Acinetobacter bacteraemia in Thailand: evidence for infections outside the hospital setting.

SUMMARY: Acinetobacter is a well-recognized nosocomial pathogen. Previous reports of community-associated Acinetobacter infections have lacked clear case definitions and assessment of healthcare-associated (HCA) risk factors. We identified Acinetobacter bacteraemia cases from blood cultures obtained <3 days after hospitalization in rural Thailand and performed medical record reviews to assess HCA risk factors in the previous year and compare clinical and microbiological characteristics between cases with and without HCA risk factors. Of 72 Acinetobacter cases, 32 (44%) had no HCA risk factors. Compared to HCA infections, non-HCA infections were more often caused by Acinetobacter species other than calcoaceticus-baumannii complex species and by antibiotic-susceptible organisms. Despite similar symptoms, the case-fatality proportion was lower in non-HCA than HCA cases (9% vs. 45%, P < 0·01). Clinicians should be aware of Acinetobacter as a potential cause of community-associated infections in Thailand; prospective studies are needed to improve understanding of associated risk factors and disease burden.

Pneumococcal antigen testing of blood culture broth to enhance the detection of Streptococcus pneumoniae bacteremia.

The purpose of this investigation was to enhance the detection of pneumococcal bacteremia cases using the Binax NOW® immunochromatographic test (ICT) on blood culture broth as part of surveillance in two rural Thailand provinces. Blood cultures were collected as clinically indicated from hospitalized patients. ICT was performed on broth from culture bottles flagged as positive by BactT/ALERT® (alarm-positive) but which failed to grow organisms on subculture. During the period May 2005-June 2007, ICT was positive on 43 (24%) of 182 alarm-positive blood cultures with no growth on subculture. Compared to pneumococcal bacteremia cases confirmed by culture, cases detected only by ICT had a longer median time from culture collection to incubation and a longer median time from alarm positivity to subculture, and were more likely to be from patients pretreated with antibiotics. In a subsequent surveillance period (July 2007-December 2009), ICT continued to detect additional pneumococcal cases, but in a lower proportion of samples (7 of 221, 3.2%). Recently, as part of a separate study, ICT applied to uninoculated blood culture broth produced weak-positive results, mandating caution if testing broth from patient blood cultures. The antigen testing of blood culture broth appears to enhance the detection of pneumococcal bacteremia, but a controlled evaluation is needed.

Incidence of respiratory pathogens in persons hospitalized with pneumonia in two provinces in Thailand.

Although pneumonia is a leading cause of death from infectious disease worldwide, comprehensive information about its causes and incidence in low- and middle-income countries is lacking. Active surveillance of hospitalized patients with pneumonia is ongoing in Thailand. Consenting patients are tested for seven bacterial and 14 viral respiratory pathogens by PCR and viral culture on nasopharyngeal swab specimens, serology on acute/convalescent sera, sputum smears and antigen detection tests on urine. Between September 2003 and December 2005, there were 1730 episodes of radiographically confirmed pneumonia (34·6% in children aged <5 years); 66 patients (3·8%) died. A recognized pathogen was identified in 42·5% of episodes. Respiratory syncytial virus (RSV) infection was associated with 16·7% of all pneumonias, 41·2% in children. The viral pathogen with the highest incidence in children aged <5 years was RSV (417·1/100,000 per year) and in persons aged ≥50 years, influenza virus A (38·8/100,000 per year). These data can help guide health policy towards effective prevention strategies.

Nuclear protein phosphatase 2A dephosphorylates protein kinase A-phosphorylated CREB and regulates CREB transcriptional stimulation.

Cyclic AMP (cAMP)-dependent protein kinase A (PKA) stimulates the transcription of many eucaryotic genes by catalyzing the phosphorylation of the cAMP-regulatory element binding protein (CREB). Conversely, the attenuation or inhibition of cAMP-stimulated gene transcription would require the dephosphorylation of CREB by a nuclear protein phosphatase. In HepG2 cells treated with the protein serine/threonine (Ser/Thr) phosphatase inhibitor okadaic acid, dibutyryl-cAMP-stimulated transcription from the phosphoenolpyruvate carboxykinase (PEPCK) promoter was enhanced over the level of PEPCK gene transcription observed in cells treated with dibutyryl-cAMP alone. This process was mediated, at least in part, by a region of the PEPCK promoter that binds CREB. Likewise, okadaic acid prevents the dephosphorylation of PKA-phosphorylated CREB in rat liver nuclear extracts and enhances the ability of PKA to stimulate transcription from the PEPCK promoter in cell-free reactions. The ability of okadaic acid to enhance PKA-stimulated transcription in vitro was entirely dependent on the presence of CREB in the reactions. The phospho-CREB (P-CREB) phosphatase activity present in nuclear extracts coelutes with protein Ser/Thr phosphatase type 2A (PP2A) on Mono Q, amino-hexyl Sepharose, and heparin agarose columns and was chromatographically resolved from nuclear protein Ser/Thr-phosphatase type 1 (PP1). Furthermore, P-CREB phosphatase activity in nuclear extracts was unaffected by the heat-stable protein inhibitor-2, which is a potent and selective inhibitor of PP1. Nuclear PP2A dephosphorylated P-CREB 30-fold more efficiently than did nuclear PP1. Finally, when PKA-phosphorylated CREB was treated with immunopurified PP2A and PP1, the PP2A-treated CREB did not stimulate transcription from the PEPCK promoter in vitro, whereas the PP1-treated CREB retained the ability to stimulate transcription. Nuclear PP2A appears to be the primary phosphatase that dephosphorylates PKA-phosphorylated CREB.

Identification of a conditionally essential heat shock protein in Escherichia coli.

Protein D48.5 was recognized as a heat-inducible protein of Escherichia coli during the screening of a group of random, temperature-inducible Mud-Lac fusion mutants. Physiological and genetic analysis demonstrated that (i) the structural gene for this protein, designated htpI, is a member of the sigma 32-dependent heat shock regulon, (ii) at 37 degrees C the synthesis of protein D48.5 is nearly constitutive, increasing slightly with growth rate in media of different composition, and (iii) this protein is essential for growth at high temperature.

Isolation and antibiotic susceptibility of Salmonella, Shigella, and Campylobacter from acute enteric infections in Egypt.

While Campylobacter, Salmonella, and Shigella remain major contributors to acute enteric infections, few studies on these pathogens have been conducted in Egypt. From January 1986 to December 1993, 869 Salmonella, Shigella and Campylobacter strains were isolated from stool specimens from 6,278 patients, presenting to the Abbassia Fever Hospital, Cairo, Egypt, with acute enteric infections. Salmonella predominated, totalling 465 isolates, followed by Shigella with 258 isolates, and Campylobacter with 146 isolates. Of the Shigella isolates, 124 were Shigella flexneri, 49 were S. sonnei, 47 were S. dysenteriae (mainly serotype 1, 2, and 3), and 38 were S. boydii. Campylobacter spp. comprised 92 Campylobacter jejuni and 54 C. coli isolates. Isolation of Salmonella was highest during the months of February-March, June-July, and October-November, while that of Shigella was maximal from July to October. Isolation of Campylobacter increased during May-June and again during August-October. Although Salmonella was sensitive to amikacin, aztreonam, ceftriaxone, and nalidixic acid, it was, however, resistant to erythromycin, streptomycin, ampicillin, chloramphenicol, and tetracycline. Shigella (> 80%) was sensitive to amikacin, ceftriaxone, cephalothin, sulphamethoxazole-trimethoprim (except S. sonnei), aztreonam, and nalidixic acid. Resistance (> 50%) was noted only for ampicillin, chloramphenicol, and tetracycline. C. jejuni and C. coli were resistant to cephalothin, aztreonam, and streptomycin. Some of the above antibiotics were employed to characterize the Egyptian isolates, but did not have any clinical utility in the treatment of diarrhoea. Significant differences (p < 0.05) were observed in the resistance profiles of Shigella and Salmonella between late 1980s and early 1990s. The results suggest the use of fluoroquinolones or a third-generation cephalosporin as an empirical treatment of enteric diseases. However, alternative control strategies, including the aggressive development of broadly protective vaccines, may be more effective approaches to curbing morbidity and mortality due to acute enteric infections.

Expression of heat shock protein D 48.5 of Escherichia coli is subject to modulation by catabolite repression.

The Escherichia coli heat shock regulon consists of approximately twenty polypeptides that are coordinately and transiently induced upon a temperature upshift under the control of an alternative sigma factor, designated sigma-32 or HtpR. Preliminary observations on one of the proteins of the heat shock response, protein D 48.5, suggested that its induction by sigma-32 during heat shock may be modulated by catabolite repression. In this study, a disk diffusion assay was used to screen the effect of several compounds on the expression of a lacZ fusion in the gene encoding protein D 48.5. This assay indicated that the expression of this protein was controlled, at least in part, by the catabolite repression response. A more indepth analysis of the expression of protein D 48.5 under both steady-state and heat shock conditions was conducted in both a wild type and cya crp background. This analysis revealed that in the cya crp background, the steady-state level of protein D 48.5 was elevated relative to the wildtype, but that the heat shock induction of the protein was reduced in magnitude relative to the wild type strain, suggesting a direct link between these two global responses. The lacZ fusion in the structural gene for protein D 48.5 should prove useful as a reporter mechanism to probe the physiology and regulation of the heat shock response.

Colonization factors of enterotoxigenic E. coli (ETEC) from residents of northern Egypt.

Infection caused by enterotoxigenic Escherichia coli (ETEC) poses a serious health problem to children in developing countries. Colonization of the small intestinal mucosa by ETEC strains is mediated by antigenically specific fimbriae, also known as colonization factor antigens (CFA). The importance of this study arises from reports that active and passive immunization with ETEC strains harboring CFAs induced protective immunity against diarrhea in animal models with preformed antibodies. In humans, ETEC containing CFA/I, II, III and IV have been identified. The aim of this study was to define CFAs of ETEC isolated in Alexandria, Egypt. One hundred and seven ETEC isolates from 132 human residents in Alexandria, Egypt were isolated during a birth cohort study. ETEC isolates were screened for heat labile (LT) and heat stable (ST) toxins using a 32P oligonucleotide hybridization probe and a GM1 ELISA. These isolates were examined using monoclonal antibodies against CFA/I, II, III, IV, and against the putative colonization antigens PCF0159 and PCF0166, CS 7 and CS 17. CFAs were found in 48% of ETEC strains. CFA/I was found in 18% of the strains, CFA/II in 10% and CFA/IV in 14%. CFA III was not found. All fifteen strains expressing CFA/IV expressed CS6 and produced ST. CFA/IV was not found in non-ST producing strains, while CFA/I was absent in ST-only producing strains.

Enteropathogens associated with diarrhea among military personnel during Operation Bright Star 96, in Alexandria, Egypt.

This study investigated the microbial causes of diarrheal disease among U.S. troops deployed near Alexandria, Egypt, during October 1995. Bacterial causes associated with 19 cases of diarrhea included: enterotoxigenic Escherichia coli (ETEC), 42% (21% heat-stable, 11% heat-labile, and 11% heat-stable/ heat-labile producers); enteropathogenic E. coli (5.3%); and enteroadherent E. coli (42%). Four cases of diarrhea were associated with enteroaggregative E. coli based on probe analysis for enteroaggregative heat-stable enterotoxin 1. Protozoan causes included; Entamoeba histolytica (11%), E. hartmanni (5%), E. nana (5%), Blastocystis hominis (5%), Chilomastix mesnili (11%), Dientamoeba fragilis (5%), Entamoeba coli (5%), and Cryptosporidium (5%). Shigella, Aeromonas, Plesiomonas, Vibrio, Campylobacter, and Salmonella were not detected. Of the eight ETEC cases, one was colonization factor antigen (CFA)/I only, one was both CFA/I and CFA/III, three were CFA/II, two were CFA/IV, and two were CFA-negative. Antibiograms of the ETEC and enteroadherent E. coli strains showed that all isolates were susceptible to norfloxacin, ciprofloxacin, and nalidixic acid but resistant to ampicillin, tetracycline, chloramphenicol, and sulfamethoxazole.

NH2-terminal modification of the phosphatase 2A catalytic subunit allows functional expression in mammalian cells.

Functional expression of recombinant wild-type phosphatase 2A catalytic subunit has been unsuccessful in the past. A nine-amino-acid peptide sequence (YP-YDVPDYA) derived from the influenza hemagglutinin protein was used to modify the NH2 and/or COOH terminus of the phosphatase 2A catalytic subunit. Addition of the nine-amino-acid sequence at the NH2 terminus allowed recombinant phosphatase 2A expression as a predominantly cytosolic phosphatase 2A enzyme. The 12CA5 monoclonal antibody that recognizes the nine-amino-acid hemagglutinin peptide sequence was used to immunoprecipitate the epitope-tagged phosphatase 2A catalytic subunit. Assay of the immunoprecipitated epitope-tagged phosphatase 2A demonstrated an okadaic acid-sensitive dephosphorylation of [32P] histone H1 and [32P]myelin basic protein similar to that measured with the wild-type enzyme. Functional phosphatase activity could be demonstrated for the NH2-terminal modified phosphatase 2A catalytic subunit following transient expression in COS cells or stable expression in Rat1a cells. In contrast, the COOH-terminal-modified phosphatase 2A catalytic subunit was very poorly expressed. The NH2-, COOH-modified subunit, having the nine-amino-acid hemagglutinin peptide sequence encoded at both termini of the polypeptide, was also expressed as a functional phosphatase 2A enzyme. Thus, NH2-terminal modification of the phosphatase 2A catalytic subunit results in a functional plasmid-expressed enzyme. The unique nine-amino-acid epitope-tag sequence also provides a method to easily resolve the recombinant phosphatase 2A from the endogenous wild-type gene product and related phosphatases expressed in cells.

Detection of enterotoxigenic Escherichia coli, Shigella and Campylobacter spp. by multiplex PCR assay.

Three oligonucleotide primers were used in a polymerase chain reaction (PCR) assay for the simultaneous amplification of regions of the invasive plasmid antigen (ipaH) of Shigella spp., flagellin gene (flaA) of Campylobacter spp., and heat-labile enterotoxin (LT) of enterotoxigenic Escherichia coli (ETEC). The multiplex assay was performed using DNA extracted by a chaotropic method directly from diarrhoeal stools. The diagnostic efficacy of the assay was analyzed by agarose gel electrophoresis. This assay shows a novel approach for the diagnosis of diarrhoea caused by Shigella spp., ETEC, and Campylobacter spp.

Characterization of an enterotoxigenic Escherichia coli strain from Africa expressing a putative colonization factor.

An enterotoxigenic Escherichia coli (ETEC) strain of serotype O114:H- that expressed both heat-labile and heat-stable enterotoxins and tested negative for colonization factors (CF) was isolated from a child with diarrhea in Egypt. This strain, WS0115A, induced hemagglutination of bovine erythrocytes and adhered to the enterocyte-like cell line Caco-2, suggesting that it may elaborate novel fimbriae. Surface-expressed antigen purified by differential ammonium sulfate precipitation and column chromatography yielded a single protein band with M(r) 14,800 when resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (16% polyacrylamide). A monoclonal antibody against this putative fimbrial antigen was generated and reacted with strain WS0115A and also with CS1-, CS17-, and CS19-positive strains in a dot blot assay. Reactivity was temperature dependent, with cells displaying reactivity when grown at 37 degrees C but not when grown at 22 degrees C. Immunoblot analysis of a fimbrial preparation from strain WS0115A showed that the monoclonal antibody reacted with a single protein band. Electron microscopy and immunoelectron microscopy revealed fimbria-like structures on the surface of strain WS0115A. These structures were rigid and measured 6.8 to 7.4 nm in diameter. Electrospray mass-spectrometric analysis showed that the mass of the purified fimbria was 14,965 Da. The N-terminal sequence of the fimbria established that it was a member of the CFA/I family, with sequence identity to the amino terminus of CS19, a new CF recently identified in India. Cumulatively, our results suggest that this fimbria is CS19. Screening of a collection of ETEC strains isolated from children with diarrhea in Egypt found that 4.2% of strains originally reported as CF negative were positive for this CF, suggesting that it is biologically relevant in the pathogenesis of ETEC.

Differentiation of Campylobacter isolates on the basis of sensitivity to boiling in water as measured by PCR-detectable DNA.

Differential sensitivity for the release of PCR-detectable genomic DNA upon boiling in water is reported for 45 Campylobacter jejuni and Campylobacter coli strains isolated in Egypt. All of the strains released PCR-detectable DNA when treated with proteinase K and sodium dodecyl sulfate. When DNA was extracted from these strains by boiling in water, nine (20%) of the strains were PCR negative or resistant to boiling, suggesting the presence of boiling-sensitive and boiling-resistant phenotypes.

Evaluation of antibodies reactive with Campylobacter jejuni in Egyptian diarrhea patients.

Serum and stool samples were collected from 128 individuals: 96 diarrhea patients and 32 apparently healthy controls. Stool specimens were cultured for enteric bacterial pathogens, while sera were screened by enzyme-linked immunosorbent assay for Campylobacter jejuni-reactive antibodies. Of 28 diarrhea patients who demonstrated C. jejuni-reactive antibodies (titers, > 100), 14 were culture positive for this organism. The 32 healthy controls showed significantly lower antibody titers (P < 0.05) with the exception of 10 subjects who were culture positive for C. jejuni and had reactive immunoglobulin M (IgM) (6 subjects) and IgG (7 subjects). IgA was not detected in those 10 individuals (asymptomatic). Avidity was expressed as the thiocyanate ion concentration required to inhibit 50% of the bound antibodies. The avidity was higher in symptomatic patients than asymptomatic healthy controls. IgG was less avid (0.92 M) compared to IgM (0.1 M) and IgA (1.1 M), with no correlation between antibody titer and avidity. However, the thiocyanate ion concentration required for the complete inhibition of IgG (5 M)-bound antibodies was higher than that of IgA (2 M) and IgM (3 M). This study also shows that C. jejuni antibodies were variably cross-reactive with Escherichia coli, Shigella flexneri, Shigella sonnei, and Neisseria meningitidis in addition to Campylobacter coli and Campylobacter rectus.

Risk and aetiology of diarrhoea at various tourist destinations.

Almost two of three tourists developed traveller's diarrhoea during 2-week stays at high-risk destinations. Large differences in infection rates between hotels were seen. Patients with milder forms of diarrhoea show a similar chronology to those more severely affected. Although enterotoxigenic Escherichia coil was the most frequent cause, viral pathogens were detected more often than in other studies.

Epidemiology of rotavirus diarrhea in Egyptian children and implications for disease control.

Reliable epidemiologic data are essential for formulating effective policy to control rotavirus disease through immunization. The objective of this study was to describe the epidemiology of rotavirus diarrhea in a population-based cohort of children under 3 years of age residing in Abu Homos, Egypt, in 1995-1996. Rotavirus diarrhea incidence rates (episodes per person-year) were 0.13 for infants aged <6 months, 0.61 for those aged 6-11 months, 0.17 for those aged 12-23 months, and 0.15 for those aged 24-35 months. Fifty-six percent of children with rotavirus diarrhea had clinical dehydration; 90% of rotavirus diarrheal episodes occurred between July and November. In infants under 1 year of age, receipt of breast milk was associated with a lower incidence of rotavirus diarrhea. No other sociodemographic or environmental factor was found to be significantly associated with rotavirus diarrhea. Of 46 rotavirus isolates with strains identified, 41 (89%) were G serotypes 1 and 2. Rotavirus diarrhea was a major cause of morbidity in this cohort. Promotion of breastfeeding may exert a protective effect in young infants in this setting, but improvements in water and sanitation are unlikely to be effective preventive measures. The use of effective immunization against rotavirus in early infancy should be considered a public health priority.

PIP: This study describes the epidemiology of rotavirus diarrhea in a population-based cohort of children under 3 years of age residing in Abu Homos, Egypt, during 1995-96. Samples consisted of a cohort of children under the age of 24 months assembled from two villages in the vicinity of Abu Homos. The age-specific incidence rates of rotavirus diarrheal episodes per person-year were 0.13 for infants aged 6 months, 0.61 for those aged 6-11 months, 0.17 for those aged 12-23 months, and 0.15 for those aged 24-35 months. No rotavirus diarrheal incidence occurred in infants under 20 weeks of age. The monthly incidence rates of rotavirus diarrhea demonstrate that 90% of the disease episodes occurred during the warmer months of July-November, with a peak incidence in August. In infants under 1 year of age, breast-feeding was associated with a lower incidence of rotavirus diarrhea. Promotion of breast-feeding may employ a protective effect in young infants in this setting, but improvements in water and sanitation are unlikely to be effective preventive measures.

Identification of Shigella flexneri subserotype 1c in rural Egypt.

In a population-based study of diarrhea in rural, northern Egypt, 60 Shigella flexneri strains were identified, of which 10 could not be definitively serotyped. Serological analysis with commercial reagents suggested that they were serotype 1, but the strains failed to react with subserotype 1a- or 1b-specific antibodies. All 10 strains reacted with MASF 1c, a monoclonal antibody specific for a provisional S. flexneri subserotype, 1c, first identified in Bangladesh and not previously detected outside of that region. Our results show that S. flexneri subserotype 1c is not unique to Bangladesh and that the inability to detect it may reflect both the limited use of suitable screening methods and the rarity of this subserotype.

Development and evaluation of serotype- and group-specific fluorogenic reverse transcriptase PCR (TaqMan) assays for dengue virus.

Five fluorogenic probe hydrolysis (TaqMan) reverse transcriptase PCR (RT-PCR) assays were developed for serotypes 1 to 4 and group-specific detection of dengue virus. Serotype- and group-specific oligonucleotide primers and fluorogenic probes were designed against conserved regions of the dengue virus genome. The RT-PCR assay is a rapid single-tube method consisting of a 30-min RT step linked to a 45-cycle PCR at 95 and 60 degrees C that generates a fluorogenic signal in positive samples. Assays were initially evaluated against cell culture-derived dengue stock viruses and then with 67 dengue viremic human sera received from Peru, Indonesia, and Taiwan. The TaqMan assays were compared to virus isolation using C6/36 cells followed by an immunofluorescence assay using serotype-specific monoclonal antibodies. Viral titers in sera were determined by plaque assay in Vero cells. The serotype-specific TaqMan RT-PCR assay detected 62 of 67 confirmed dengue virus-positive samples, for a sensitivity of 92.5%, while the group-specific assay detected 66 of 67 confirmed dengue virus-positive samples, for a sensitivity of 98.5%. The TaqMan RT-PCR assays have a specificity of 100% based on the serotype concordance of all assays compared to cell culture isolation and negative results obtained when 21 normal human sera and plasma samples were tested. Our results demonstrate that the dengue virus TaqMan RT-PCR assays may be utilized as rapid, sensitive, and specific screening and serotyping tools for epidemiological studies of dengue virus infections.

Pathogenicity and convalescent excretion of Campylobacter in rural Egyptian children.

Campylobacter infection in developing countries has not received much public health attention because of the observation that infections are not associated with disease beyond the first 6 months of life. A cohort of 397 Egyptian children aged less than 3 years, who were observed twice weekly during 1995--1998, experienced an incidence of 0.6 episodes of Campylobacter diarrhea per child-year. A total of 13% of the Campylobacter diarrheal episodes were characterized by severe dehydration. Age-specific incidence rates (episodes per year) were 0.9 in infants aged less than 6 months, 1.5 in those 6--12 months, and 0.4 and 0.2 in the second and third years of life, respectively. Convalescent excretion of Campylobacter after a diarrheal episode might be enhancing transmission and contributing to this high incidence. Observed risk factors for Campylobacter diarrhea were poor hygienic conditions and the presence of animals in the house. Regardless of the child's age, a first infection by Campylobacter was associated with diarrhea (odds ratio = 2.45; 95% confidence interval: 1.61, 3.71); however, subsequent infections were associated with diarrhea only in children aged less than 6 months. This observation that natural infection did not confer protection during the first 6 months of life poses a challenge to vaccine development.