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Helicobacter pylori , invades the gastric mucosa and is one of the causative agents of stomach cancer and peptic ulcers. Origanum vulgare , is a flavouring herb used worldwide. But little is known about the effects of extracts prepared by maceration in cold PBS. This study was aimed at determining the superoxide dismutase (SOD)- and peroxidase (Px)-like antioxidant activities as well as the immunomodulatory activity (anti-inflammatory/pro-inflammatory) of an aqueous extract of O. vulgare by evaluating the production of nitric oxide (NO) in macrophages stimulated with H. pylori derivatives. The cold extract presented SOD-like and Px-like activities with effective concentration 50 (EC50) values of Px = 489.7 ± 48 µg/ml and SOD= 384.7 ± 30 µg/ml. The extract was also capable of modulating the production of NO in macrophages stimulated by H. pylori derivatives by exerting a pro-inflammatory activity at high concentrations and an anti-inflammatory activity at low concentrations.

Helicobacter pylori is a Gram negative bacterium most frequently associated with human gastrointestinal infections worldwide. The increasing occurrence of antibiotic-resistant isolates of H. pylori constitutes a challenge. The eradication of the microorganism is currently being considered a "high priority" by the World Health Organization (WHO). In this context, bioactive compounds found in natural products seem to be an effective therapeutic option to develop new antibiotics against the pathogen. In this study, we investigated the effect of asclepain cI, the main purified proteolytic enzyme of the latex of petioles and stems from Asclepia curassavica L. (Asclepiadaceae), a South American native plant, against H. pylori ; in order to obtain a natural therapeutic adjuvant and a safe nutraceutical product. Asclepain cI showed antibacterial activity against reference strains and drug-resistant clinical isolates of H. pylori in vitro . A range of minimal inhibitory concentration (MIC) from 1 to 2 μg/ml and minimal bactericidal concentration (MBC) from 2 to 4 μg/ml was obtained, respectively. The action of asclepain cI on the transcription of omp 18, ure A, fla A genes showed a significantly decreased expression of the selected pathogenic factors. Furthermore, asclepain cI did not induce toxic effects at the concentrations assayed. Asclepain cI could be considered a highly feasible option to be used as a natural therapeutic adjuvant and a safe nutraceutical product against H. pylori ., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Salinas Ibáñez, Origone, Liggieri, Barberis and Vega.)

Helicobacter pylori infection is widespread worldwide, with more than a half of the world population infected. H. pylori antibiotic-resistant strains and non-compliance to therapy are the major causes of H. pylori eradication failure. The search for new therapies based on plant extracts is a scientific interest field. The present study was conducted to evaluate the effect in vitro of extra virgin olive oil (EVOO), hydroxytyrosol (HT), and oleuropein (Olp) against two H. pylori strains and the effect in vivo of the oral administration of EVOO on the gastric mucosa of BALB/c mice infected with this microorganism. The broth microdilution method assayed the antibacterial in vitro activity of EVOO, HT, and Olp against H. pylori strains. For in vivo studies, male BALB/c mice were infected orally with an H. pylori suspension every 72 h. Four groups were used: (1) Control, (2) H. pylori -infected (HP), (3) EVOO, and (4) HP + EVOO. Mice were sacrificed at 7, 15, and 30 days. The stomachs were removed and observed under a microscope. Scoring of the degree of erosion was determined. Samples were processed by histological techniques for light microscopy. Macroscopic analysis showed that the presence of small erosions increased, both in number and size, in the infected group. Animals infected and treated with EVOO exhibited the presence of fewer erosions, which decreased in number as the treatment progressed. The mucosa of the control and EVOO groups showed normal histological characteristics at the three times studied. The mucosa of animals infected with H. pylori showed disruptions of the lining epithelium, damage to gastric glands, and vasodilation. The mucosa of animals infected with H. pylori and treated with EVOO showed morphological characteristics similar to those of normal and EVOO mucosa. For the first time, the current study showed the effect in vitro and in vivo of EVOO and combined administration of HT and Olp against H. pylori using an animal model. Future studies are needed to establish the mechanism of EVOO's action at the gastric mucosa level to propose this product as a natural antimicrobial agent for the treatment of gastric H. pylori infections., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Arismendi Sosa, Mariani, Vega and Penissi.)

Helicobacter pylori is a gram-negative, helix-shaped, and microaerophilic bacteria that colonizes the human gastric mucosa, causing chronic infections, gastritis, peptic ulcer, lymphomas associated with lymphoid mucosa tissue, and gastric cancer. H. pylori is considered a Type 1 human carcinogen by WHO. The prevalence of the infection is estimated in more than half of the world population. Treatment of H. pylori infection includes antibiotics and proton pump inhibitors, but the increasing antibiotic resistance promotes the research of novel, more effective, and natural antibacterial compounds. The aim of this work was to study the effect of the partially purified proteolytic extract (RAP) of the fruits from Solanum granuloso-leprosum (Dunal), a South American native plant, and a purified fraction named granulosain I, against H. pylori , to obtain natural food additives for the production of anti- H. pylori functional foods. Furthermore, granulosain I and RAP could be used as natural adjuncts to conventional therapies. Granulosain I and RAP antibacterial activity was evaluated as minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) against H. pylori NCTC 11638 (reference strain) and twelve H. pylori wild strains, using a microdilution plating technique (Clinical and Laboratory Standards Institute). All the strains tested were susceptible to granulosain I with MIC from 156.25 to 312.5 μg/mL and MBC from 312.5 to 625 μg/mL, respectively. Besides, all the strains tested were susceptible to the RAP with MIC from 312.5 to 625 μg/mL and MBC from 625 to 1,250 μg/mL, respectively. The effect of granulosain I and RAP on the transcription of H. pylori genes encoding pathogenic factors, omp 18, ure A, and fla A, with respect to a housekeeping gene ( 16S rRNA), was evaluated by RT-PCR technique. The band intensity between pathogenic factors and control gene was correlated under treated or untreated conditions, using the ImageJ program. Granulosain I and RAP significantly decreased the expression of pathogenic factors: omp 18, ure A, and fla A. The combined inhibitory effect of granulosain I or RAP and an antibiotic such as, amoxicillin (AML, 10 μg), clarithromycin (CLA, 15 μg), levofloxacin (LEV, 5 μg), and metronidazole (MTZ, 5 μg) was evaluated, using the agar diffusion technique. Granulosain I and RAP showed significant synergistic effect on AML, CLA, and LEV, but no significant effect on MTZ was observed. Besides, granulosain I and RAP did not show toxicological effects at the concentrations studied. Finally, granulosain I and RAP could be used as safe natural food additives and as adjuvants for conventional therapies against H. pylori ., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Salinas Ibáñez, Vallés, Adaro, Barberis and Vega.)

This paper focuses on studying how mineral oil, sunflower, soybean, and corn lubricants influence friction and wear effects during the manufacturing of aluminum parts via the single point incremental forming (SPIF) process. To identify how friction, surface roughness, and wear change during the SPIF of aluminum parts, Stribeck curves were plotted as a function of the SPIF process parameters such as vertical step size, wall angle, and tool tip semi-spherical diameter. Furthermore, lubricant effects on the surface of the formed parts are examined by energy dispersive spectroscopy (EDS) and scanning electron microscope (SEM) images, the Alicona optical 3D measurement system, and Fourier-transform infrared spectroscopy (FTIR). Results show that during the SPIF process of the metallic specimens, soybean and corn oils attained the highest friction, along forces, roughness, and wear values. Based on the surface roughness measurements, it can be observed that soybean oil produces the worst surface roughness finish in the direction perpendicular to the tool passes ( Ra =1.45 μm) considering a vertical step size of 0.25 mm with a 5 mm tool tip diameter. These findings are confirmed through plotting SPIFed Stribeck curves for the soybean and corn oils that show small hydrodynamic span regime changes for an increasing sample step-size forming process. This article elucidates the effects caused by mineral and vegetable oils on the surface of aluminum parts produced as a function of Single Point Incremental Sheet Forming process parameters.

Helicobacter pylori is a microorganism that in the last years has been associated with extragastric disorders such as respiratory diseases, however, its impact on lung is partially understood. The aim of this work was to study infection impact of H. pylori on the inflammatory markers expression at the pulmonary level using an animal model. Infection was performed by BALB/c wild type (WT) mice orotracheal instillation with 20 μl of 1 × 10 8 H. pylori reference strain suspension once per day throughout 3 days. Inflammatory response was evaluated at 3, 7, 14, 21 and 30 days post infection. Lung was aseptically removed and pulmonary edema index values showed a significant change at 30 days of infection. Hematoxylin-Eosin (H-E) stain allowed to visualizing H. pylori presence in lung samples at 3 days of infection near the phagocytic cells or in the alveoli lumen. Bronchoalveolar lavage (BAL) was used for inflammatory response evaluation. Lactate dehydrogenase values showed a gradual increase in infected animals along infection time. Protein concentrations in mg/ml from BAL increased significantly at 7 days in infected animals. Macrophages viability obtained from BAL, decreased at the first moment of infection, maintaining constant values along contamination time. Results obtained demonstrate an inflammatory response in lung after orotracheal H. pylori infection and suggest that the pathogenic mechanism is strongly evidenced by tissue damage, endothelial dysfunction inflammatory mediators and markers expression at the pulmonary level., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Helicobacter pylori infection has been reported to be associated with extra-digestive disorders such as respiratory diseases; however, the impact of H. pylori on lung is incompletely understood. Inflammatory response is mediated by the release of cytokines, interferon, and enzymes such as metalloproteinases (MMPs). This may contribute to collagen accumulation during the early phase of infection. MMP expression is an important factor for the proliferation and infiltration of lung cells in the process of fibrosis formation. The aim of this work was to study the impact of the infection with H. pylori on lung using a mouse model. We looked for histological lesions of lung infected with the microorganism as well as the expression of inflammatory and of endothelial dysfunction markers. C57BL/6 wild type (WT) mice were infected by orotracheal instillation with 20 μl of 1 × 10 8 H. pylori reference strain suspension once per day for 3 days. Animals infected and controls were sacrificed at 3, 7, 14, 21 and 30 days. The lung from mice were stained with Hematoxylin-Eosin (H-E), Masson's Trichromic and Periodic Acid Schifft (PAS) for histological study. Also, lipid hydroperoxides and enzime catalase (CAT) activity were determined. Expression level of multiple markers implicated in inflammation (tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-4, IL-6, IL-8, IL-10; metalloproteinase MMP-9) and markers of endothelial dysfunction (I-CAM and V-CAM) was determined from lung tissues mRNA using RT-PCR. Results showed that H. pylori induced morphological changes in the lung tissue with recruitment of inflammatory cells and lung parenchymal cell degradation. The mRNA of IL-1β and TNF-α; MMP-9, I-CAM and V-CAM increased at 3-7 days infections. Also, iNOS, IL-8 and Phosphocholine cytidyltransferase (CCT) increased with lung injury. Anti-inflammatory interleukin as: IL-4, and IL-10 increased at 7 and 14 days post infection respectively. The results obtained suggest that the pathogenic mechanism of H. pylori on lung could be strongly associated with lung injury as indicate the expression increased of inflammatory mediators and markers of endothelial dysfunction., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Ethnopharmacological Relevance: Lithraea molleoides (Vell.) Engl. (Anacardiaceae) is a medicinal plant traditionally used in South America to treat various ailments, including diseases of the digestive system., Aim of the Study: To evaluate the in vivo antiulcer and antimicrobial activities against Helicobacter pylori of L. molleoides and its isolated compounds., Materials and Methods: Methanolic extract 250 and 500 mg/kg, (LmE 250 and LmE 500, respectively) and infusions, 10 g and 20 g en 100mL (LmI 10 and LmI 20, respectively) of L. molleoides was evaluated for antiulcer activity against 0.6N HCl, 0.2N NaOH, 200mg/kg acetilsalicilic acid and absolute ethanol-induced gastric ulcers in rats. The degree of erosion in the glandular part of the stomach was assessed from a scoring system. Acute toxicity in mice was also evaluated. The antiulcer effect of the isolated compounds (catechol, mannitol, rutin, gallic acid, ferulic acid and caffeic acid, 100mg/kg) was evaluated against absolute ethanol-induced gastric ulcers in rats. The anti-Helicobacter pylori activity of L. molleoides and isolated compounds was performed using broth dilution methods., Results: The LmE 250, LmE 500, LmI 10 and LmI 20 produced significant inhibition on the ulcer index in 0.6N HCl, 0.2N NaOH, 200mg/kg acetilsalicilic acid and absolute ethanol- induced gastric ulcers in rats. The isolated compounds, catechol, mannitol, rutin, ferulic acid and caffeic acid were active in absolute ethanol- induced gastric ulcers in rats. L. molleoides and different compounds showed antimicrobial activity in all strains tested. The lowest MIC value (0. 5 μg/mL) was obtained with catechol in six of eleven strains assayed. No signs of toxicity were observed with doses up to 2g/kg in an acute toxicity assay., Conclusion: These findings indicate that L. molleoides displays potential antiulcerogenic and antimicrobial activities and the identification of active principles could support the use of this plant for the treatment of digestive affections., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

The purpose of this work was to evaluate the use of an alternative transport medium supplemented with a cyanobacterial extract (CE), free of animal derivatives, to preserve the viability of Helicobacter pylori strains during long-term transportation and allow its recovery from biopsy samples. The transport media evaluated were Mueller-Hinton broth 0.3% agar (MH) and 0.3% of CE (MH-CE). MH broth 5% fetal calf serum (FCS) was used as the reference medium (MH-FCS). Biopsy samples from 134 patients, H. pylori NCTC 11638 and six clinical isolates were studied. A higher recovery (p ≤ 0.001) at 4°C was obtained in MH-CE than in MH-FCS after 96 h of storage. Only MH-CE allowed recovery after 120 h. The H. pylori recovery at room temperature after 96 h was higher (p ≤ 0.005) in MH-CE than in MH-FCS. Similar survival rates were observed in biopsy samples conserved in MH-CE and MH-FCS at 4°C. The recovery after 48 h at room temperature in MH-CE was higher (p ≤ 0.05) than MH-FCS and was the only medium allowing recovery after 72 h. The MH-CE medium is a simple, inexpensive and animal derivatives-free transport medium that can be used to preserve H. pylori viability and its recovery from biopsy samples.

Objective: To characterize Helicobacter pylori isolates from western Argentina using virulence markers and antimicrobial susceptibility patterns in order to assess the association between virulent genotypes, antimicrobial resistance, and disease. DNA fingerprinting was also evaluated for the segregation of virulent or resistant strain clusters., Methods: Genotyping of 299 H. pylori isolates was performed by PCR using specific primers for the cagA, vacA and iceA genes. Random amplification of polymorphic DNA (RAPD)-PCR and rep-PCR genetic clustering were assessed using five random primers and BOXA1R and ERIC primers, respectively. Resistance to clarithromycin (Cla) and metronidazole (Mtz) was assessed by the agar dilution method., Results: It was observed that 40.8% of the genotypes were cagA-positive; 66.9% were vacA s1m1 genotype and the iceA1 allele was found in 40.8%. A significant correlation (p=0.0000) was observed between cagA positivity and vacA s1m1/iceA1 genotypes. Triple virulent genotypes were statistically associated with peptic ulcer (PU) (p=0.0001) and Cla resistance (p=0.0000). RAPD fingerprints obtained with AO2 primers identified clusters that were strongly associated with PU, virulence markers, and resistance to Cla and Mtz., Conclusions: The H. pylori isolates that harbored two or three virulence markers were more resistant to Cla and Mtz. Combined analysis of virulent genotypes and resistance patterns may permit identification of high-risk patients to prevent PU later in life or to avoid antimicrobial treatment failure., (Copyright © 2010 International Society for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

Ethnopharmacological Relevance: The increasing resistance of Helicobacter pylori to antibiotics demands the search for novel compounds from plant based sources. Artemisia douglasiana Besser is widely used in Cuyo region (Argentina) as folk medicine for the treatment of gastric ailments., Aim of Study: Based on our previous studies that Artemisia douglasiana exert cytoprotective actions against ethanol-induced gastric mucosal injury we assayed the anti-Helicobacter pylori effect of the Artemisia douglasiana extract and its active compound, dehydroleucodine., Materials and Methods: The in vitro anti-bacterial activity of Artemisia douglasiana extract and its active compound, dehydroleucodine were determined against one standard strain and six clinical isolates of Helicobacter pylori by using the agar dilution methods., Results: The results showed that both dehydroleucodine and Artemisia douglasiana extract had activity against the microorganism with MICs between 1-8 and 60-120 mg/L, respectively., Conclusions: Artemisia douglasiana may be a useful alternative treatment strategy principally in eradication of metronidazole and clarithromycin-resistant strain.

Clarithromycin resistance is an important factor of eradication failure. A commercially available fluorescent in situ hybridization (FISH) kit (creaFAST) was used to detect H. pylori infection and the resistance to clarithromycin in frozen biopsies. A total of 33 biopsies, H. pylori culture-positive, obtained from pediatric patients were retrospectively studied. Clarithromycin resistance was compared with MICs detected by E-test from H. pylori clinical isolates. All culture-positive biopsies were positive by FISH. Detection of clarithromycin resistance showed sensitivity of 90%, specificity of 100%, positive predictive value of 100%, and negative predictive value of 86.7% compared with results obtained by E-test. Discrepant results were 2 biopsies, clarithromycin-susceptible by FISH but intermediate by E-test. In conclusion, FISH technology is a rapid, easy-to-implement, and reliable cultivation-independent method for routine application; however, when frozen biopsies are studied, some modification of the recommended procedure should be used to obtain better results.

The vacA and cagA genotypes of Helicobacter pylori exhibited distinct geographic distribution and correlation with severity of disease. In the above genotypes (obtained from 150 H. pylori-positive patients--139 with gastritis, 10 with ulcer and 1 patient with gastric cancer) combinations vacA s1/m1 and s2/m2 were detected using PCR in 75 and 25% of isolates, respectively, in patients with chronic gastritis. The of s1/m1 and s2/m2 combinations were also detected from ulcers (60 and 40%, respectively). The cagA was detected in 30% of isolates. Concentrated culture supernatants of 7 (64%) out of 11 H. pylori strains induced vacuolization in Vero cells in titers ranging from 1:5 to 1:40. The vacA s1 genotype was significantly associated with, but not predictive of the presence of vacuolating cytotoxin activity and the cagA gene.

We studied here the effect of aqueous extracts of Larrea divaricata Cav on the growth of Helicobacter pylori. Results show that cold extract, infusion, decoction and simulated digestion had inhibitory activity at 0.04-0.1 mg/l against clarithromycin and metronidazole susceptible and resistant H. pylori strains. These results support the popular use of L. divaricata Cav in gastric disturbances and prompt further research to characterize these compounds with a therapeutic potential against gastric ulcers and gastric cancer associated with H. pylori.

Solid and liquid media supplemented only with a cyanobacterial extract (CE) and free of fetal calf serum (FCS), blood, and its derivatives support the growth of Helicobacter pylori. A total of 11 strains of H. pylori isolated from gastric biopsy samples were successfully subcultured in Mueller-Hinton agar supplemented with 0.4% CE. When this medium was used for primary isolation of H. pylori, a low isolation rate (30%) was observed because of the abundant growth of contaminants. The growth kinetics of eight isolates and H. pylori reference strain NCTC 11638 in Mueller-Hinton broth (MHB) supplemented with 0.7% CE were estimated by use of growth parameters, and the results were compared with those obtained with MHB-5% FCS. For four strains the cellular concentrations obtained with CE were statistically higher (P < 0.05) than those obtained with FCS, and in some cases these values were similar to the highest values reported in the literature. Depending on the strain, the specific growth rates obtained with CE were similar to or increased compared with those obtained with FCS. The replacement of FCS by CE in H. pylori cultures would facilitate the retrieval of cultures with high cellular densities as a source of cellular and extracellular proteins free of serum. Also, CE has advantages over conventional supplements, such as easier conservation and compliance with the pressing tendency at present to avoid the use of products derived from animals.

Several methods have been used to detect clarithromycin resistance. Agar dilution is now recommended by the NCCLS (susceptible to clarithromycin: MIC<=0.25 mg/l; intermediate resistance: MIC=0.5 mg/l; resistant: MIC>=1 mg/l), and the detection of mutations involved in resistance is used in many laboratories. We analyzed 36 clarithromycin-resistant strains isolated from children and 30 from adults. In vitro susceptibility to clarithromycin was determined by an agar dilution method. DNA from the isolates was extracted using the method published by Ge and Taylor. A2142G and A2143G mutations were identified by PCR-RFLP. A 1.4 Kpb of the 23S rRNA gene was amplified and digested using MboII or BsaI restriction enzymes to detect mutations. The prevalence of the A-G transition mutation at position 2143 was higher in the children (80.55%) than in the adult patients (46.66%) (p<0.05); however, the prevalence of the mutation at position 2142 was higher in adults than in children (36.66% vs. 5.55%; p<0.05). In children, a higher MIC (2-64 mg/l) was observed when the A-G mutation was detected at position 2143. However, in adult patients higher MICs were observed when the A-G mutation was detected at position 2142.

Clarithromycin and metronidazole resistance was 29.1 and 23.9%, respectively, in 96 Helicobacter pylori strains obtained from pediatric patients. No resistance to amoxicillin was observed. Resistance according to patients' ages to clarithromycin and metronidazole was 45.4 and 18.2% in 22 patients from 4 to 8 years old, 30.2 and 20.7% in 53 patients from 9 to 13 years old, and 9.5 and 38.1% in 21 patients from 14 to 18 years old, respectively. The A2143G mutation was the most prevalent (82.1%) among clarithromycin-resistant strains.

Aim: To study microbiological factors of Helicobacter pylori, such as antimicrobial susceptibility and virulence, which affect the eradication of the microorganism in gastric mucosa of adult and pediatric patients., Methods: Fifty-five H. Pylori strains were isolated from culture of biopsy specimens from 39 adult and 16 pediatric patients. Antibiotic susceptibility was determined by agar dilution and study of virulence factors (cagA gene and alleles s1 and s2 of the vacA gene) by PCR. Outcome of treatment with amoxicillin (AMX), clarithromycin (CLR) and omeprazole was assessed by the urea breath test. We studied the relation of minimum inhibitory concentration (MIC) of AMX and CLR and presence of virulence factors with eradication of the microorganism.Results. The eradication rate was 69% (38/55), 71.7% in adults and 62.5% in children. Resistance to CLR and AMX was 14.5% and 0%, respectively. Overall eradication rates with respect to the variables studied were: 75% and 53% in strains with AMX MICs of < or = 0.01 6mg/L and > or = 0.032 mg/L (range < or = 0.008-0.5) (p > 0.05), 79% and 12% in strains with clarithromicina MICs of < 1 mg/L and > or = 1mg/L (range # 0.008-64) (p < 0.05), 79% and 54% in cagA+ and cagA strains (p > 0.05) and 82% and 62% in s1 and s2 strains (p > 0.05), respectively., Conclusions: H. pylori strains with higher amoxicillin and clarithromycin MICs, and cagA and vacA s2 strains were related with lower rates of eradication in both adult and pediatric populations treated with amoxicillin, clarithromycin and omeprazole.

In this study we present a relationship between different gastroduodenal pathologies and Helicobacter pylori infection. We used four diagnosis invasive methods for H. pylori infection: urease test (UT), histopathology (H), Gram stain (G) and culture (C). The upper gastrointestinal endoscopy of 300 dyspeptic patients showed that 71.6% had erosive congestive gastropathies, 13.6% had duodenopathies, 5.6% had gastric ulcer, 6.3% had duodenal ulcer and 2.6% had probable gastric neoplasia. We also correlated the data of water intake source with the pathologies. The percentage of infected patients with H. pylori was determined using: a) two simultaneous reference tests (UT and H), 54.3%, b) each test UT = 55.0%, H = 59.0%, G = 51.3%, and C = 43.0%. Sex, age and the source of water ingested did not show statistically significant differences.

Digitoxin is very strongly bound to serum albumin. Although free digitoxin is pharmacologically active, it is not monitored because of the lack of a sufficiently sensitive technique. The concentration of free digitoxin in the protein-free ultrafiltrate is usually below the detection limit of digitoxin immunoassays. A modified technique is described by which free digitoxin can be routinely monitored using commercially available immunoassays. The fluorescence polarization immunoassay for determining total digitoxin concentration requires that 100 microL of serum be treated with 300 microL of methanol to precipitate proteins. It is demonstrated that free digitoxin can easily be measured by adding 100 microL of methanol to 300 microL of ultrafiltrate, thus improving the sensitivity of the assay three-fold. The free digitoxin concentration can easily be calculated by dividing the observed value by 3. An attempt to use only ultrafiltrate (no methanol added) caused significant bias in the result, probably as a result of a matrix problem. The chemiluminescent assay for digitoxin does not require any specimen pretreatment and requires only 10 microL of serum. The program was modified and used 50 microL of ultrafiltrate to improve the sensitivity of the free digitoxin assay. If the chemiluminescent assay is used to measure free digitoxin, the true free digitoxin concentration can be calculated by dividing the observed value by 4.3. The free digitoxin concentrations were comparable in eight patients receiving digitoxin as measured by both methods. To show an application of this technique, two serum pools were prepared from patients receiving digitoxin and supplemented with various concentrations of phenytoin. A significant increase in free digitoxin concentration was observed because of the displacement of digitoxin from protein binding sites by phenytoin.

It has been suggested that oral dissemination might be the major transmission vehicle for Helicobacter pylori, and that dental plaque might act as its reservoir. The presence of H. pylori was investigated in 62 odontological male and female patients (average age: 35 years old). Samples were taken from supragingival plaque, placed in 0.3 ml of thioglycolate broth, cultured within 12 h in Mueller-Hinton agar with the addition of 5-7% of sheep blood and antibiotic supplement, and incubated at 37 degrees C in microaerophilia for 5-7 days. Typical colonies were identified by gram, urease, oxidase and catalase. H. pylori was detected in a 15 year-old patient suffering from gastric acidity (1.61% positivity index). The medium used facilitated recovery of the agent from a sample abundant in germs. H. pylori was not recovered from the same patient 12 months later, suggesting that there might have been a transitory passage by gastric reflux or that the bacterium was acquired from an exogenous source.