Aim: This study aimed to develop and evaluate an in-house enzyme-linked immunosorbent assay (ELISA) based on autochthonous antigens to detect immunoglobulin G (IgG) antibodies against Helicobacter pylori (H. pylori) in adult sera. Methods: Whole-cell antigens from three genetically characterized clinical isolates of H. pylori were mixed and used as coating antigens. This assay was validated with a panel of human sera samples of H. pylori seropositive and seronegative patients. Likewise, sera samples from patients with uninvestigated dyspepsia, who were also evaluated by invasive and noninvasive tests (i.e., histopathology, rapid urease test, and stool antigen test), blood donors and patients with confirmed viral and parasitic diseases were also collected. The IgG response against H. pylori was detected by the in-house assay using the commercial ELISA IBL (Germany), as a reference test. Statistical analysis was performed with GraphPad Prism version 5.01. Results: The in-house ELISA showed high repeatability and reproducibility. Sensitivity was 91.1%; 95% confidence interval (CI): 87.2–94.0, specificity was 94.8% (95% CI: 85.0–94.8), and accuracy was 91.6% (95% CI: 88.5–94.6). The in-house ELISA showed an excellent area under the curve (0.96; 95% CI: 0.93–0.98) and a better IgG detection by the inverse cumulative distribution. The frequency of seropositivity in patients with dyspepsia (76.0%) was significantly higher (P < 0.05) than in healthy individuals (57.7%) and patients with other infectious diseases resembling H. pylori infection symptoms (54.4%). The H. pylori seroprevalence was estimated to be 62.7%. A good correlation was found between IgG seropositivity and H. pylori infection diagnosed by histopathology, rapid urease test, and stool antigen test in Cuban adults with dyspepsia. Conclusions: The in-house ELISA demonstrated good diagnostic accuracy and potential usefulness for estimating H. pylori exposure in the adult population, henceforward, this method could be used as an alternative for H. pylori diagnosis in the Cuban setting.

Currently, diagnosis of Neisseria meningitidis in nasopharyngeal samples can be made by culture and nucleic acid amplification techniques.1 In Cuba, molecular diagnosis of meningococcal meningitis was introduced at the National Reference Laboratory for Neisseria (NRLN), in 2010, through a PCR that amplifies a fragment of the ctrA gene using the protocol described by Taha, 2000.2 This gene codes for a capsule protein that regulates the adhesion of N. meningitidis to the host, and 16 to 28% of meningococci isolates, especially in the nasopharynx, lack this gene.2 In contrast, the sodC gene, related to the production of superoxide-dismutase of this organism, is less sensitive to antigenic variation, hence its importance for molecular diagnosis in patients and asymptomatic carriers.3 Information on the meningococcal carriage is essential for public health policy,4. Still, the high number of invasive meningococci disease (IMD) affecting Cuba during the 1980s and the absence of molecular tools prevented its accurate microbiological diagnosis in carriers.

TO THE EDITOR—We read with interest the recent publication by Prazuck et al. concerning the effectiveness of a pooled methodology to detect Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG), and Mycoplasma genitalium (MG) in the 3 anatomical sites among men who have sex with men (MSM) using the Allplex STI Essential Assay (Seegene, Seoul, South Korea) [1]. Due to the increasing evidence that pooling of samples to detect sexually transmitted infections (STIs) is cost-effective with limited reduction in performance characteristics, we decided to validate a similar pooling method in Cuba. Although extragenital sampling among MSM is recommended to curb the STI epidemic in this population, extragenital sampling for STIs is still not recommended in Cuba. One of the main reasons is the additional cost of extragenital STI testing. As such, a pooling methodology that can be used in Cuba is urgently needed. STI pooling methodologies described in the literature mostly use American platforms such as Abbott (Abbott Technologies, Illinois, USA), Aptima (Hologic, San Diego, California, USA), and GeneXpert (Cepheid, California, USA), which are unfortunately not available in Cuba [2–5]. The Allplex STI Essential Assay pooling method described by Prazuck et al. uses Seegene-specific instruments or the CFX96 real-time polymerase chain reaction (PCR) instrument from Bio-Rad (CA, USA), which is also difficult to purchase in Cuba. In Cuba, the National Reference Center for STIs (IPK, Havana, Cuba) uses the multiplex Real-Time PCR kit from Sacace to detect Neisseria gonorrhoeae/ Chlamydia trachomatis/Mycoplasma genitalium/Trichomonas vaginalis (Sacace Biotechnologies, Italy). This system is compatible with the Rotor-Gene Q instrument, Qiagen (Hilden, Germany), which is available at our institute. In this initial

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.

Synergic Effect of Curcuma longa L. Extract with Antimicrobials against Cuban Helicobacter pylori Isolates

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.

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.

The investigation involved 63 consecutive adult patients with dyspeptic symptoms (42females and 21 males), who were diagnosed with H. pylori infection at the outpatient clinicof the Institute ¨Pedro Kouri (IPK), in Havana, Cuba, between November–December 2005.Patients were excluded if they had received any antibiotics or proton pump inhibitors in the3 weeks prior to the study. All patients were submitted to endoscopy analysis; two antralbiopsies, one for the RUT and the other for culture and two PCR methods were collected.Written informed consent was obtained from all individuals as per the recommendations ofthe Ethics Committee of the IPK.RUT: It was performed by a non-commercial test, consisting of 0.02% urea solution,plus phenol red in phosphate buffer. A color change from yellow to pink observed up to 4hours indicates a positive result at 37°C.Culture: A biopsy sample was placed in Eppendorf tubes containing 0.5 ml of sterilesaline solution kept at 4°C and sent to the microbiology laboratory in less than 4 hours.Each specimen was smeared onto Columbia agar medium base plus 10% sheep blood,1% fetal calf serum (Gibco, USA) and Dent supplement (Oxoid, UK). Plates were incubatedunder microaerophilic conditions at 37°C, and were read on day 5. A culture was consideredpositive if typical

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.

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. (C) 2005 IMSS. Published by Elsevier Inc.

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.

5 methods of utilization of sugars were evaluated in 25 strains previously identified as N. gonorrhoeae: CTA agar, modified CTA agar, gelatin starch agar, Mueller Hinton agar plus bromotimol blue and rapid method. 100% of the strains of N. gonorrhoeae were identified by the CTA and rapid methods, whereas 96% were identified by the modified CTA and gelatin starch agar methods. No strain of gonoccocus was identified by the Mueller Hinton agar method plus bromotimol blue. The cystine tripticase agar medium (CTA) is the elective method to confirm the isolates of Neisseria gonorrhoeae. As it is mainly made by U.S. companies, it is difficult for Cuba to acquire it. The gelatin-starch agar method and the rapid method are useful alternative of the CTA medium, so their use is proposed in this paper.

P64k is a meningococcal protein from Neisseria meningitidis that has been obtained by recombinant DNA technology. Recombinant P64k has been extensively characterized by physicochemical and immunological methods. Lately this protein has been found to act as a versatile immunological carrier for weak antigens in mice. In the present work, a Phase I clinical trial was carried out in healthy volunteers who received three inoculations of either placebo or recombinant P64k (20 or 50 μg). No severe adverse events occurred during the trial. Only mild adverse events in ten volunteers were observed. At 1 month after the third dose, 15 out of 18 volunteers (83.3%) who received the recombinant antigen had a P64k‐specific antibody titre 1:100, as detected by ELISA. A fourth dose, given 9 months after the third one, elicited a potent booster immune response in P64k vaccinees. Accordingly, these P64k formulations were considered safe and immunogenic in healthy human volunteers.

The susceptibility to penicillin of 111 Neisseria meningitidis strains was assessed by the agar-dilution procedure and serosubtypes were determined by a whole-cell enzyme-linked immunoassay using monoclonal antibodies reagents. Thirty-five isolates showed reduced sensitivity to penicillin (MIC > or = 0.1 mg/l and < or = 1 mg/l) and no resistant strains were detected. The most common phenotype was B:4:P1.15 (77.5%) and a rising trend of non-typeable and non-subtypeable strains was detected. The increase in levels of minimal inhibitory concentrations of meningococci to penicillin gives cause for concern and the increase in non-typeable and non-subtypeable isolation demand the use of molecular biology techniques for their typing.