Demonstration of pneumococcal capsule under immunoelectron microscopy

It is challenging to demonstrate the capsule of Streptococcus pneumoniae (S. pneumoniae) under immunoelectron microscopy because of the thick mucopeptide cell wall hampering proper fixation. A novel rapid freeze fixation method was established to observe the capsule of S. pneumoniae. A strain of serotype 3 of S. pneumoniae isolate was analyzed after rapid freezing. An ethanol freezing-substitution fixing method was applied and immunohistochemical staining with osmium tetroxide was tested. The capsule was confirmed using the serotype 3 specific polyclonal antibodies labeled with colloidal gold particles. To the best of our knowledge, this is the first report of S. pneumoniae capsule by immunoelectron microscope

Community-acquired pneumonia in Ugandan adults: short-term parenteral ampicillin therapy for bacterial pneumonia

A hospital-based prospective study of 99 patients with community-acquired pneumonia (CAP) was carried out in Kampala, Uganda. We evaluated microbiological etiologies, clinical features and effectiveness of short-term parenteral ampicillin followed by oral amoxicillin for these patients in relation to HIV-status. We demonstrated a very high prevalence (75%) of HIV-1 infection. No significant difference was observed with respect to age, gender, prior antibiotic usage, symptoms, laboratory data or bacterial etiology between HIV-1-infected and HIV-uninfected CAP patients. Most strains of Streptococcus pneumoniae (n = 19) and Haemophilus influenzae (n = 8) isolated from HIV-1-infected patients were penicillin-resistant (95%) and beta-lactamase producing (75%) strains, respectively. A high percentage of good clinical response was found in both HIV-1-infected (81%) and HIV-uninfected (86%) among 39 patients with CAP due to a defined bacterial pathogen. These data support the use of short-term parenteral ampicillin for patients with bacterial CAP irrespective of HIV-status

A novel high-throughput method for molecular serotyping and serotype-specific quantification of Streptococcus pneumoniae using a nanofluidic real-time PCR system

Serotype-specific quantification data are essential for elucidating the complex epidemiology of Streptococcus pneumoniae and evaluating pneumococcal vaccine efficacy. Various PCR-based assays have been developed to circumvent the drawback of labour-intensive and time-consuming culture-based procedures for serotype determination and quantification of pneumococcus. Here, we applied a nanofluidic real-time PCR system to establish a novel assay. Twenty-nine primer pairs, 13 of which were newly designed, were selected for the assay to cover 50 serotypes including all currently available conjugate and polysaccharide vaccine serotypes. All primer pairs were evaluated for their sensitivity, specificity, efficiency, repeatability, accuracy and reproducibility on the Fluidigm Biomark HD System, a nanofluidic real-time PCR system, by drawing standard curves with a serial dilution of purified DNA. We applied the assay to 52 nasopharyngeal swab samples from patients with pneumonia confirmed by chest X-ray to validate its accuracy. Minimum detection levels of this novel assay using the nanofluidic real-time PCR system were comparable to the conventional PCR-based assays (between 30 and 300 copies per reaction). They were specific to their targets with good repeatability (SD of copy number of 0.1), accuracy (within ±0.1 fold difference in log10 copy number) and reproducibility (SD of copy number of 0.1). When artificially mixed DNA samples consisting of multiple serotypes in various ratios were tested, all the serotypes were detected proportionally, including a minor serotype of one in 1000 copies. In the nasopharyngeal samples, the PCR system detected all the culturepositive samples and 22 out of 23 serotypes identified by the conventional method were matched with PCR results. We conclude that this novel assay, which is able to differentially quantify 29 pneumococcus groups for 45 test samples in a single run, is applicable to the large-scale epidemiological study of pneumococcus. We believe that this assay will facilitate our understanding of the roles of serotype-specific bacterial loads and implications of multiple serotype detections in pneumococcal diseases

Risk for Death among Children with Pneumonia, Afghanistan

In Afghanistan, childhood deaths from pneumonia are high. Among 639 children at 1 hospital, the case-fatality rate was 12.1%, and 46.8% of pneumococcal serotypes detected were covered by the 13-valent vaccine. Most deaths occurred within 2 days of hospitalization; newborns and malnourished children were at risk. Vaccination could reduce pneumonia and deaths

Population Based Cohort Study for Pediatric Infectious Diseases Research in Vietnam

A population-based cohort study on pediatric infectious diseases was established at Khanh Hoa Province, central Vietnam in 2006, to determine the etiology and risk factors for severe pediatric infectious diseases (SPID) such as acute respiratory infection (ARI), diarrhea and dengue which are the major causes of under 5 mortality. A population census survey was conducted in Nha-Trang and Ninh-Hoa to collect demographic, social-behavioral data and disease burden on SPID. The study site covered a population of 353,525 residing in 75,826 households with 24,781 children less than 5 years. Hospital databases from two hospitals covering the region were obtained. Linking the census and hospital databases, we were able to investigate on a variety of SPID such as environmental tobacco smoking exposure and increased risked of pediatric pneumonia hospitalization, population density, water supply and risk of dengue fever and animal livestock and risk of hospitalized diarrhea. To determine incidence, viral etiology and risk factors for pediatric ARI/pneumonia, we setup a population based prospective hospitalized Pediatric ARI surveillance at Khanh Hoa General Hospital, Nha-Trang in February 2007. The study has revealed RSV, rhinovirus and influenza A as major viral pathogens, role of multiple viral infection and its interaction with bacteria in the development of pneumonia. In addition, we are also conducting a birth cohort study to investigate the incidence of congenital infection and its impact on physical-neurological development, and role of host genetic polymorphism on SPID hospitalization in Vietnam. Population mobility, high cost of regular census update and low mortality are the challenges

Arachis hypoaea (PNA) Lectin Binding Affinity to Candida Parapsilosis in Histologic Sections

A battery of 8 peroxidase-labeled lectins was tested on sections of paraffin-embedded human tissues to determine which lectin could be used in the microscopic diagnosis of Candida parapsilosis. The peroxidase-labeled lectin histochemisry was ore clearly in identifying Candida parapsilosis than fluorescein isothiocyanate (FITC)-labeled lectins. One lectin, from Archis hypoaea (PNA) was found to react with Candida parapsilosis. Seven lectins, from Triticum vulgare (WGA), Ulex europaeus (UEA-1), Ricinus communis (RCA-120), Dolichos biflorus (DAB), Phaseolus vulgaris (PHA-E), Lens culinaris (LCA), and Canavalin eniformis (Con A) did not react stained with Candida parapsilosls

Antimicrobial Susceptibility and Genetic Characteristics of Streptococcus pneumoniae Isolates Indicating Possible Nosocomial Transmission Routes in a Community Hospital in Japan▿

A clinical study was designed to study Streptococcus pneumoniae isolates recovered from a community hospital in Japan from April 2001 to November 2002. A total of 73 isolates were defined as derived from inpatient, outpatient, and hospital staff groups. The MIC results showed that 20 strains (27.4%) were susceptible to penicillin G, 39 strains (53.4%) had intermediate resistance, and 14 strains (19.2%) had full resistance. Low susceptibility to macrolides was also detected: 32.9%, 32.9%, and 34.2% of all strains were resistant to erythromycin, clarithromycin, and azithromycin, respectively. Thirty strains (41%) were resistant to at least two different kinds of antibiotics. Nineteen disparate serotypes were detected besides two nontypeable strains, and the predominant serotypes were 19F and 23F. Pulsed-field gel electrophoresis (PFGE) pattern A was dominant in the serotype 19F group; this pattern was similar to that of the international clone Taiwan 19F. A total of 10 different patterns were detected in the 23F group and were distinguishable from those of the international clones Spain 23F and Taiwan 23F. Pattern b strains were identified in the same ward, and pattern d strains were found both in patients with nosocomial pneumococcal infections (NPI) and in outpatients. In conclusion, drug-resistant S. pneumoniae was spreading rapidly, especially isolates of the serotype 19F and 23F groups. PFGE data revealed interpatient transmission and suggested that there might be some association between NPI patient strains and outpatient strains

Possible High Rate of Transmission of Nontypeable Haemophilus influenzae, Including β-Lactamase-Negative Ampicillin-Resistant Strains, between Children and Their Parents

The possible transmission of nontypeable Haemophilus influenzae between children and their parents was evaluated in 18 pairs of subjects from 15 families. Of the 33 isolates, 31 were found to be β-lactamase negative, including 10 β-lactamase-negative, ampicillin (AMP)-resistant (BLNAR) strains (AMP MIC, ≥1.0 μg/ml) and 2 were β-lactamase producing. Molecular typing by pulsed-field gel electrophoresis (PFGE) showed that 10 BLNAR isolates had 6 patterns, 23 non-BLNAR isolates had 13 patterns, and these patterns were different from each other, except for 1 pattern. As a result, the PFGE patterns in 14 of 18 pairs were indistinguishable and those in 4 pairs were different. These data indicate a possible high rate of intrafamilial transmission of nontypeable H. influenzae, including BLNAR strains, between children and their parents