Alarin but not its alternative-splicing form, GALP (Galanin-like peptide) has antimicrobial activity.

Alarin is an alternative-splicing form of GALP (galanin-like peptide). It shares only 5 conserved amino acids at the N-terminal region with GALP which is involved in a diverse range of normal brain functions. This study seeks to investigate whether alarin has additional functions due to its differences from GALP. Here, we have shown using a radial diffusion assay that alarin but not GALP inhibited the growth of Escherichia coli (strain ML-35). The conserved N-terminal region, however, remained essential for the antimicrobial activity of alarin as truncated peptides showed reduced killing effect. Moreover, alarin inhibited the growth of E. coli in a similar potency as human cathelicidin LL-37, a well-studied antimicrobial peptide. Electron microscopy further showed that alarin induced bacterial membrane blebbing but unlike LL-37, it did not cause hemolysis of erythrocytes. In addition, alarin is only active against the gram-negative bacteria, E. coli but not the gram-positive bacteria, Staphylococcus aureus. Thus, these data suggest that alarin has potentials as an antimicrobial and should be considered for the development in human therapeutics.

Diagnostic Testing Accuracy for Helicobacter pylori Infection among Adult Patients with Dyspepsia in Cuba's Primary Care Setting.

Evidence of the effectiveness of the tests used to diagnose Helicobacter pylori (H. pylori) in primary healthcare is limited. This cross-sectional study aims to assess the accuracy of tests used for to diagnose H. pylori infection in primary care patients and its relationship with gastroduodenal pathologies. Over 12 months, 173 primary care patients with dyspeptic symptoms were referred for upper gastrointestinal endoscopy to obtain gastric biopsies, and venous blood was extracted from them. H. pylori infection was detected using a rapid urease test (RUT), real-time polymerase chain reaction (RT-PCR), H. pylori-IgG ELISA, and Western blot (WB). The culture and histological findings were used as the reference standard for H. pylori infection. H. pylori prevalence was 50%. There were no significant differences between men and women overall or by age group. The presence of H. pylori was associated with chronic moderate gastritis and its absence with chronic inactive gastritis, as well as the combination of gastritis and gastric lesions (p < 0.05). RUT and ELISA H. pylori -IgG tests showed the highest overall performance (accuracy 98.9% and 84.4%), followed by WB and RT-PCR (accuracy 79.3% and 73.9%). These findings support the notion that combined invasive and noninvasive methods, such as RUT and H. pylori-IgG ELISA, can be a primary diagnostic screening tool for detecting H. pylori among adult dyspeptic patients in Cuba's primary care setting.

Low prevalence of Helicobacter pylori among symptomatic children from a hospital in Havana, Cuba.

The aims of this study were to assess the prevalence of Helicobacter pylori infection and to introduce a new algorithm to improve its diagnosis in Cuban symptomatic children. One hundred and thirty-three consecutive children with upper gastrointestinal symptoms were studied. Patients were endoscoped and antral biopsies were obtained for rapid urease test (RUT), culture and histology. Prevalence of H. pylori infection was 30.8%. No statistical differences were found concerning demographic, socio-economic factors or chief clinical complaints, between H. pylori-positive and negative children, except for haematemesis, which was significantly higher in infected children (p = 0.003). Histologically, there was statistical association between moderate chronic gastritis in infected children (p = 0.04). Culture and RUT had the highest specificity and sensitivity, respectively. The prevalence of H. pylori infection in Cuban symptomatic children is similar to the one observed in developed countries. Culture and RUT is a useful combination to diagnose H. pylori infection in paediatric patients.

Direct binding of gangliosides to Helicobacter pylori vacuolating cytotoxin (VacA) neutralizes its toxin activity.

Gangliosides are target receptors for bacterial entry, yet those present in human milk exhibit a protective role against bacterial infection. Here, we show that treatment with ganglioside mixture at a concentration of 100 microg/mL resulted in significant inhibition of the vacuole formation activity of Helicobacter pylori vacuolating cytotoxin (VacA) in gastric epithelial cancer AZ-521 cells. All gangliosides (GM1, GM2, GM3, GD1a, GD1b, GD3 and GT1b) examined showed good neutralizing capacity against VacA. A pull-down assay was performed using lyso-GM1 coupled to Sepharose as the tagged polysaccharide polymer to capture VacA from H. pylori culture supernatant. GM1-VacA complexes were successfully precipitated, suggesting that GM1 binds directly to VacA. The hydrodynamic binding of lyso-GM1 and VacA measured by fluorescence correlation spectroscopy had a K(d) value of 190 nM. VacA also bound to lyso-GM1 at pH 2 corresponding to the physiological pH of human stomach. Collectively, these results showed that direct binding of H. pylori VacA to free gangliosides neutralizes the toxin activity of VacA. These findings offer an alternative insight into the role of gangliosides in VacA toxicity and the pathogenesis of H. pylori.

Phenotypic and genetic characterization of antimicrobial profiles of Helicobacter pylori strains in Cuba.

The study evaluated the antibiotic resistance patterns of Helicobacter pylori strains against metronidazole and clarithromycin in a hospital in Havana, Cuba. Eighty-five percent, 22.5%, and 10% of 40 H. pylori strains investigated were resistant to metronidazole, ciprofloxacin, and clarithromycin respectively but all were susceptible to amoxicillin and tetracycline. RdxA truncation was found only in metronidazole-resistant strains. In such strains, reported are eight and two novel mutations in the rdxA and frxA genes respectively. Two-point mutations in the 23S rRNA genes of clarithromycin-resistant strains were detected. A high prevalence of metronidazole resistance was found in Cuban H. pylori strains. Mutations in the rdxA gene may contribute more significantly than frxA gene to the high level of resistance to metronidazole. This study supports the need to continue monitoring the antibiotic susceptibility in H. pylori in Cuba to guide the treatment of such infection.

Safety and preliminary immunogenicity of MenC/P64k, a meningococcal serogroup C conjugate vaccine with a new recombinant carrier.

This study reports the preliminary assessment of the safety and immunogenicity of the first serogroup C conjugate vaccine candidate that includes meningococcal P64k recombinant protein as the carrier (MenC/P64k). Twenty volunteers were recruited for a double-blind, randomized, controlled phase I clinical trial, receiving a single dose of MenC/P64k (study group) and a single dose of the commercial polysaccharide vaccine AC (control group). Only mild reactions were observed. No statistical differences were detected between the antipolysaccharide C IgG responses of both groups as well as between bactericidal serum titre (P > 0.05). The MenC/P64k vaccine was found to have a good safety profile, to be well tolerated and immunogenic.

Low prevalence of Vibrio cholerae O1 versus moderate prevalence of intestinal parasites in food-handlers working with health care personnel in Haiti.

Food-handlers with poor personal hygiene working in food-service establishments could be potential sources of infection due to pathogenic organisms. In May 2011, a cross-sectional study was undertaken to determine the prevalence of bacteria and intestinal parasites among food-handlers working with Cuban health personnel in Haiti. Stool specimens were collected from 56 food-handlers and samples were examined using standard procedures. Of the food handlers, 26.8% had one bacterial or intestinal parasite. The most prevalent species of organism found were Blastocystis spp. (9%), followed by Vibrio cholerae O1 serotype Ogawa, Aeromonas spp. and Giardia intestinalis, each one with 4%. The prevalence of intestinal parasites was 19.7%. Five out of 56 food handlers had diarrhea at the time the study was conducted. It was found that there was a lower prevalence of V. cholerae O1 serotype Ogawa in comparison to intestinal parasites. The study highlights the importance of the precautions that must be taken in cholera-affected countries by medical teams and their organizations, with emphasis on the preparation, processing, and serving of meals. The recommendation is to intensify continuing education programs, periodical laboratory examinations to detect carriers and food-handlers reporting sick, and to observe strict adherence to hygienic food-handling practices. In addition, food handlers with diarrhea should refrain from preparation or delivery of food.

Nasopharyngeal colonization by Moraxella catarrhalis and study of antimicrobial susceptibility in healthy children from Cuban day-care centers.

The prevalence of nasopharyngeal carriage of Moraxella catarrhalis was determined for the first time in Cuba. One-hundred fifty healthy children attending three day-care centers in the municipality of Marianao, Havana City were studied. The percentage of recovering bacteria in nasal and pharyngeal swabs was compared. Antimicrobial susceptibilities to ampicillin, trimethoprim-sulfamethoxazole, tetracycline, cefotaxime, ceftriaxone, chloramphenicol, erythromycin, azithromycin, amoxicillin/clavulanate, and norfloxacin were determined by the disk diffusion method according to recommendations of the National Committee for Clinical Laboratory Standards. Sixty-five percent of the children studied carried Moraxella catarrhalis. The nasal cavity was the main isolation site for this organism (81% of positive cultures). Most strains were highly susceptible to the antimicrobial agents tested, except to ampicillin (53.6% resistance). This study provides evidence of the need for continued surveillance of antimicrobial susceptibility of Moraxella catarrhali, in order to determine optimal empiric therapy for community-acquired respiratory tract infections produced by this pathogen.

Evaluation of the BIOCEN GC agar medium base in antimicrobial susceptibility testing of Neisseria gonorrhoeae.

BACKGROUND: Since 1995, the Cuban Reference Laboratory for Neisseria has been monitoring the antibiotic susceptibility of gonococci, following the methodology of the National Committee for Clinical Laboratory Standards, which uses GC agar medium base supplemented with 1% Vitox. We evaluated three lots of GC agar medium base produced by BIOCEN, Cuba, in antibiotic susceptibility testing of reference and wild strains of gonococci. METHODS: The susceptibilities to five antibiotics were evaluated five times on three lots of GC agar medium base from BIOCEN. Four and one gonococcal reference strains were tested by MIC dilution and disc diffusion methods, respectively. Later, the antimicrobial susceptibilities of ten wild Neisseria gonorrhoeae strains were tested in triplicate. As internal control, a GC agar medium from Difco was used. RESULTS: All antibiotic MICs obtained on four lots of GC agar medium from different manufacturers fell within the proposed quality control limits for reference strains analyzed. The disc diffusion data for the reference strain of N. gonorrhoeae ATCC 49226 to five antibiotics provided essentially identical results in all lots of GC agar medium base. For wild strains of gonococci, identical modal MIC values and zone size diameters within a 3-mm range were observed in all the antibiotics tested. CONCLUSIONS: Excellent agreement in susceptibility testing methods among different lots of GC agar medium base from BIOCEN and Difco was obtained for all reference and wild gonococcal strains and antibiotics tested. We proposed that GC medium from BIOCEN can be used in antimicrobial susceptibility testing of N. gonorrhoeae by MIC dilution and disc diffusion tests.

Sentinel Surveillance Detects Low Circulation of Vibrio cholerae Serotype Inaba in Haiti, 2011-2012.

Over 700,000 cases of cholera were reported in Haiti between October 2010 and February 2015. In November 2011, the Cuban Medical Team serving in Haiti established a laboratory-supported sentinel surveillance system for cholera in 10 public hospitals (one in each of Haiti's 10 departments), to estimate the proportion of hospitalized patients with cholera and detect emergence of new Vibrio cholerae serotypes. Each month, the first ten stool samples collected from patients admitted with acute watery diarrhea were studied in all hospitals involved. Surveillance system findings from November 1, 2011, to October 30, 2012 showed that acute watery diarrhea was caused by V. cholerae serogroup O1 in 45.9% (210/458) of patients: Serotype Ogawa was found in 98.6% of this isolates (207/210) and serotype Inaba in 1.4% (3/210), indicating low circulation level of the latter in Haiti. Continuing laboratory sentinel surveillance of V. cholerae is needed to monitor the spread of the disease and prevent and contain outbreaks, particularly of new serotypes. It is important to ensure that these findings are systematically integrated with data available to MSPP from other surveillance sources. KEYWORDS Vibrio cholerae, serotype Inaba, serotype Ogawa, epidemiological surveillance, medical cooperation, Haiti, Cuba.

Prevalence of Helicobacter pylori vacA, cagA, and iceA Genotypes in Cuban Patients with Upper Gastrointestinal Diseases.

Virulence factors of Helicobacter pylori can predict the development of different gastroduodenal diseases. There are scarce reports in Cuba about H. pylori isolates genotyping. The aim of the present investigation was to identify allelic variation of the virulence genes vacA, cagA, and iceA in sixty-eight patients diagnosed as H. pylori positive by culture. In seven out of 68 patients, strains from both gastric regions were obtained and considered independent. DNA was extracted from all the H. pylori strains and evaluated by PCR-genotyping. The vacA s1 allele, cagA gene, and iceA2 allele were the most prevalent (72.0%, 56.0%, and 57.3%, respectively). Alleles from m-region showed a similar frequency as s1a and s1b subtypes. The presence of multiple H. pylori genotypes in a single biopsy and two gastric region specimens were found. Significant statistical association was observed between iceA2 allele and patients with non-peptic ulcer dyspepsia (NUD) (P = 0.037) as well as virulence genotypes (s1, s1m2) and patients over 40 years old (P < 0.05). In conclusion, the results demonstrated a high prevalence of H. pylori virulent genotypes in Cuban patients over 40 years old while iceA2 alleles demonstrated a good specificity in patients with NUD.

Co-infection with Cyclospora cayetanensis and Salmonella typhi in a patient with HIV infection and chronic diarrhoea.

A 45-year-old-Haitian male patient with fever, abdominal cramping, chronic diarrhoea and weight loss of about 3 kg was investigated. Stool examination revealed Salmonella typhi and Cyclospora cayetanensis. The HIV test was positive with a CD4 count of 130 cells/mm(3). We provided the first report of co-infection Cyclospora cayetanensis and Salmonella typhi in a HIV patient with chronic diarrhoea. The patient was treated with oral ciprofloxacin, 500 mg, twice daily for two weeks, with a good clinical outcome.

Laboratory-based surveillance of Streptococcus pneumoniae invasive disease in children in 10 Latin American countries: a SIREVA II project, 2000-2005.

BACKGROUND: For the last 14 years the Pan American Health Organization has been promoting surveillance of invasive pneumococcal disease in Latin American children for better understanding of the disease tendencies regarding capsular types circulation in each country and susceptibility to antimicrobials. METHODS: Laboratory-based surveillance data from 10 Latin American countries collected from 2000 to 2005 were analyzed, including serotype distribution and susceptibility to beta-lactam antibiotics. RESULTS: Although 61 different capsular types were identified during the 6-year surveillance, 13 serotypes accounted for 86% of all isolates. These were consistently the most prevalent throughout the study period with serotype 14 predominating. Diminished susceptibility to penicillin was detected in 38% of all Streptococcus pneumoniae isolates, with the highest prevalence in Dominican Republic and Mexico. Decreased susceptibility to penicillin increased in Brazil and Colombia whereas decreased high resistance rates was recorded in Chile. CONCLUSIONS: These data indicate that 10 countries of the Region continue to have high quality laboratory-based surveillance for pneumococcal disease thus generating valuable information so that healthcare decision makers may prioritize interventions. The heptavalent vaccine will potentially cover from 52.4% to 76.5% of strains causing invasive pneumococcal disease and the 13 valent from 76.7% to 88.3%.

[Development of a method to determine the minimum inhibitory concentration in reference strains from leptospiras]. / Desarrollo de un método para determinar la concentración mínima inhibitoria en cepas de referencias de leptospiras.

A method to determine the minimum inhibitory concentration for leptospiras was developed, since there is not a standard method to measure it at the international level. Reference strains from the pathogenic complex L. interrogans and L. biflexa were used against penicillin, cyprofloxacine, chloramphenicol, rifampicine and tetracycline. The minimum inhibitory concentration was defined as the lowest concentration of antibiotic where it was observed the inhibition of the bacterial mobility by direct examination in dark field. Ranges for penicillin were from 0.095 to 152 microg/mL, for tetracycline from 0.156 to 3.13 microg/mL, for chloramphenicol, from 0.08 to 12.52 microg/mL, for rifampicine from 0.08 to 1.56 microg/mL, and for cyprofloxacine from 0.15 to 2.4 microg/mL. The antibiotics that showed the lowest values were cyprofloxacine, rifampicine and tetracycline, whereas the most elevated value was obtained against chloramphenicol and penicillin. The strains from the serogroups circulating more frequently in Cuba were used in this research. This study will allow in a near future to determine the antimicrobial susceptibility in autochthonous strains isolated from patients with Leptospirosis at the national level.

High percentages of resistance to tetracycline and penicillin and reduced susceptibility to azithromycin characterize the majority of strain types of Neisseria gonorrhoeae isolates in Cuba, 1995-1998.

BACKGROUND: In many regions the susceptibility of Neisseria gonorrhoeae isolates to antimicrobial agents is rarely tested. The Gonococcal Antimicrobial Surveillance Program (GASP) in Cuba was established as part of a larger regional GASP program to facilitate the collection and reporting of antimicrobial susceptibility data for N gonorrhoeae isolates. GOAL: The goal was to retrospectively determine the antimicrobial susceptibility and molecular epidemiology of 91 isolates of N gonorrhoeae isolated from 11 centers in Cuba. STUDY DESIGN: Isolates of N gonorrhoeae were collected and presumptively identified from 11 Cuban provincial health centers. They were then forwarded to the National Laboratory of Pathogenic Neisseria Havana for confirmatory identification and were subsequently analyzed at the Center for GASP in Ottawa. Isolates were tested for susceptibility to penicillin, tetracycline, spectinomycin, ceftriaxone, ciprofloxacin, and azithromycin by the agar dilution method. To establish baseline data for molecular epidemiologic profiles, the auxotype (A), serovar (S), plasmid content (P), and TetM type of the isolates were determined. Certain A/S/P classes were further analyzed by pulsed field gel electrophoresis (PFGE). RESULTS: High percentages of the 91 N gonorrhoeae isolates were resistant to penicillin (68%) and tetracycline (83.5%), with 56% being penicillinase-producing (PPNG) and 64% carrying plasmid-mediated tetracycline resistance (TRNG; 50% were PP/TRNG). An additional 14% of the isolates carried chromosomal resistance (CMRNG) to either tetracycline or penicillin or both antibiotics. All isolates were susceptible to spectinomycin, ceftriaxone, and ciprofloxacin. However, nine isolates were resistant to azithromycin (MIC, > or = 1.0 microgram/ml), and 43 other isolates displayed reduced susceptibility to this antibiotic (MIC, 0.25-0.5 microgram/ml). Although a total of 21 different A/S classes were identified, most of the isolates (61) belonged to three A/S classes: NR/IA-6 (35 isolates), NR/IB-1 (15 isolates), and P/IA-6 (11 isolates). Thirty-two of 45 PP/TRNG were A/S class NR/IA-6, and nine of the P/IA-6 isolates were TRNG. By contrast, most of A/S class NR/IB-1 (8) were CMRNG. PFGE analysis following digestion with NheI or SpeI further clustered the isolates into separate groups. CONCLUSIONS: This study demonstrates high percentages of N gonorrhoeae isolates with penicillin and tetracycline resistance in Cuba. As has been noted in other studies in the Caribbean region and Latin America, resistance and reduced susceptibility to azithromycin are developing as emerging problems. Since penicillin and tetracycline continue to be widely used for the treatment of gonococcal infections in Cuba, this study indicates the importance of antimicrobial susceptibility surveillance so that effective antibiotics may be recommended for treatment of gonococcal infections.

Evaluation of four methods for detecting the beta-lactamase activity in Neisseria gonorrhoeae isolated in Cuba.

Four methods (chromogenic, acidimetric, inhibition, and iodometric) for demonstration of the beta-lactamase production by 70 isolates of Neisseria gonorrhoeae, were evaluated in Cuba. There was 100% correlation between all beta-lactamase methods and the standardized penicillin dilution susceptibility test for penicillinase-non-producing N. gonorrhoeae. For penicillinase-producing N. gonorrhoeae strains, there was a perfect correlation between the chromogenic method and penicillin susceptibility testing, but one and two strains failed to give a positive result for beta-lactamase with the inhibition/acidimetric and the iodometric methods, respectively. There was a high concordance between the chromogenic method, considered as gold standard and the rest of penicillinase tests evaluated: Kappa Index (KI) = 0.98 for inhibition/acidimetric methods and KI = 0.97 for the iodometric method. The four methods evaluated were accurate, reproducible, easily readable, economical, and ease to use for screening primary isolates of N. gonorrhoeae in Cuba. We recommended the use of the inhibition method, when testing the penicillinase activity in gonococcal isolates in provincial and municipal reference laboratories.

Antimicrobial susceptibility of Neisseria gonorrhoeae in Cuba (1995-1999): implications for treatment of gonorrhea.

BACKGROUND: Antibiotic-resistant strains of Neisseria gonorrhoeae, especially those resistant to penicillin and tetracycline, have spread with remarkable rapidity in many Caribbean countries. GOAL: The goal of the study was to survey the antibiotic susceptibilities of N gonorrhoeae strains isolated from 1995 to 1999 in Cuba and to discuss the impact of antimicrobial resistance on the management of gonorrhea in the country. STUDY DESIGN: Susceptibility of the strains to penicillin, tetracycline, cefuroxime, ceftriaxone, ciprofloxacin, spectinomycin, and azithromycin were determined by an agar dilution method. RESULTS: Penicillin and tetracycline resistance was noted in 60.8% and 54.2%, respectively, of the N gonorrhoeae strains tested. A total of 63.35 (76/120) of the N gonorrhoeae strains exhibited plasmid-mediated resistance to penicillin, tetracycline, or both. Strains with chromosomally mediated resistance to these antibiotics accounted for 10% (12/120) of the strains. The strains were susceptible to ceftriaxone, cefuroxime, spectinomycin, and ciprofloxacin. One strain's ciprofloxacin MIC was 0.125 mircog/ml. Of the 52 strains tested, 23.1% displayed intermediate resistance to azithromycin. CONCLUSIONS: N gonorrhoeae strains exhibited a high frequency of resistance and multiresistance to penicillin and tetracycline. Therefore, these antibiotics should no longer be used to treat gonococcal infections in Cuba and should be substituted with effective drugs such as third-generation cephalosporins, spectinomycin, and fluoroquinolones. The detection of intermediate resistance to azithromycin and ciprofloxacin underlines the importance of periodic surveillance for susceptibility of N gonorrhoeae strains to antimicrobials agents used as primary therapy for gonorrhea.