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.

The relationship between biofilm formations and capsule in Haemophilus influenzae.

To evaluate the biofilm formation of non-typeable Haemophilus influenzae (NTHi) and H. influenzae type b (Hib) clinical isolates, we conducted the following study. Serotyping and polymerase chain reaction were performed to identify ß-lactamase-negative ampicillin (ABPC)-susceptible (BLNAS), ß-lactamase-negative ABPC-resistant (BLNAR), TEM-1 type ß-lactamase-producing ABPC-resistant (BLPAR)-NTHi, and Hib. Biofilm formation was investigated by microtiter biofilm assay, as well as visually observation with a scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM) in a continuous-flow chamber. As a result, totally 99 strains were investigated, and were classified into 4 groups which were 26 gBLNAS, 22 gBLNAR, 28 gBLPAR-NTHi and 23 Hib strains. The mean OD600 in the microtiter biofilm assay of gBLNAS, gBLNAR, gBLPAR-NTHi, and Hib strains were 0.57, 0.50, 0.34, and 0.08, respectively. NTHi strains were similar in terms of biofilm formations, which were observed by SEM and CLSM. Five Hib strains with the alternated type b cap loci showed significantly increased biofilm production than the other Hib strains. In conclusion, gBLNAS, gBLNAR, and gBLPAR-NTHi strains were more capable to produce biofilms compared to Hib strains. Our data suggested that resistant status may not be a key factor but capsule seemed to play an important role in H. influenzae biofilm formation.

Incidence of radiologically-confirmed pneumonia and Haemophilus influenzae type b carriage before Haemophilus influenzae type b conjugate vaccine introduction in Central Vietnam.

OBJECTIVES: To determine the incidence of radiologically-confirmed pneumonia (RCP) and Haemophilus influenzae type b (Hib) carriage in central Vietnam as a baseline data before Hib conjugate vaccine introduction. STUDY DESIGN: In the context of ongoing population-based prospective, hospitalized acute respiratory infection surveillance study, a cross-sectional Hib carriage study was conducted among 1000 children < 5 years of age living in NhaTrang, Vietnam in June 2010, 1 month before the nationwide introduction of Hib conjugate vaccine in Vietnam. RESULTS: The incidence of RCP hospitalizations among children < 5 years of age was 3.3 per 1000 children. The highest incidence was observed among children 12-23 month age group (8.3 per 1000). Haemophilus influenzae carriage was detected in 37% of the children and Hib carriage rate was 3%. Eighty-two percent of the Haemophilus influenzae had TEM ß-lactamase resistance gene. The presence of 6 or more family members was associated with an increased rate of Hib carriage (P = .04). CONCLUSIONS: Incidence of RCP and Hib carriage in this cross-sectional survey are lower compared with other studies. Continued surveillance for invasive Hib disease and sequential Hib carriage surveys are needed to support future assessments of the impact of Hib conjugate vaccine in Vietnam.

A prospective study of intrafamilial transmission and antimicrobial susceptibility of Moraxella catarrhalis.

Moraxella catarrhalis has been recognized as a particularly threatening respiratory tract pathogen in humans. A prospective study was performed to investigate which strains of M. catarrhalis can be transmitted within families; the study also addressed features of antimicrobial susceptibility. Seventy-five strains were isolated from six participants between July 2002 and February 2004, including 73 that were verified as beta-lactamase-producing strains. Antimicrobial susceptibility was tested for six types of antibiotics and no treatment issues were found. Pulsed-field gel electrophoresis (PFGE) was performed on all strains and 25 independent PFGE patterns were detected. The dominant pattern L (defined in the present study) was found in 21 (28%) of strains that were continuously recovered from children from the same family over an 8-month period. Strains with the patterns G, J, L, M, R, S, U, and W seemed to spread among the children, but there was no evidence of child-parent transmission. In the present study, the characteristics of M. catarrhalis within families have been documented, and PFGE profiles found to reveal alternating colonization and intrafamilial transmission.

Bactericidal activity in filtrated supernatant of Streptococcus sanguinis against multidrug-resistant Pseudomonas aeruginosa.

In the past decade, multidrug-resistant Pseudomonas aeruginosa (MDRP) infection has become a serious clinical problem, due to the limitation of drug choices to fight against the bacteria. Here we explored the bactericidal activity in the filtrated supernatant of Streptococcus (S.) sanguinis against Pseudomonas (P.) aeruginosa. S. sanguinis is one of the alpha-hemolytic streptococci that commonly reside in the human oral cavity. A strain of S. sanguinis, isolated from the sputum of a pulmonary-disease patient, was cultured for overnight. The filtered supernatant was tested for bactericidal effect using the minimum bactericidal concentration method on 20 strains of P. aeruginosa, including two MDRP and five mucoid-type strains. The viable number of P. aeruginosa was decreased with time after exposing to the filtrated supernatant of S. sanguinis, and collapsed bacteria were detected with electron microscopy. Of the 20 strains, 19 (95%) strains of P. aeruginosa were affected by bactericidal effect. Among other species of bacteria examined, the filtrated supernatant of S. sanguinis showed remarkable bactericidal effect on 49% of indole-positive Proteus species (4/9 strains) and 60% of Acinetobacter (A.) baumannii (6/10 strains). We next investigated the property of bactericidal activity in filtrated supernatant by treating with proteinase K or autoclave. There was no change in the bactericidal activity of the filtrated supernatant after each treatment, excluding the involvement of protein and plasmid. Here, we identify the bactericidal activity in the filtrated supernatant of S. sanguinis against MDRP. This unexpected observation may contribute to the development of a novel therapeutic drug against P. aeruginosa.

Drug-resistant pneumococci in children with acute lower respiratory infections in Vietnam.

BACKGROUND: Acute lower respiratory infections (ALRI), primarily pneumonia, are the leading cause of death in children under 5 years of age. Most of these deaths occur in Africa and southeast Asia. Increasing rates of drug resistance in pneumococcal strains emphasize the necessity of prevention of pneumococcal vaccines. The aim of the present study was to determine the frequency of drug resistance and the distribution of serotype of pneumococcal strains isolated from pediatric patients with ALRI in Vietnam. METHODS: Two hundred and twenty pediatric patients with ALRI under 5 years of age were enrolled in Hanoi, Vietnam between 2001 and 2002. Bacterial pathogens with a heavy growth (10(6) c.f.u./mL) were isolated from nasopharyngeal secretions on quantitative culture. Fifty-three pneumococcal strains isolated from the nasopharynx of pediatric patients were examined for antibiotic susceptibility including drug-resistant genes and serotyping. RESULTS: A total of 73.6% of pneumococcal strains were genotypic penicillin-resistant Streptococcus pnemoniae (gPRSP), possessing altered penicillin-binding protein genes pbp 1a + 2x + 2b; 67.9% of these strains were gPRSP and simultaneously had the ermB gene, which is responsible for high resistance to erythromycin. The majority of gPRSP strains were serotype 19F or 23F. CONCLUSION: gPRSP strains with serotype 19F or 23F are highly prevalent among pediatric patients with ALRI under 5 years of age in Hanoi, Vietnam.

Metallo-beta-lactamase IMP-1 in Providencia rettgeri from two different hospitals in Japan.

In 2002, 495 indole-positive proteae strains were isolated from patients at 60 hospitals in Japan. Nine indole-positive proteae strains had reduced susceptibility to imipenem (MIC > or = 8 microg ml(-1)) and were identified as Providencia rettgeri by BD Phoenix. Eight of the nine Prov. rettgeri isolates were confirmed as metallo-beta-lactamase producers by the double-disc synergy test. All the metallo-beta-lactamases were classified as IMP-1 by PCR and DNA sequence analysis. These bla(IMP-1) genes were encoded in the integron structure on conjugative plasmids. These plasmids could transfer from Prov. rettgeri clinical isolates to Escherichia coli ML4903 at a frequency between 1.5 x 10(-5) and 5.5 x 10(-7). The eight bla(IMP)-positive strains were isolated from two hospitals, and showed two different PFGE patterns, two different integron structures and two different incompatibility groups, which corresponded to the two hospitals. These results strongly suggest the possibility of nosocomial infections by bla(IMP-1)-producing Prov. rettgeri isolates.

Molecular characterization of the transmission between the colonization of methicillin-resistant Staphylococcus aureus to human and environmental contamination in geriatric long-term care wards.

OBJECTIVE: Transmission between human and environmental contamination from colonized methicillin-resistant Staphylococcus aureus (MRSA) remains a controversial issue. We, therefore, investigated the differences between MRSA types which colonize in humans and in the environment. METHODS: A 4-week prospective culture survey for MRSA was performed for 12 patients as well as for the environment of the room of MRSA carriers in quarantine in the geriatric long-term care ward of a 270-bed hospital. RESULTS: A total of 97 S. aureus strains (80 MRSA and 17 methicillin-sensitive Staphylococcus aureus [MSSA]) was isolated during the periods of September 8 to 10, 23 to 25 and October 5 to 7, 1998; 25 strains were from the respiratory tract, 4 strains from feces and 11 strains from decubitus ulcers. Fifty-seven strains were from the patients' environment. Molecular typing by pulsed-field gel electrophoresis (PFGE) with the Sma I restriction enzyme demonstrated that the predominant type of MRSA isolated from the environment changed by the minute. The patterns of 42 MRSA strains isolated from the environment were identical in 26 (61.9%), closely related in 15 (35.7%) and possibly related in 1 (2.4%) of the cases of those isolated from patients simultaneously. There was no correlation between patients and the environment with the 17 MSSA isolates. CONCLUSION: Our results demonstrated that MRSA from patients can contaminate the environment, whereas MRSA from the environment might be potentially transmitted to patients via health care workers under unsatisfactory infection control.

Decreased serum opsonic activity against Streptococcus pneumoniae in human immunodeficiency virus-infected Ugandan adults.

Type-specific immunoglobulin G (IgG) to pneumococcal capsular polysaccharide (CPS) and opsonic activity against Streptococcus pneumoniae were evaluated in serum samples from 36 Ugandan adults with community-acquired pneumonia and 58 asymptomatic Ugandan adults with or without human immunodeficiency virus type 1 (HIV-1) infection. The levels of serum IgG to CPS were significantly higher in HIV-1-infected subjects than in HIV-uninfected subjects. Serum samples from HIV-1-infected subjects that had lower IgG titers demonstrated higher opsonic activity against type 3 (titers of 7) and type 9 (titers of 7-11) pneumococcal strains. Plasma HIV-1 load also correlated inversely with serum opsonic activity against these strains, and peripheral blood CD4+ lymphocyte numbers also tended to correlate with serum opsonic activity in asymptomatic HIV-1-infected adults. Our findings suggest that the opsonic activity of type-specific IgG is impaired in the serum of HIV-1-infected African adults, which may expose them to a serious risk of invasive pneumococcal infections.

[A case report of pulmonary nocardiosis associated with bronchiectasis after pulmonary tuberculosis successfully treated with sparfloxacin].

A 48-year-old female with bronchiectasis after pulmonary tuberculosis was admitted to our hospital because of bloody sputum and multiple cavity formation in the right lung. Chest X-ray & CT films revealed diffuse nodular shadows and cavity formation with a thick wall. Nocardia asteroides was isolated from the stum but no other pathogenic bacteria was isolated. Administration of sulfame-thoxazole-trimethoprim is not effective. The results of an MIC test for antimicrobial agents led to treatment with sparfloxacin and the clinical symptoms and cavity formations in the right lung improved. Nocardia asteroides may cause exacerbation of bronchiectasis.

[Three adult cases with Corynebacterium propinquum respiratory infections in a community hospital].

Corynebacterium propinquum, which is included in Corynebacterium group ANF-3, exists as a commensal in the oral flora. This organism has not yet been fully recognized as a respiratory pathogen. We previously reported that the first case with respiratory infection caused by C. propinquum. On the other hand, Corynebacterium pseudodiphtheriticum is recognized as a causative organism in respiratory infections. Recently we experienced two cases with C. propinquum respiratory infections in our hospital. Three types of the onset such as a community-acquired infection, a hospital-acquired infection, and a nursing home acquired infections were observed. Our analysis indicated that gram staining of the purulent sputum is an essential tool to evaluate whether C. propinquum is a respiratory pathogen or not, because this organism is a commensal bacteria.

[A case of community-acquired pneumonia caused by Corynebacterium propinquum].

Corynebacterium propinquum, which is classified as Corynebacterium ANF-3, has not yet been described as a cause of the respiratory infections. In this paper, we reported a case of 67-year-old immunocompetent male with community-acquired pneumonia caused by C. propinquum. Our study suggested that Gram staining of the pulurent sputum was the most important diagnostic tool to determine the pathogenecity of this organism.

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 culture-positive 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.

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.

Development of a single-tube loop-mediated isothermal amplification assay for detection of four pathogens of bacterial meningitis.

Several loop-mediated isothermal amplification (LAMP) assays have been developed to detect common causative pathogens of bacterial meningitis (BM). However, no LAMP assay is reported to detect Streptococcus agalactiae and Streptococcus suis, which are also among common pathogens of BM. Moreover, it is laborious and expensive by performing multiple reactions for each sample to detect bacterial pathogen. Thus, we aimed to design and develop a single-tube LAMP assay capable of detecting multiple bacterial species, based on the nucleotide sequences of the 16S rRNA genes of the bacteria. The nucleotide sequences of the 16S rRNA genes of main pathogens involved in BM were aligned to identify conserved regions, which were further used to design broad range specific LAMP assay primers. We successfully designed a set of broad range specific LAMP assay primers for simultaneous detection of four species including Staphylococcus aureus, Streptococcus pneumoniae, S. suis and S. agalactiae. The broad range LAMP assay was highly specific without cross-reactivity with other bacteria including Haemophilus influenzae, Neisseria meningitidis and Escherichia coli. The sensitivity of our LAMP assay was 100-1000 times higher compared with the conventional PCR assay. The bacterial species could be identified after digestion of the LAMP products with restriction endonuclease DdeI and HaeIII.

Association between nasopharyngeal load of Streptococcus pneumoniae, viral coinfection, and radiologically confirmed pneumonia in Vietnamese children.

BACKGROUND: The interplay between nasopharyngeal bacterial carriage, viral coinfection, and lower respiratory tract infections (LRTIs) is poorly understood. We explored this association in Vietnamese children aged less than 5 years. METHODS: A hospital-based case-control study of pediatric LRTIs was conducted in Nha Trang, Vietnam. A total of 550 hospitalized children (274 radiologically confirmed pneumonia [RCP] and 276 other LRTIs) were enrolled and 350 healthy controls were randomly selected from the community. Polymerase chain reaction-based methods were used to measure bacterial loads of Streptococcus pneumoniae (SP), Haemophilus influenzae, and Moraxella catarrhalis and to detect 13 respiratory viruses and bacterial serotypes in nasopharyngeal samples of study participants. RESULTS: The median nasopharyngeal bacterial load of SP was substantially higher in children with RCP compared with healthy controls or children with other LRTIs (P < 0.001). SP load was 15-fold higher in pneumonia children with viral coinfection compared with those children without viral coinfection (1.4 x 107/mL vs. 9.1 x 105/mL; P 0.0001). SP load was over 200-fold higher in serotypeable SP compared with nontypeable SP (2.5 x 106/mL vs. 1 x 104/mL; P < 0.0001). These associations were independent of potential confounders in multiple regression models. No clear association was found between nasopharyngeal load of Haemophilus influenzae or Moraxella catarrhalis and viral coinfection in either RCP or other LRTIs groups. CONCLUSIONS: An increased load of SP in the nasopharynx was associated with RCP, viral coinfection, and presence of pneumococcal capsule.

Molecular epidemiological study of Moraxella catarrhalis isolated from nosocomial respiratory infection patients in a community hospital in Japan.

BACKGROUND: Moraxella catarrhalis, occasionally, plays the essential role in nosocomial respiratory infection (NRI). Few studies have reported the route by which this organism spreads in a nosocomial infection outbreak. We identified characteristics of the strains isolated from NRI and attempted to reveal the potential nosocomial transmission routes. METHODS: A follow-up study has been performed in a Japanese community hospital between July 2002 and January 2003. M. catarrhalis clinical isolates were identified and beta-lactamase production test as well as the minimal inhibitory concentrations (MICs) have been examined. Pulsed-field gel electrophoresis (PFGE) and the multi locus sequence typing method (MLST) have been introduced as the effective "fingerprinting" methods. RESULTS: A total of 29 strains were isolated from 17 participants; 7 independent DNA fragment patterns were detected by PFGE. Pattern B (defined in this study) was dominant, and was detected both in strains from a health care worker (HCW) and inpatients. In the 9 selected strains analyzed by MLST, 7 unique MLST types were identified, which showed the congruence with the results of PFGE results. CONCLUSION: Epidemiological analysis proved the transmission route from patient to patient, and suggested that more studies should be focused on identifying the possible transmission route between HCWs and inpatients.

Nosocomial bacteremia caused by biofilm-forming Bacillus cereus and Bacillus thuringiensis.

OBJECTIVE: Bacterial biofilms cause serious problems, such as antibiotic resistance and medical device-related infections. Recent reports indicate that Bacillus species potentially form biofilms and cause nosocomial bacteremia via catheter infection. Our objective was to investigate the relationship between nosocomial bacteremia caused by Bacillus species and biofilm formations. METHODS: Between 2001 and 2006, Bacillus cereus and Bacillus thuringiensis were isolated from blood samples of 21 patients with nosocomial bacteremia in two hospitals. The patients had underlying diseases such as cerebrovascular damage, malignant disease, or chronic obstructive lung disease and had high fever at the onset of bacteremia. After investigation, B. cereus and B. thuringiensis were isolated from patient's catheter tip, gauze, and hospital environment. Pulsed-field gel electrophoresis (PFGE) on 32 B. cereus and 7 B. thuringiensis isolates, microtiter biofilm assay and scanning electron microscopy (SEM) on 22 B. cereus isolates from patient's blood were performed. RESULTS: Molecular analysis by PFGE showed that 32 B. cereus strains had 21 patterns and 7 B. thuringiensis strains had 3 patterns. The PFGE patterns of B. thuringiensis and B. cereus in blood samples from 2 patients blood were similar to those from the same patient's catheter tip. The PFGE pattern of B. cereus from a hospital environment was similar to that from 2 patients' blood samples, and the PFGE pattern of B. thuringiensis from 2 hospital environments was similar to that from 2 patients' blood. The biofilm formations by 22 B. cereus isolates from patients' blood were confirmed by microtiter biofilm assay and SEM even at 24 hours. CONCLUSION: Our data indicate that various types of Bacillus species exist in hospital environments and the biofilm-forming strains potentially cause nosocomial bacteremia by catheter infection.

Antimicrobial effect of fluoroquinolones for the eradication of nontypeable Haemophilus influenzae isolates within biofilms.

Biofilms can be defined as communities of microorganisms attached to a surface. Those bacterial biofilms cause serious problems, such as antibiotic resistance and medical device-related infections. Nontypeable Haemophilus influenzae (NTHi) is an important pathogen in respiratory infections, as it forms biofilms both in vitro and in vivo such as human middle ear. Recent reports indicate that otitis media, paranasal sinusitis and lower respiratory tract infections caused by Haemophilus influenzae have become more difficult to treat with oral antibiotic therapy. However, there has been no attention given to antibiotic eradication of NTHi biofilm. To investigate the antimicrobial effect of various antibiotics against NTHi biofilm formation, we conducted the following comparative study using both beta-lactamase-negative ampicillin (AMP)-susceptible (BLNAS) and AMP-resistant (BLNAR) NTHi strains. In a microtiter biofilm assay, both levofloxacin and gatifloxacin, of the fluoroquinolone antibiotic group, significantly inhibited biofilm formation by BLNAS and BLNAR NTHi in a dose-dependent fashion compared to ampicillin of the penicillin antibiotic group, cefotaxime of the cephalosporin antibiotic group, and both erythromycin and clarithromycin of the macrolide antibiotic group. Furthermore, in flow cell chamber studies, confocal laser scanning microscopy counted survival bacteria in mature biofilm had been treated with gatifloxacin, ampicillin, cefotaxime and erythromycin. Only gatifloxacin completely killed the BLNAR NTHi isolates within biofilms without regard to the thickness of biofilm formation. The results of this study suggest that fluoroquinolones potentially have a role in therapy against diseases caused by both BLNAS and BLNAR NTHi isolates within biofilms.