P5 peptide showed significant IgG immunoreactivity mostly in the sera of patients with dengue without warning signs in comparison with patients with dengue with warning signs, correlating with mild disease. This finding suggests that the low antibody response against P5 epitope could be a risk factor for higher susceptibility to dengue virus infection with warning signs, and that P5 could be a potential antigen for vaccine development.

Cytokine profile as diagnostic and prognostic factor in neonatal sepsis.

Antecedents: The serum levels of some cytokines can be useful in the diagnosis of neonatal sepsis; the prognostic value of a cytokine profile has not, to our knowledge, been explored in this disease. Objective: The objective of this study is to evaluate the diagnostic value of the serum levels of cytokines IL-1, -2, -4, -5, -6, -7, -8, -10, -12, -13, and -17, TNF, IFNγ, G-CSF, GM-CSF, MCP1, and MIP1ß in neonates with high risk of developing sepsis. Methods: Sepsis was evaluated in 96 high-risk neonates. We assessed cytokine levels on hospital admission and during or not during sepsis. Results: Fifty (52%) presented sepsis (26 early and 24 late). Sepsis was associated with high levels of IL-6, IL-10, G-CSF, and MCP1 and low levels of IFNγ, early sepsis with high levels of IL-6 and G-CSF, severe sepsis with high levels of IL-6 and IL-10, while deaths or sequelae was associated with low levels of IL-4, IL-12, IFNγ, and high levels of GM-CSF. IL-6 values of ≥40.1 pg/mL were associated with the development of any type of sepsis (relative risk [RR]: 1.70; 95% confidence interval [95% CI]: 1.18-2.24; p = .01), while IL-6 values of ≥44.9 pg/mL were associated with early sepsis (RR: 1.29; 95% CI: 1.29-4.56; p = .01). Conclusion: In neonates with high risk for the development of sepsis, there is an association between levels of IL-6, IL-10, and G-SCF and the disease development/outcome.

Healthcare delay in breast cancer patients: a case study in a low-density population region from Mexico.

AIM: To describe delay intervals, their impact on clinical stage and initiation of first oncologic treatment, and evaluate associated factors in breast cancer patients in Yucatan, Mexico, a low-density population region. PATIENTS & METHODS: A retrospective analysis was done of 92 medical records, and bivariate and multivariate models applied to identify associations between healthcare delay and several factors. RESULTS: System delay accounted for most of the delay (median: 86 days; 61% of delay). Socioeconomic status and delivery to tertiary-care hospital predicted delay. Clinical stage determined initiation of first oncologic treatment. CONCLUSION: Delay in treatment was largely due to system delay. Only a few variables explained this delay. Clinical stage had the strongest effect on initiation of first oncologic treatment.

Serum levels of IFN-ß are associated with days of evolution but not with severity of dengue.

The serum levels of beta interferon (IFN-ß) were evaluated in clinical samples taken in the acute phase of dengue fever for 107 patients during the 2011 dengue outbreak in Yucatán, México. Dengue diagnoses were confirmed by NS1 or IgM/IgG serology in all patients. Average serum IFN-ß levels in patients with dengue fever without warning signs (n = 53) were 140 pg/ml, and 105 pg/ml for patients with warning signs (n = 54). There was no statistically significant difference between IFN-ß levels for the two groups (Mann-Whitney U Test P > 0.05) and no association with warning or mild signs (OR: 0.57; 95%CI: 0.26-1.22) was indicated. Thrombocytopenia was the most prevalent warning sign (91%) in one group of patients (49) with mean 113 pg/ml IFN-ß levels. In contrast, the patients without thrombocytopenia (50) had 126 pg/ml of IFN-ß, but this level was not statistically significant (Mann-Whitney U Test P > 0.05). The average levels of IFN-ß were also found to have statistically similar results, using the 1997 WHO classification system. The amount of IFN-ß at 1-3, 4-6, and 7-9 days after onset of illness, however, did show significant differences (ANOVA P = 0.038) between patients for the 1-3 and 4-6 days pair (Scheffe post-hoc P = 0.043). These results suggest that serum levels of IFN-ß do not correlate well with the severity of dengue illness, but there is a clear association between changes in IFN-ß levels and the days of evolution during the acute phase of the disease.

Intestinal parasites and genotyping of Giardia duodenalis in children: first report of genotype B in isolates from human clinical samples in Mexico

Giardia duodenalis is one of the most prevalent enteroparasites in children. This parasite produces several clinical manifestations. The aim of this study was to determine the prevalence of genotypes of G. duodenalis causing infection in a region of southeastern Mexico. G. duodenalis cysts were isolated (33/429) from stool samples of children and molecular genotyping was performed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis, targeting the triosephosphate isomerase ( tpi ) and glutamate dehydrogenase ( gdh ) genes. The tpi gene was amplified in all of the cyst samples, either for assemblage A (27 samples) or assemblage B (6 samples). RFLP analysis classified the 27 tpi -A amplicons in assemblage A, subgenotype I. Samples classified as assemblage B were further analysed using PCR-RFLP of the gdh gene and identified as assemblage B, subgenotype III. To our knowledge, this is the first report of assemblage B of G. duodenalis in human clinical samples from Mexico.

Intestinal parasites and genotyping of Giardia duodenalis in children: first report of genotype B in isolates from human clinical samples in Mexico.

Giardia duodenalis is one of the most prevalent enteroparasites in children. This parasite produces several clinical manifestations. The aim of this study was to determine the prevalence of genotypes of G. duodenalis causing infection in a region of southeastern Mexico. G. duodenalis cysts were isolated (33/429) from stool samples of children and molecular genotyping was performed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis, targeting the triosephosphate isomerase ( tpi ) and glutamate dehydrogenase ( gdh ) genes. The tpi gene was amplified in all of the cyst samples, either for assemblage A (27 samples) or assemblage B (6 samples). RFLP analysis classified the 27 tpi -A amplicons in assemblage A, subgenotype I. Samples classified as assemblage B were further analysed using PCR-RFLP of the gdh gene and identified as assemblage B, subgenotype III. To our knowledge, this is the first report of assemblage B of G. duodenalis in human clinical samples from Mexico.

13C-urea breath test for the diagnosis of Helicobacter pylori infection in children: a systematic review and meta-analysis.

BACKGROUND: The (13) C-urea breath test ((13) C-UBT) is a safe, noninvasive and reliable method for diagnosing H. pylori infection in adults. However, the test has shown variable accuracy in the pediatric population, especially in young children. We aimed to carry out a systematic review and meta-analysis to evaluate the performance of the (13) C-UBT diagnostic test for H. pylori infection in children. METHODS: We conducted a systematic review of the PubMed, Embase and Liliacs databases including studies from January 1998 to May 2009. Selection criteria included studies with at least 30 children and reporting the comparison of (13) C-UBT against a gold standard for H. pylori diagnosis. Thirty-one articles and 135 studies were included for analysis. Children were stratified in subgroups of <6 and ≥6 years of age, and we considered variables such as type of meal, cutoff value, tracer dose, and delta time for the analysis. DISCUSSION: The (13) C-UBT performance meta-analyses showed 1, good accuracy in all ages combined (sensitivity 95.9%, specificity 95.7%, LR+ 17.4, LR- 0.06, diagnostic odds ratio (DOR) 424.9), 2, high accuracy in children >6 years (sensitivity 96.6%, specificity 97.7%, LR+ 42.6, LR- 0.04, DOR 1042.7), 3, greater variability in accuracy estimates and on average a few percentage points lower, particularly specificity, in children ≤6 years (sensitivity 95%, specificity 93.5%, LR+ 11.7, LR- 0.12, DOR 224.8). Therefore, the meta-analysis shows that the (13) C-UBT test is less accurate for the diagnosis of H. pylori infection in young children, but adjusting cutoff value, pretest meal, and urea dose, this accuracy can be improved.

Utility of stool sample-based tests for the diagnosis of Helicobacter pylori infection in children.

OBJECTIVE: Helicobacter pylori antigen or DNA in stool are meant to detect the bacteria; however, in children the colonization of the gastric mucosa by H pylori is usually weak and fecal excretion of antigen or DNA varies considerably, challenging the utility of these tests in this age group. The aim of the present study was to carry out a systematic review and meta-analysis to evaluate the performance of stool H pylori DNA and antigen tests for the diagnosis of infection in children. METHODS: We conducted a systematic review and meta-analysis to assess the accuracy of stool tests for diagnosis of H pylori infection in children. We searched PubMed, EMBASE, and LILACS databases. Selection criteria included participation of at least 30 children and the use of a criterion standard for H pylori diagnosis. In a comprehensive search, we identified 48 studies. RESULTS: Regarding antigen-detection tests, enzyme-linked immunosorbent assay (ELISA) monoclonal antibodies showed the best performance, with sensitivity and specificity of 97%, positive likelihood ratio (LR+) of 29.9, and negative likelihood ratio (LR-) of 0.03. ELISA polyclonal antibodies had sensitivity of 92%, specificity of 93%, LR+ of 16.2, LR- of 0.09, and high heterogeneity (P < 0.0001). One-step monoclonal antibody tests demonstrated sensitivity of 88%, specificity of 93%, LR+ of 10.6, and LR- of 0.11. For DNA detection, polymerase chain reaction-based test showed sensitivity of 80.8%, specificity of 98%, LR+ of 17.1, and LR- of 0.18. CONCLUSIONS: Detection of H pylori antigen in stools with ELISA monoclonal antibodies is a noninvasive efficient test for diagnosis of infection in children. One-step tests showed low accuracy and more studies are needed to obtain a useful office-based screening test. The available molecular tests are still unreliable.

Synthesis of benzologues of Nitazoxanide and Tizoxanide: a comparative study of their in vitro broad-spectrum antiprotozoal activity.

We have synthesized two new benzologues of Nitazoxanide (NIT) and Tizoxanide (TIZ), using a short synthetic route. Both compounds were tested in vitro against six protozoa (Giardia intestinalis, Trichomonas vaginalis, Entamoeba histolytica, Plasmodium berghei, Leishmania mexicana and Trypanosoma cruzi). Compound 1 (benzologue of NIT) showed broad antiprotozoal effect against all parasites tested, showing IC(50)'s<5 µM. This compound was five-times more active than NIT, and 18-times more potent than metronidazole against G. intestinalis. It was 10-times more active than pentamidine against L. mexicana, and it was sevenfold more potent than benznidazole versus T. cruzi. This compound could be considered as a new broad spectrum antiprotozoal agent.

Design, synthesis, and in vitro antiprotozoal, antimycobacterial activities of N-{2-[(7-chloroquinolin-4-yl)amino]ethyl}ureas.

We have synthesized a new series of quinoline tripartite hybrids from chloroquine, ethambutol, and isoxyl drugs, using a short synthetic route. Compounds 1-8 were tested in vitro against five protozoa (Giardia intestinalis, Trichomonas vaginalis,Entamoeba histolytica, Leishmania mexicana and Trypanosoma cruzi) and Mycobacterium tuberculosis. N-(4-Butoxyphenyl)-N'-{2-[(7-chloroquinolin-4-yl)amino]ethyl}urea (6) was the most active compound against all parasites tested. Compound 6 was 670 times more active than metronidazole, against G. intestinalis. It was as active as pentamidine against L. mexicana, and it was twofold more potent than ethambutol and isoxyl versus M. tuberculosis. This compound could be considered as a new broad spectrum antimicrobial agent.

Medicinal potions used against infectious bowel diseases in Mayan traditional medicine.

AIM OF THE STUDY: Since one of the main health problems of the indigenous population are infectious bowel diseases, we decided to test Mayan medicinal potions used to treat these conditions against some of the causal agents. MATERIALS AND METHODS: Thirty-one herbal formulations used for the treatment of infectious bowel diseases were prepared according to the collected ethnobotanical data. Their activities were tested against some of the causal agents of diarrheic symptoms, such as Entamoeba histolytica, Giardia lamblia, Escherichia coli, Klebsiella pneumoniae, Salmonella typhi and Shigella flexneri. RESULTS: Nine formulations were active against bacteria (MIC=0.5 mg/ml), four on Entamoeba histolytica, and seven on Giardia lamblia (IC(50)≤20 µg/ml). CONCLUSIONS: This work supports the use of the traditional Mayan formulations against some infectious bowel diseases, and it is the first step towards their study.

Immunogenicity of novel Dengue virus epitopes identified by bioinformatic analysis.

We used T cell epitope prediction tools to identify epitopes from Dengue virus polyprotein sequences, and evaluated in vivo and in vitro the immunogenicity and antigenicity of the corresponding synthetic vaccine candidates. Twenty-two epitopes were predicted to have a high affinity for MHC class I (H-2Kd, H-2Dd, H-2Ld alleles) or class II (IAd alleles). These epitopes were conserved between the four virus serotypes, but with no similarity to human and mouse sequences. Thirteen synthetic peptides induced specific antibodies production with or without T cells activation in mice. Three synthetic peptides induced mostly IgG antibodies, and one of these from the E gene induced a neutralizing response. Ten peptides induced a combination of humoral and cellular responses by CD4+ and CD8+ T cells. Twelve peptides were novel B and T cell epitopes. These results indicate that our bioinformatics strategy is a powerful tool for the identification of novel antigens and its application to human HLA may lead to a potent epitope-based vaccine against Dengue virus and many other pathogens.

Synthesis and antimicrobial and nitric oxide synthase inhibitory activities of novel isothiourea derivatives.

The reaction of substituted benzylhalides, or of halomethyl derivatives of thiophene or furane, with thiourea or its derivatives yielded the respective isothioureas as hydrohalide salts. The products (a total of 17, including 16 novel compounds) were tested for activity against five Gram-positive and nine Gram-negative bacterial strains, six yeast species and two protozoan species. The most active against Gram-positive bacteria were S-(2,4-dinitrobenzyl)isothiourea hydrochloride (MIC range for four out of five strains tested: 12.5-25 microg/mL) and S-(2,3,4,5,6-pentabromobenzyl)isothiourea hydrobromide (MIC range: 12.5-50 microg/mL). The lowest MICs of novel isothioureas for yeast and Gram-negative bacteria ranged between 50 and 100 microg/mL. Nine novel isothioureas showed appreciable genotoxicity in the Bacillus subtilis 'rec-assay' test, the most potent being S-2-(5-nitrofuran-2-ylmethyl)isothiourea and S-(2-nitrobenzyl) isothiourea. At 10 muM concentration, S-(3,4-dichlorobenzyl)isothiourea hydrochloride and S-(2,3,4,5,6-pentabromobenzyl)isothiourea hydrobromide inhibited Ca(2+)/calmodulin-dependent (non-inducible) nitric oxide synthase activity in normal rat brain homogenates stronger (p < 0.05) than the reference drug 7-nitroindazole (by 78, 76 and 60%, respectively); ten other new isothiourea derivatives significantly inhibited the activity to a lower extent (by 28-60%). These results extend the list of promising isothioureas with substantial activity in vitro and suggest that an in-depth study of toxicity, antimicrobial properties in vivo and nitric oxide synthase isoform selectivity of selected novel compounds is warranted.

Synthesis and in vitro trichomonicidal, giardicidal and amebicidal activity of N-acetamide(sulfonamide)-2-methyl-4-nitro-1H-imidazoles.

Two new series of imidazole derivatives (acetamides: 1-8 and sulfonamides: 9-15) were synthesized using a short synthetic route. Compound 1 as well as the intermediate 16g were characterized by X-ray crystallography. Imidazole derivatives 1-15 were tested in vitro against three unicellular parasites (Giardia intestinalis, Trichomonas vaginalis and Entamoeba histolytica) in comparison with benznidazole (Bzn) and metronidazole. Compound 1 [N-benzyl-2-(2-methyl-4-nitro-1H-imidazol-1-yl)acetamide] was 2 times more active than Bzn against T. vaginalis and G. intestinalis and it was as active as Bzn against E. histolytica. Sulfonamides showed selective toxicity against E. histolytica over the other parasites. Toxicity assay showed that all compounds are non-cytotoxic against MDCK cell line. The results revealed that compounds 1-15 have antiparasitic bioactivity in the micromolar range against the parasites tested, and could be considered as benznidazole bioisosteres.

Seroepidemiology of giardiasis in Mexico.

Prevalence of antibodies against Giardia duodenalis was determined by enzyme-linked immunosorbent assay in serum samples from a national serologic survey of Mexico that included all geographic areas and socioeconomic and demographic data for each person sampled. The country was divided into four regions on the basis of development (high, medium high, medium low, and low). Of 3,461 serum samples tested, 1,914 (55.3%) were positive for IgG antibodies against Giardia duodenalis. Seropositivity was age-specific; the probability of seropositivity increased 4.9-fold (95% confidence interval [CI] = 3.16-7.64) in adolescents 10-19 years of age, 8.0-fold (95% CI = 5.19-12.53) in young adults 20-39 years of age, and 12.6-fold (95% CI = 7.93-20.28) in adults more than 40 years of age. Giardia duodenalis seropositivity was associated with male sex (odds ratio = 1.40, 95% CI = 1.22-1.61). No association was found between seropositivity and socioeconomic variables or regional development status.

Antibody-based detection tests for the diagnosis of Helicobacter pylori infection in children: a meta-analysis.

BACKGROUND: Numerous serologic tests are available for the diagnosis of H. pylori infection in children. Common designs of antibody-based detection tests are ELISA and Western Blot (WB). For developing countries with limited laboratory resources and access, ELISA would be the preferred method because of its simplicity, lower cost and speed. Although in adults ELISA has proven to be highly accurate in diagnosing H. pylori infection; in children, it has shown variable accuracy. METHODS/FINDINGS: We conducted a systematic review and meta-analysis to assess the accuracy of antibody-based detection tests for the diagnosis of H. pylori infection in children. Selection criteria included participation of at least 30 children and the use of a gold standard for H. pylori diagnosis. In a comprehensive search we identified 68 studies. Subgroup analyses were carried out by technique, immunoglobulin class, and source of test (commercial and in-house). The results demonstrated: 1) WB tests showed high overall performance, sensitivity 91.3% (95% CI, 88.9-93.3), specificity 89% (95% CI, 85.7-91.9), LR+ 8.2 (95% CI, 5.1-13.3), LR- 0.06 (95% CI, 0.02-0.16), DOR 158.8 (95% CI, 57.8-435.8); 2) ELISA-IgG assays showed low sensitivity 79.2% (95% CI, 77.3-81.0) and high specificity (92.4%, 95% CI, 91.6-93.3); 3) ELISA commercial tests varied widely in performance (test for heterogeneity p<0.0001); and 4) In-house ELISA with whole-cell antigen tests showed the highest overall performance: sensitivity 94% (95% CI, 90.2-96.7), specificity 96.4% (95% CI, 94.2-97.9), LR+ 19.9 (95% CI, 7.9-49.8), LR- 0.08 (95% CI, 0.04-0.15) DOR 292.8 (95% CI, 101.8-841.7). CONCLUSIONS/SIGNIFICANCE: WB test and in-house ELISA with whole-cell antigen tests are the most reliable tests for the diagnosis of H. pylori infection in children. Antigens obtained from local strains of the community could partially explain the good overall accuracy of the in-house ELISA. Because of its cost and technical demands, in-house ELISA might be more suitable for use in developing countries.

Design, synthesis and in vitro antiprotozoal activity of benzimidazole-pentamidine hybrids.

A series of ten novel hybrids from benzimidazole and pentamidine were prepared using a short synthetic route. Each compound was tested in vitro against the protozoa Trichomonas vaginalis, Giardia lamblia, Entamoeba histolytica, Leishmania mexicana, and Plasmodium berghei, in comparison with pentamidine and metronidazole. Some analogues showed high bioactivity in the low micromolar range (IC(50)<1 microM) against the first four protozoa, which make them significantly more potent than either standard. 1,5-bis[4-(5-methoxy-1H-benzimidazole-2-yl)phenoxy]pentane (2) was 3- and 9-fold more potent againstG. lamblia than metronidazole and pentamidine, respectively. This compound was 23-, 108-, and 13-fold more active than pentamidine against T. vaginalis, E. histolytica and L. mexicana, respectively. Studying further structure-activity relationships through the use of bioisosteric substitution in these hybrids should provide new leads against protozoal diseases.

21beta-Hydroxy-oleanane-type triterpenes from Hippocratea excelsa.

Stem bark of Hippocratea excelsa afforded six pentacyclic triterpenes, five oleanane and one ursane types. They were identified as 11beta,21beta-dihydroxy-olean-12-ene-3-one (2), 3alpha,11alpha,21beta-trihydroxy-olean-12-ene (3), 3alpha,21beta-dihydroxy-11alpha-methoxy-olean-12-ene (4), 3alpha,21beta-dihydroxy-olean-9(11),12-diene (5), 3alpha,21beta-dihydroxy-olean-12-ene (6) and 3alpha,21beta-dihydroxy-11alpha-methoxy-urs-12-ene, isolated as its diacetate derivative (7), as well as 3alpha,21beta-dihydroxy-olean-12-ene (1) previously isolated from the root bark. The known alpha- and beta-amyrin, oleanoic and ursolic acids, trans-polyisoprene, and the ubiquitous beta-sitosterol were also isolated. Structures were elucidated on the basis of spectroscopic analyses, including homo- and heteronuclear correlation NMR experiments (COSY, ROESY, HSQC and HMBC) and comparison with literature data. The antigiardial activity of compounds 2-5 was not significant.

Antiprotozoal activity of Senna racemosa.

Methanol extracts of leaves, roots and bark of Senna racemosa (Mill.) H.S. Irwin & Barneby (syn. Cassia racemosa Mill.) were tested for antiprotozooal activity against Giardia intestinalis and Entamoeba histolytica. All of the tested extracts showed good activity against both protozoa species. Extracts from stem bark and leaves were most active, with an IC(50) of 2.10 microg/mL for Giardia intestinalis and 3.87 microg/mL for Entamoeba histolytica. Of the previously isolated compounds from Senna racemosa, the piperidine alkaloid cassine had greater activity against Giardia intestinalis with an IC(50) of 3.28 microg/mL and chrysophanol, a 1,8-dihydroxy-anthraquinone, was the most active agent against Entamoeba histolytica, with an IC(50) of 6.21 microg/mL.