Discovery and Contribution of Nontypeable Haemophilus influenzae NTHI1441 to Human Respiratory Epithelial Cell Invasion.

Nontypeable Haemophilus influenzae (NTHi) is the primary cause of bacterially induced acute exacerbations of chronic obstructive pulmonary disease (COPD). NTHi adheres to and invades host respiratory epithelial cells as a means to persist in the lower airways of adults with COPD. Therefore, we mined the genomes of NTHi strains isolated from the airways of adults with COPD to identify novel proteins to investigate their role in adherence and invasion of human respiratory epithelial cells. An isogenic knockout mutant of the open reading frame NTHI1441 showed a 76.6% ± 5.5% reduction in invasion of human bronchial and alveolar epithelial cells at 1, 3, and 6 h postinfection. Decreased invasion of the NTHI1441 mutant was independent of either intracellular survival or adherence to cells. NTHI1441 is conserved among NTHi genomes. Results of whole-bacterial-cell enzyme-linked immunosorbent assay (ELISA) and flow cytometry experiments identified that NTHI1441 has epitopes expressed on the bacterial cell surface. Adults with COPD develop increased serum IgG against NTHI1441 after experiencing an exacerbation with NTHi. This study reveals NTHI1441 as a novel NTHi virulence factor expressed during infection of the COPD lower airways that contributes to invasion of host respiratory epithelial cells. The role in host cell invasion, conservation among strains, and expression of surface-exposed epitopes suggest that NTHI1441 is a potential target for preventative and therapeutic interventions for disease caused by NTHi.

Heart rate variability after bariatric surgery: The add-on value of exercise.

PURPOSE: To assess the impact of bariatric surgery and an added supervised exercise training programme on heart rate variability (HRV) in patients with severe obesity. METHODS: Fifty-nine patients who underwent bariatric surgery were randomised in the post-operative period to a 12-week supervised exercise training programme (moderate intensity combination aerobic/resistance exercise training programme) or a control group. Indices of HRV including time-domain, spectral-domain, and nonlinear parameters were measured preoperatively, and at 3, 6, and 12 months. RESULTS: After the surgical procedure, both groups improved anthropometric parameters. Type 2 diabetes, hypertension, and dyslipidemia resolutions were similar between groups. Total body weight loss at 6 and 12 months were also comparable between groups (6 months: 28 ± 6 vs. 30 ± 6%; 12 months: 38 ± 9 vs. 38 ± 10%; control vs. intervention group respectively). Bariatric surgery improved HRV parameters at 12 months compared to the pre-operative values in the intervention group: standard deviation of R-R interval (SDNN) (156.0 ± 46.4 vs. 122.6 ± 33.1 ms), low frequency (LF) (6.3 ± 0.8 vs. 5.8 ± 0.7 ms2), and high frequency (HF) (5.1 ± 0.8 vs. 4.7 ± 0.9 ms2) (all p<0.001). For the control patients, similar improvements in SDNN (150.0 ± 39.4 vs. 118.8 ± 20.1 ms), LF (6.1 ± 0.9 vs. 5.7 ± 0.8 ms2), and HF (5.0 ± 0.9 vs. 4.7 ± 0.9 ms2) were obtained (all p<0.001). However, there was no add-on impact of the supervised exercise training programme on HRV after 12 months (p>0.05 for all HRV parameters). CONCLUSION: Bariatric surgery is associated with an improvement in HRV. A supervised exercise training programme in the post-operative period did not modulate further the benefits of bariatric surgery regarding HRV parameters.

Comparison of the Respiratory Resistomes and Microbiota in Children Receiving Short versus Standard Course Treatment for Community-Acquired Pneumonia.

Pediatric community-acquired pneumonia (CAP) is often treated with 10 days of antibiotics. Shorter treatment strategies may be effective and lead to less resistance. The impact of duration of treatment on the respiratory microbiome is unknown. Data are from children (n = 171), ages 6 to 71 months, enrolled in the SCOUT-CAP trial (NCT02891915). Children with CAP were randomized to a short (5 days) versus standard (10 days) beta-lactam treatment strategy. Throat swabs were collected at enrollment and the end of the study and used for shotgun metagenomic sequencing. The number of beta-lactam and multidrug efflux resistance genes per prokaryotic cell (RGPC) was significantly lower in children receiving the short compared to standard treatment strategy at the end of the study (Wilcoxon rank sum test, P < 0.05 for each). Wilcoxon effect sizes were small for beta-lactam (r: 0.15; 95% confidence interval [CI], 0.01 to 0.29) and medium for multidrug efflux RGPC (r: 0.23; 95% CI, 0.09 to 0.37). Analyses comparing the resistome at the beginning and end of the trial indicated that in contrast to the standard strategy group, the resistome significantly differed in children receiving the short course strategy. Relative abundances of commensals such as Neisseria subflava were higher in children receiving the standard strategy, and Prevotella species and Veillonella parvula were higher in children receiving the short course strategy. We conclude that children receiving 5 days of beta-lactam therapy for CAP had a significantly lower abundance of antibiotic resistance determinants than those receiving standard 10-day treatment. These data provide an additional rationale for reductions in antibiotic use when feasible. IMPORTANCE Antibiotic resistance is a major threat to public health. Treatment strategies involving shorter antibiotic courses have been proposed as a strategy to lower the potential for antibiotic resistance. We examined relationships between the duration of antibiotic treatment and its impact on resistance genes and bacteria in the respiratory microbiome using data from a randomized controlled trial of beta-lactam therapy for pediatric pneumonia. The randomized design provides reliable evidence of the effectiveness of interventions and minimizes the potential for confounding. Children receiving 5 days of therapy for pneumonia had a lower prevalence of two different types of resistance genes than did those receiving the 10-day treatment. Our data also suggest that children receiving longer durations of therapy have a greater abundance of antibiotic resistance genes for a longer period of time than do children receiving shorter durations of therapy. These data provide an additional rationale for reductions in antibiotic use.

Streptococcus pneumoniae serotype 6C: an intra- and interclonal complex comparison.

We report the annotated draft genome sequences of four serotype 6C Streptococcus pneumoniae isolates of differing genetic backgrounds. Serotype 6C isolates are increasing in prevalence and becoming progressively more resistant to antibiotics. As a result, these strains are likely to become more important in the near future.

Antibiotic resistance among pathogens causing disease in Jamaican children with HIV/AIDS.

OBJECTIVE: There are limited data regarding the antimicrobial resistance patterns of pathogens in children with HIV/AIDS from developing countries. We aimed to determine the prevalence and antibiotic susceptibility patterns of bacterial pathogens causing urinary tract infections (UTIs) and sepsis in a cohort of 219 HIV-infected Jamaican children. METHODS: This cross-sectional study examined clinical and microbiological data for children enrolled in the Kingston Paediatric/Perinatal HIV/AIDS programme from September 1, 2002 to May 31, 2007. Cases were defined as physician-diagnosed, laboratory confirmed UTIs and sepsis based on Centers for Disease Control and Prevention (CDC) criteria. Only isolates from urine, blood and sterile sites were considered. RESULTS: Forty-four patients (20.1%) accounted for 74 episodes of UTIs and sepsis. Mean number of infections was 1.7 +/- 1.3 per patient. There were 31 males (70.5%) and mean age at time of infection was 5.6 +/- 4.7 years. Bacterial infections comprised cystitis (n = 52, 70.3%), bacterial pneumonia (n = 15, 20.3%), meningitis (n = 4, 5.4%), septicaemia (n = .2, 2.7%) and bone infection (n = 1, 1.4%). Among 52 UTIs, 39 were caused by a single organism. The most common UTI isolates included Escherichia coli (n = 21, 53.8%) and Enterobacter spp (n = 5, 12.8%). Among 22 cases of sepsis, isolates included Streptococcus pneumoniae (n = 8, 36.4%) and coagulase negative Staphylococcus (n = 6, 27.3%). All E coli isolates at two of three clinical sites were resistant to cotrimoxazole. There were 79.7% (n = 51) of infectious episodes with a cotrimoxazole-resistant organism occurring among those on cotrimoxazole prophylaxis. CONCLUSIONS: Escherichia coli was the most frequent bacterial isolate. Cotrimoxazole is a poor choice for empiric treatment of sepsis and UTIs in this clinical setting.

Comparative net cost impact of the utilization of panitumumab versus cetuximab for the treatment of patients with metastatic colorectal cancer in Canada.

OBJECTIVE: Clinical practice guidelines support the use of the epidermal growth factor receptor (EGFR) inhibitors panitumumab and cetuximab for the treatment of metastatic colorectal cancer (mCRC) after failure of other chemotherapy regimens, based on significant clinical benefits in patients with wild-type KRAS. The purpose of the analysis was to compare provincial hospital costs when using panitumumab vs cetuximab with or without irinotecan in this patient population using a Net Impact Analysis (NIA) approach. METHODS: The NIA determined the total per patient cost of the reimbursed regimens of panitumumab vs cetuximab in British Columbia, Alberta, Manitoba, Ontario, and Québec. Utilization of healthcare resources related to EGFR inhibitor infusions, follow-up monitoring, and treatment of adverse events (AEs) were also included. Healthcare resource use including drugs, medical supplies, laboratory testing, oncology infusion time, and healthcare professionals' time was obtained through expert consultation and the use was then multiplied by the province-specific cost of each resource. Numerous sensitivity analyses were conducted. RESULTS: Based on the dosing regimens in place in each province, the total annual per patient cost of panitumumab ranged from $22,203-$32,600, while the total annual per patient cost of cetuximab treatment varied from $30,321-$40,908. Treatment with panitumumab resulted in lower costs in all cost categories including drug acquisition, infusion preparation/administration, patient monitoring, and AE management. Per patient savings with panitumumab ranged from a low of $3815 in British Columbia to a high of $10,603 in Ontario. In sensitivity analyses, panitumumab remained cost saving in all scenarios where the savings ranged from $150-$16,006 per patient. CONCLUSIONS: Treating chemorefractory mCRC patients with panitumumab rather than cetuximab reduced healthcare resource costs. Provincial healthcare savings achieved with the use of panitumumab could potentially be re-allocated to other cancer treatments, although further study would be needed to validate this assumption.

Cost-effectiveness implications based on a comparison of nursing home and home health case mix.

Case-mix differences between 653 home health care patients and 650 nursing home patients, and between 455 Medicare home health patients and 447 Medicare nursing home patients were assessed using random samples selected from 20 home health agencies and 46 nursing homes in 12 states in 1982 and 1983. Home health patients were younger, had shorter lengths of stay, and were less functionally disabled than nursing home patients. Traditional long-term care problems requiring personal care were more common among nursing home patients, whereas problems requiring skilled nursing services were more prevalent among home health patients. Considering Medicare patients only, nursing home patients were much more likely to be dependent in activities of daily living (ADLs) than home health patients. Medicare nursing home and home health patients were relatively similar in terms of long-term care problems, and differences in medical problems were less pronounced than between all nursing home and all home health patients. From the standpoint of cost-effectiveness, it would appear that home health care might provide a substitute for acute care hospital use at the end of a hospital stay, and appears to be a more viable option in the care of patients who are not severely disabled and do not have profound functional problems. The Medicare skilled nursing facility, however, is likely to continue to have a crucial role in posthospital care as the treatment modality of choice for individuals who require both highly skilled care and functional assistance.

Comparison of NHS and private patients undergoing elective transurethral resection of the prostate for benign prostatic hypertrophy.

OBJECTIVES: To compare the operative thresholds and clinical management of men undergoing elective transurethral resection of the prostate for benign prostatic hypertrophy in the NHS and privately. DESIGN: Cohort study of patients recruited by 25 surgeons during 1988. SETTING: Hospitals in Oxford and North West Thames regions. PATIENTS: Of 400 consecutive patients, 129 were excluded because of open surgery (nine), lack of surgeons' information (three), and emergency admission (117) and three failed to give information, leaving 268 patients, 214 NHS patients and 54 private patients. MAIN MEASURES: Sociodemographic factors, prevalence and severity of symptoms, comorbidity, general health (Nottingham health profile) obtained from patient questionnaire preoperatively and reasons for operating, and operative management obtained from surgeons perioperatively. RESULTS: NHS and private patients were similar in severity of symptoms and prevalence of urinary tract abnormalities. They differed in four respects: NHS patients' general health was poorer as a consequence of more comorbid conditions (49, 23% v 7, 13% in severe category); the condition had a greater detrimental effect on their lives (36, 17% v 2, 4% severely affected; p < 0.01); private patients received more personalised care more quickly and were investigated more before surgery, (29, 54% v 60, 20% receiving ultrasonography of the urinary tract); and NHS patients stayed in hospital longer (57, 27% v 3, 6% more than seven days; p < 0.001). CONCLUSIONS: Private patients' need for surgery, judged by symptom severity, was as great as that of NHS patients, and there was no evidence of different operative thresholds in the two sectors, but, judged by impact on lifestyle, NHS patients' need was greater.

Rectal prolapse during pregnancy. A case report.

BACKGROUND: Rectal prolapse is a bothersome surgical problem that is relatively infrequent. It usually occurs in the fifth to seventh decades of life and is more common in women. CASE: A 33-year-old woman, gravida 3, para 2, was found to have a large rectal prolapse at 33 weeks' gestation. Manual reduction was successfully performed after injecting 2% lidocaine into and around the anal sphincter. Because she could not undergo definitive surgical repair during her pregnancy, the patient was managed with an aggressive stool softening regimen and self-reduction techniques. Labor was induced in the 40th gestational week. Epidural anesthesia was employed, and delivery was accomplished via low-outlet forceps application. The patient underwent definitive surgical repair of the rectal prolapse eight weeks postpartum. CONCLUSION: Rectal prolapse is a rare condition during the childbearing years. We found no prior case reports of rectal prolapse occurring during pregnancy. While childbirth itself is not considered a risk factor for rectal prolapse, a prior history of perineal lacerations may be a risk factor. To manage rectal prolapse that occurs during pregnancy, consideration should be given to passive forceps delivery under epidural anesthesia to avoid the possibility of worsening the prolapse.

Antibiotic resistance among pathogens causing disease in Jamaican children with HIV/AIDS / Resistencia antibiótica entre los patógenos que causan enfermedades en los niños jamaicanos con VIH/SIDA

OBJECTIVE: There are limited data regarding the antimicrobial resistance patterns of pathogens in children with HIV/AIDS from developing countries. We aimed to determine the prevalence and antibiotic susceptibility patterns of bacterial pathogens causing urinary tract infections (UTIs) and sepsis in a cohort of 219 HIV-infected Jamaican children. METHODS: This cross-sectional study examined clinical and microbiological data for children enrolled in the Kingston Paediatric/Perinatal HIV/AIDS programme from September 1, 2002 to May 31, 2007. Cases were defined as physician-diagnosed, laboratory confirmed UTIs and sepsis based on Centers for Disease Control and Prevention (CDC) criteria. Only isolates from urine, blood and sterile sites were considered. RESULTS: Forty-four patients (20.1%) accounted for 74 episodes of UTIs and sepsis. Mean number of infections was 1.7 ± 1.3 per patient. There were 31 males (70.5%) and mean age at time of infection was 5.6 ± 4.7 years. Bacterial infections comprised cystitis (n = 52, 70.3%), bacterial pneumonia (n = 15, 20.3%), meningitis (n = 4, 5.4%), septicaemia (n = 2, 2.7%) and bone infection (n = 1, 1.4%). Among 52 UTIs, 39 were caused by a single organism. The most common UTI isolates included Escherichia coli (n = 21, 53.8%) and Enterobacter spp (n = 5, 12.8%). Among 22 cases of sepsis, isolates included Streptococcus pneumoniae (n = 8, 36.4%) and coagulase negative Staphylococcus (n = 6, 27.3%). All E coli isolates at two of three clinical sites were resistant to cotrimoxazole. There were 79.7% (n = 51) of infectious episodes with a cotrimoxazole-resistant organism occurring among those on cotrimoxazole prophylaxis. CONCLUSIONS: Escherichia coli was the most frequent bacterial isolate. Cotrimoxazole is a poor choice for empiric treatment of sepsis and UTIs in this clinical setting.

OBJETIVO: Los datos existentes en relación con los patrones de resistencia antimicrobiana en los ninos con VIH/SIDA de los países en vías de desarrollo, son limitados. Nuestro objetivo fue determinar la prevalencia y los patrones de susceptibilidad antibiótica de los patógenos bacterianos que causan infecciones de las vías urinarias (IVU) y sepsis en una cohorte de 219 ninos jamaicanos infectados con VIH. MÉTODOS: Este estudio transversal examinó datos clínicos y microbiológicos de ninos enrolados en el programa KPAIDS del 1ero. de septiembre de 2002 al 31 de mayo de 2007. Los casos se definieron como IVU y sepsis de diagnóstico médico, confirmada en el laboratorio, a partir de criterios de los Centros de Control y Prevención de Enfermedades (CCE). Solamente se tuvieron en cuenta aislados de orina, sangre y sitios estériles. RESULTADOS: Cuarenta y cuatro pacientes (20.1%) dieron lugar a 74 episodios de IVU y sepsis. El número promedio de infecciones fue 1.7 ± 1.3 por paciente. Hubo 31 varones (70.5%) y la edad promedio en el momento de la infección fue 5.6 ± 4.7 anos. Las infecciones bacterianas abarcaron: cistitis (n = 52, 70.3%), pulmonía bacteriana (n = 15, 20.3%), meningitis (n = 4, 5.4%), septicemia (n = 2, 2.7%) e infección ósea (n = 1, 1.4%). De las 52 IVU, 39 fueron causadas por un solo microorganismo. Los aislados más comunes de IVU incluyeron Escherichia coli (n = 21, 53.8%) y Enterobacter spp (n = 5, 12.8%). De los 22 casos de sepsis, los aislados incluyeron Streptococcus pneumoniae (n = 8, 36.4%) y Staphylococcus coagulasa negativo (n = 6, 27.3%). Todos los aislados de E coli en dos o tres sitios clínicos eran resistentes al cotrimoxazol. Se produjeron 79.7% (n = 51) episodios infecciosos con un organismo resistente al cotrimoxazol entre los pacientes que se hallaban bajo profilaxis con cotrimoxazol. CONCLUSIONES: Escherichia coli fue el aislado bacteriano más frecuente. El cotrimoxazol es una opción pobre para el tratamiento empírico de sepsis e IVU en esta situación clínica.

Influence of hydrocortisone on gentamicin-induced nephrotoxicity in rats.

Many risk factors associated with aminoglycoside nephrotoxicity have been identified in humans and experimental animals. They include an initial high rate of creatinine clearance, high initial peak levels in serum, age, sex, duration of therapy, liver disease, and renal infection. The concomitant administration of steroids has never been investigated. We evaluated the role of hydrocortisone on gentamicin-induced nephroxicity in a model of infused rats. We showed that hydrocortisone given over 3 days after the infusion did not modify the gentamicin half-life in the renal cortex, gentamicin-induced lysosomal phospholipidosis, or histopathology but did reduce significantly the 3H/DNA ratio on day 4 after gentamicin infusion. We concluded that hydrocortisone interferes with the postnecrotic cellular regeneration process, an important step that is responsible for the recovery of normal kidney structure following toxic injuries associated with aminoglycoside therapy.

Hospital-based and freestanding home health case mix: implications for medicare reimbursement policy.

This project assessed case mix differences between 299 hospital-based and 354 freestanding clients randomly selected from 20 home health agencies in nine states in 1982. Similarities between hospital-based and freestanding clients outweighed their differences, suggesting that no reimbursement differential is warranted for the two types of providers at this time. Medicare's prospective payment system for hospitals may result in more pronounced case mix differences between the two modalities in the future. Basing payment on case mix is therefore appropriate in order to provide incentives for treating different types of Medicare home health beneficiaries.

Wolbachia neither induces nor suppresses transcripts encoding antimicrobial peptides.

Wolbachia are intracellular maternally inherited microorganisms that are associated with reproductive abnormalities such as cytoplasmic incompatibility (CI), feminization and parthenogenesis in the various arthropod species they infect. Surveys indicate that these bacteria infect more than 16% of all insect species as well as isopods, mites and nematodes, making Wolbachia one of the most ubiquitous parasites yet described. However, nothing is known about the interactions of this bacterium with the host's immune system. We studied the expression of inducible antimicrobial markers in the adults of two Wolbachia infected insect species, Drosophila simulans and Aedes albopictus. The lack of available immune markers in the mosquito species led us to clone part of the defensin gene from this species, which was found to be very similar to the other mosquito defensins cloned from Anopheles gambiae and Aedes aegypti. Comparisons of the expression pattern of the antibacterial markers between Wolbachia-infected and cured lines, and also between bacteria-challenged and unchallenged adults indicated that Wolbachia does not either constitutively induce or suppress the transcription of these antibacterial genes. In addition, no difference in the transcription of these genes was found between double and single Wolbachia-infected strains or between strains in which Wolbachia has different tissue tropisms.

Semliki Forest virus as an expression vector in insect cell lines.

Studies were undertaken to determine if replication-deficient Semliki Forest virus expression vectors could be successfully used to express foreign gene constructs in insect cell lines. Using green fluorescent protein (GFP) as a marker we recorded infection levels of nearly 100% in the Aedes albopictus cell lines C6/36 and Aa23T, as well as in the Ae. aegypti cell line MOS20. The virus was capable of infecting an Anopheles gambiae cell line MOS55. The amount of GFP protein produced in each cell line was quantified. Northern analysis of viral transcription revealed the presence of novel transcripts in Aa23T, C6/36, and MOS55 cell lines, but not in the BHK or MOS20. The initial characterization of these transcripts is described.

Practical use of the pessary.

The pessary is an effective tool in the management of a number of gynecologic problems. The pessary is most commonly used in the management of pelvic support defects such as cystocele and rectocele. Pessaries can also be used in the treatment of stress urinary incontinence. The wide variety of pessary styles may cause confusion for physicians during the initial selection of the pessary. However, an understanding of the different styles and their uses will enable physicians to make an appropriate choice. Complications can be minimized with simple vaginal hygiene and regular follow-up visits.

Effects of daptomycin and vancomycin on tobramycin nephrotoxicity in rats.

Daptomycin is a new biosynthetic antibiotic which belongs to a new class of drugs known as lipopeptides. The objective of this study was to evaluate the effects of daptomycin and vancomycin on tobramycin-induced nephrotoxicity. Female Sprague-Dawley rats were treated during 4 and 10 days with either saline (NaCl, 0.9%) or tobramycin at doses of 4 and 40 mg/kg per day (given every 12 h [q12h] intraperitoneally). Each treatment was combined with saline, daptomycin at a dose of 20 mg/kg per day (given q12h subcutaneously), and ancomycin at a dose of 50 mg/kg per day (given q12h subcutaneously). Daptomycin and vancomycin had no effect on the intracortical accumulation of tobramycin. Daptomycin did not accumulate in renal tissue even after 10 days of treatment. Tobramycin given at a dose of 40 mg/kg per day during 10 days induced a significant inhibition of sphingomyelinase activity in the renal cortex (P less than 0.01) and increased cellular regeneration (P less than 0.01), as measured by the incorporation of [3H]thymidine into DNA of the renal cortex. These changes were minimal when daptomycin was combined with tobramycin. Histologically, signs of tobramycin toxicity were also less severe in the presence of daptomycin. The intracortical accumulation of vancomycin was not modified by tobramycin. The sphingomyelinase activity was significantly more inhibited (P less than 0.01) when vancomycin was associated with tobramycin (4 and 40 mg/kg) without affecting the rate of [3H]thymidine incorporation into DNA. Histologically, signs of tobramycin toxicity were not affected by vancomuycin, but the cellular vacuolizations which were also observed in vancomycin-treated animals were still present in the proximal tubular cells of animals that were treated with the combination vancomycin-tobramycin. This study strongly suggests that daptomycin protects animals from tobramycin-induced nephrotoxicity but that vancomycin may enhance the effect of tobramycin. We conclude that daptomycin is safe and protects kidney cells from tobramycin-induced nephrotoxicity.

Use of pulsed-field gel electrophoresis, enterobacterial repetitive intergenic consensus typing, and automated ribotyping to assess genomic variability among strains of nontypeable Haemophilus influenzae.

We compared 75 nontypeable (NT) Haemophilus influenzae isolates by pulsed-field gel electrophoresis (PFGE), enterobacterial repetitive intergenic consensus (ERIC)-PCR, and automated ribotyping. PFGE was the most discriminatory of the techniques. ERIC-PCR provides a useful screen but should not replace other techniques as the sole method to group NT H. influenzae strains.

Identification of the lipooligosaccharide biosynthesis gene lic2B as a putative virulence factor in strains of nontypeable Haemophilus influenzae that cause otitis media.

Nontypeable (NT) strains of Haemophilus influenzae are an important cause of acute otitis media (OM). The pathogenic process by which NT H. influenzae strains cause OM is poorly understood. In order to identify specific virulence factors important for OM pathogenesis, genomic subtraction of the NT H. influenzae middle ear isolate G622 against H. influenzae strain Rd was conducted and the resulting subtraction products were used to screen a panel of H. influenzae isolates. Subtraction identified 36 PCR fragments unique to strain G622, which were used in a preliminary screen of 48 middle ear isolates and 46 nasopharyngeal and throat isolates to identify genes found more frequently among middle ear isolates. These experiments identified a PCR fragment with high homology to the lipooligosaccharide biosynthesis gene lic2B (originally identified in an H. influenzae type b strain) among 52% of the middle ear isolates and 9% of nasopharyngeal and throat isolates. The lic2B gene cloned from NT H. influenzae strain G622 was 99% identical at the amino acid level to that of the H. influenzae type b strain RM7004. The lic2B gene was used to screen a larger panel of H. influenzae isolates including the original 48 middle ear isolates, 40 invasive type b isolates, 90 NT H. influenzae throat isolates from children attending day care, and 32 NT H. influenzae nasopharyngeal clinical isolates. The lic2B gene was found 3.7 times more frequently among middle ear isolates than in throat isolates from children attending day care. These data suggest that a specific NT H. influenzae gene is associated with OM.

Mathematical analysis of perifusion data: models predicting elution concentration.

System models are constructed and analyzed for combined convective flow and for dispersion in distorting concentrations of a chemical signal as it passes through a packed column. We derive general analytical solutions for these models. The results have applications to analyses such as in biological experiments involving hormonal stimulation of perifused cells, elution chromatography, adsorption columns, and studies of groundwater flow. The models reveal that the column distorts an incoming signal (such as a change in solute concentration in the flowing liquid) at the inlet. This distortion is greatest at low values of the Peclet number of the flow and is small at larger values. We explore the effects of the approximations inherent in the mathematical models of the system. Specification of the boundary conditions of the problem are shown to be particularly important. With the use of incorrect models, it is possible to obtain accurate interpolations to data obtained from perfusion experiments. However, the parameters derived (in particular the dispersion constant and the peak concentration of a solute concentration pulse) may be considerably in error. This may lead to errors when these parameter estimates are used to predict results in other experimental situations.

In vitro cultivation of Wolbachia pipientis in an Aedes albopictus cell line.

A continuous cell line, Aa23, was established from eggs of a strain of the Asian tiger mosquito, Aedes albopictus, naturally infected with the intracellular symbiont Wolbachia pipientis. The resulting cell line was shown to be persistently infected with the bacterial endosymbiont. Treatment with antibiotics cured the cells of the infection. In the course of establishing this cell line it was noticed that RFLPs in the PCR products of two Wolbachia genes from the parental mosquitoes were fixed in the infected cell line. This indicates that the mosquito host was naturally superinfected with different Wolbachia strains, whereas the infected cell line derived from these mosquitoes only contained one of the original Wolbachia strains. The development of an in vitro culture system for this fastidious microorganism should facilitate molecular analysis of the reproduction distorting phenotypes it induces in natural arthropod hosts.