A strain-independent method to induce progressive and lethal pneumococcal pneumonia in neutropenic mice.

BACKGROUND: Experimental models of pneumonia with penicillin non-susceptible Streptococcus pneumoniae (PNSSP) are hard to reproduce because the majority of strains with clinical relevance (like serotypes 6B, 9 V and 19 F) have low murine virulence. By optimization of culture and inoculum conditions of PNSSP (using porcine mucin), our aim was to develop a suitable, reliable and reproducible pneumonia mouse model for anti-infective pharmacology research. RESULTS: Seven PNSSP strains, including serotypes 6B, 9 V, 14 and 19 F were included. Strain INS-E611 displayed the highest murine virulence and was chosen to validate the lung model. Nose-instilled pneumococci grew between 2.1 and 2.5 log10 CFU/g of lung in 24 hours when an optimized culture of bacterial cells was used, but animals were all alive and recovered of infection after 36 h. In contrast, inoculum supplementation with mucin led to 100% mortality related to a successful lung infection confirmed by histopathology. These findings were reproduced with all seven PNSSP strains in neutropenic mice. Immunocompetent animals cleared all strains spontaneously. CONCLUSIONS: This pneumonia model produces a progressive and uniformly fatal lung infection with diverse serotypes of PNSSP independently of their intrinsic murine virulence.

The OLGA-OLGIM staging and the interobserver agreement for gastritis and preneoplastic lesion screening: a cross-sectional study.

Stomach cancer (SC) incidence and mortality are relevant public health issues worldwide. In Colombia, screening for preneoplastic lesions (PNL) and the presence of H. pylori is not routinely performed. Therefore, the aim of this study was to evaluate OLGA-OLGIM staging and the interobserver agreement in gastritis and preneoplastic lesions in patients with gastroduodenal symptoms from Colombia. A cross-sectional study was conducted in 272 patients with gastroduodenal symptoms. Gastric biopsies were taken following the Updated Sydney System with the OLGA-OLGIM classification, and the results were evaluated by two pathologists. Chronic gastritis and PNL were reported in 76% and 24% of the patients, respectively. Furthermore, 25% of the patients with PNL displayed gastric atrophy (GA) and 75% intestinal metaplasia (IM). Agreement in the histopathological reading for IM was good, whereas for OLGA was variable, and for the H. pylori quantity was poor. OLGA-OLGIM stages 0-II were the most frequent (96%), while stage III (4%) and SC (4%) were the least frequent. Age and coffee consumption were associated with a higher prevalence of PNL. This work determined that 4% of the population is at high risk of developing SC and would benefit from follow-up studies. Reinforcement of training programs to improve the agreement in histopathology readings is required.

Generic vancomycin products fail in vivo despite being pharmaceutical equivalents of the innovator.

Generic versions of intravenous antibiotics are not required to demonstrate therapeutic equivalence with the innovator because therapeutic equivalence is assumed from pharmaceutical equivalence. To test such assumptions, we studied three generic versions of vancomycin in simultaneous experiments with the innovator and determined the concentration and potency of the active pharmaceutical ingredient by microbiological assay, single-dose pharmacokinetics in infected mice, antibacterial effect by broth microdilution and time-kill curves (TKC), and pharmacodynamics against two wild-type strains of Staphylococcus aureus by using the neutropenic mouse thigh infection model. The main outcome measure was the comparison of magnitudes and patterns of in vivo efficacy between generic products and the innovator. Except for one product exhibiting slightly greater concentration, vancomycin generics were undistinguishable from the innovator based on concentration and potency, protein binding, in vitro antibacterial effect determined by minimal inhibitory or bactericidal concentrations and TKC, and serum pharmacokinetics. Despite such similarities, all generic products failed in vivo to kill S. aureus, while the innovator displayed the expected bactericidal efficacy: maximum antibacterial effect (Emax) (95% confidence interval [CI]) was 2.04 (1.89 to 2.19), 2.59 (2.21 to 2.98), and 3.48 (2.92 to 4.04) versus 5.65 (5.52 to 5.78) log10 CFU/g for three generics and the innovator product, respectively (P<0.0001, any comparison). Nonlinear regression analysis suggests that generic versions of vancomycin contain inhibitory and stimulatory principles within their formulations that cause agonistic-antagonistic actions responsible for in vivo failure. In conclusion, pharmaceutical equivalence does not imply therapeutic equivalence for vancomycin.

Etiologic diagnosis of chronic osteomyelitis: a prospective study.

BACKGROUND: Although bone specimens were established 25 years ago as the gold standard for etiologic diagnosis of chronic osteomyelitis, recent studies suggest that nonbone specimens are as accurate as bone to identify the causative agent. We examined concordance rates between cultures from nonbone and bone specimens in 100 patients. METHODS: Prospective study conducted at Hospital Universitario San Vicente de Paul, a 750-bed university-based hospital located in Medellín, Colombia. We included patients with chronic osteomyelitis who had been free of antibiotic therapy for at least 48 hours, excluding those with diabetic foot and decubitus ulcers. At least 1 nonbone and 1 bone specimen were taken from each individual and subjected to complete microbiologic analysis. RESULTS: Bone cultures allowed agent identification in 94% of cases, including anaerobic bacteria in 14%. Cultures of nonbone and bone specimens gave identical results in 30% of patients, with slightly better concordance in chronic osteomyelitis caused by Staphylococcus aureus (42%) than by all other bacterial species (22%). However, statistical concordance determined by the Cohen kappa statistic was less than 0 (-0.0092+/-0.0324), indicating that the observed concordance was no better than that expected by chance alone (P>.99). CONCLUSIONS: Appropriate diagnosis and therapy of chronic osteomyelitis require microbiologic cultures of the infected bone. Nonbone specimens are not valid for this purpose.

Neutropenia induced in outbred mice by a simplified low-dose cyclophosphamide regimen: characterization and applicability to diverse experimental models of infectious diseases.

BACKGROUND: For its low cost and ease of handling, the mouse remains the preferred experimental animal for preclinical tests. To avoid the interaction of the animal immune system, in vivo antibiotic pharmacodynamic studies often employ cyclophosphamide (CPM) to induce neutropenia. Although high doses (350-450 mg/kg) are still used and their effects on mouse leukocytes have been described, a lower dose (250 mg/kg) is widely preferred today, but the characteristics and applicability of this approach in outbred mice have not been determined. METHODS: Fifteen female ICR mice were injected intraperitoneally with 150 and 100 mg/kg of CPM on days 1 and 4, respectively. Blood samples (approximately 160 microL) were drawn from the retro-orbital sinus of each mouse on days 1, 4, 5, 6, 7 and 11. Leukocytes were counted manually and the number of granulocytes was based on microscopic examination of Wright-stained smears. The impact of neutropenia induced by this method was then determined with a variety of pathogens in three different murine models of human infections: pneumonia (Klebsiella pneumoniae, Streptococcus pneumoniae, Staphylococcus aureus), meningoencephalitis (S. pneumoniae), and the thigh model (S. aureus, Escherichia coli, Bacteroides fragilis). RESULTS: The basal count of leukocytes was within the normal range for outbred mice. On day 4, there was an 84% reduction in total white blood cells, and by day 5 the leukopenia reached its nadir (370 +/- 84 cells/mm3). Profound neutropenia (< or =10 neutrophils/mm3) was demonstrated at day 4 and persisted through days 5 and 6. Lymphocytes and monocytes had a 92% and 96% decline between days 1 and 5, respectively. Leukocytes recovered completely by day 11. Mice immunosupressed under this protocol displayed clinical and microbiological patterns of progressive and lethal infectious diseases after inoculation in different organs with diverse human pathogens. CONCLUSION: A CPM total dose of 250 mg/kg is sufficient to induce profound and sustained neutropenia (<10 neutrophils/mm3) at least during 3 days in outbred mice, is simpler than previously described methods, and allows successful induction of infection in a variety of experimental models.

Optimization of culture conditions to obtain maximal growth of penicillin-resistant Streptococcus pneumoniae.

BACKGROUND: Streptococcus pneumoniae, particularly penicillin-resistant strains (PRSP), constitute one of the most important causes of serious infections worldwide. It is a fastidious microorganism with exquisite nutritional and environmental requirements to grow, a characteristic that prevents the development of useful animal models to study the biology of the microorganism. This study was designed to determine optimal conditions for culture and growth of PRSP. RESULTS: We developed a simple and reproducible method for culture of diverse strains of PRSP representing several invasive serotypes of clinical and epidemiological importance in Colombia. Application of this 3-step culture protocol consistently produced more than 9 log10 CFU/ml of viable cells in the middle part of the logarithmic phase of their growth curve. CONCLUSION: A controlled inoculum size grown in 3 successive steps in supplemented agar and broth under 5% CO2 atmosphere, with pH adjustment and specific incubation times, allowed production of great numbers of PRSP without untimely activation of autolysis mechanisms.

Fundación del primer bioterio MPF funcional de Colombia / Foundation of a functional pathogen animal facility in Colombia

Cualquier investigación donde se experimente con modelos animales exige que el estado microbiológico y genético de los mismos sea definido desde el principio y verificado periódicamente para garantizar la fiabilidad y certeza de los datos. Buscando producir ratones libres de patógenos murinos en Colombia, se importaron 173 reproductores no emparentados suizos albinos de la cepa ICR que se hospedaron bajo condiciones protocolizadas de aislamiento microbiológico en el Bioterio MPF de la Facultad de Medicina de la Universidad de Antioquia. Tras dos años de funcionamiento ininterrumpido, el control microbiológico demostró que los progenitores mantuvieron inalterada la flora con la cual fueron exportados a Colombia, que sus crías portan flora murina idéntica, y que la misma corresponde a la exigida internacionalmente. Diversas pruebas a la integridad de la barrera microbiológica condujeron a la detección y erradicación de un brote infeccioso por Parvovirus murino. El control reproductivo permitió mantener intacta la eterocigocidad de la cepa, denominada Udea:ICR(CD-1).

In order to guarantee the accuracy of the data, experimentation with animal models requires well defined microbiological and genetic statuses with periodical verification. To produce murine pathogen free mice, an outbreeding program was set up by importing 173 non-related Swiss albino ICR breeders that were hosted under strict conditions of microbiological isolation in the animal facilities of the University of Antioquia Medical Faculty. After two years of uninterrupted reproduction, microbiological control demonstrated unaltered murine flora for both parents and offspring, the same with which original breeders were dispatched to Colombia, complying with current international standards. An outbreak of Murine Parvovirus (MPV) was timely detected and erradicated thanks to strict testing of microbiological barriers. The breeding program allowed the production of animals free of genetic contamination.