Detection of enteropathogens in fatal and potentially fatal diarrhea in Cairo, Egypt

A 1-year study of the etiology of acute diarrhea complicated by severe (10%) dehydration, active bleeding, shock and cardiovascular collapse, pneumonia, acute renal failure, or seizures in infants under 18 months of age was performed in Cairo, Egypt. Of 145 infants, 19 (13%) died or left the hospital moribund; the remaining 126 patients were classified as having potentially fatal illness. A variety of enteropathogens were identified with approximately equal frequency in the fatal and nonfatal complicated cases as well as in 135 controls with severe uncomplicated diarrhea. The agents most frequently detected in infants with severe diarrhea in this population which were felt to be etiologically important were rotavirus (33%), heat-stable enterotoxin-producing Escherichia coli (20%), heat-labile enterotoxin-producing E. coli (11%), enteropathogenic E. coli (8%), and Salmonella spp. (5%). The high rate of occurrence of Giardia lamblia (35%) probably represented the high carriage rate of the protozoan in this population. Complicated (fatal and potentially fatal) cases differed from control cases in a number of ways: the onset of diarrhea was more sudden, the course was progressive and of greater initial intensity, vomiting occurred more frequently, the patients more often had visited another physician before coming to the hospital, the patients more often had respiratory symptoms and pulmonary abnormalities on auscultation, hypoactive bowel sounds and abdominal distention were more common, as was oliguria, and the patients showed lower mean body weights.     

Molecular, biochemical, and morphometric characterization of Fasciola species potentially causing zoonotic disease in Egypt

Fascioliasis is an important disease caused by Fasciola hepatica and Fasciola gigantica. The distributions of both species overlap in many areas of Asia and Africa including Egypt. Fifty adult Fasciola worms were collected from livers of cattle and sheep slaughtered in abattoirs, Cairo, Egypt. They were subjected to morphological and metric assessment of external features of fresh adults, morphological and metric assessment of internal anatomy of stained mounted worms, determination of electrophorezed bands of crude adult homogenates using SDS-PAGE, and molecular characterization of species-specific DNA segments using RFLP-PCR. It was found that the correlation between conventional morphology and its morphotype was statistically significant (P value?=?0.00). Using SDS-PAGE, 13 bands were detected among both genotypes of Fasciola (35.7, 33.6, 32.4, 29.3, 27.5, 26, 24.4, 23, 21.45, 19, 16.75, 12.5, and 9.1?kDa).The most prevalent bands were that with a molecular weight of 29.3, 26, and 19?kDa. Bands detected were common for both species, but protein bands could not distinguish between F. hepatica and F. gigantica. The result of PCR for the amplification of the selected 28S rDNA fragment with the designed primer set yielded 618?bp long PCR products for F. hepatica and F. gigantica. Different band patterns generated after digestion of the 618?bp segment by the enzyme AvaII obtained with F. hepatica showed segments of the length 529, 62, 27?bp, while with F. gigantica 322, 269, 27?bp bands were obtained. Genotyping revealed no equivocal results. The conventional morphological parameters for species determination of Fasciola spp. endemic in Egypt were evaluated versus protein bands characterization and genotyping. It was concluded that conventional morphological and metric assessments were not useful for differentiation between F. gigantica and F. hepatica due to extensive overlap in the relative ranges. Similar conclusion was reached concerning protein band characterization where the patterns of protein banding were mostly similar. In contrast, genotyping using RFLP-PCR gave consistent results and clear differentiation between the two species. Considering the implications of proper speciation of endemic parasites on clinical evaluation, therapy, epidemiology, and control measures, speciation of parasites is currently revised on molecular basis. The presently used molecular tool is therefore recommended for further study to help draw a proper map for geographical distribution of Fasciola species.     

Isolation and molecular characterization of potentially pathogenic Acanthamoeba genotypes from diverse water resources including household drinking water from Khyber Pakhtunkhwa, Pakistan

Acanthamoeba, an opportunistic protozoan pathogen, is ubiquitous in nature, and therefore plays a predatory role and helps control microbial communities in the ecosystem. These Acanthamoeba species are recognized as opportunistic human pathogens that may cause blinding keratitis and rare but fatal granulomatous encephalitis. To date, there is not a single report demonstrating Acanthamoeba isolation and identification from environmental sources in Pakistan, and that is the aim of this study. Acanthamoeba were identified by morphological characteristics of their cysts on non-nutrient agar plates seeded with Escherichia coli. Additionally, the polymerase chain reaction (PCR) was performed with genus-specific primers followed by direct sequencing of the PCR product for molecular identification. Furthermore, our PCR and sequencing results confirmed seven different pathogenic and nonpathogenic genotypes, including T2–T10, T4, T5, T7, T15, T16, and T17. To the best of our knowledge, we have identified and isolated Acanthamoeba sp., for the first time, from water resources of Khyber Pakhtunkhwa, Pakistan. There is an urgent need to address (1) the pathogenic potential of the identified genotypes and (2) explore other environmental sources from the country to examine the water quality and the current status of Acanthamoeba species in Pakistan, which may be a potential threat for public health across the country.     

Thermophilic potentially pathogenic amoebae isolated from natural water bodies in Poland and their molecular characterization

The free-living amoebae (FLA) may live in the environment and also within other organisms as parasites and then they are called amphizoic. They are potentially pathogenic for humans and animals and are found in water that is a source of infection. The aim of this study was molecular detection and identification of these FLA in natural water bodies in North-Western Poland to evaluate the risk of the pathogenic amoebae infections. We examined surface water samples collected from 50 sites and first, the tolerance thermic test was performed in order to select thermophilic, potentially pathogenic strains. For molecular identification of FLA, regions of 18S rDNA, 16S rDNA and intergenic spacers were amplified. Acanthamoeba T4 and T16 genotypes of 18S rDNA gene and 18S rDNA of H. vermiformis were detected. We identified two variants of Acanthamoeba T4 genotype, two variants of Acanthamoeba T16 genotype and one variant of H. vermiformis. Identification of the T16 genotype and H. vermiformis in water was for the first time in Poland. Additionally, we made attempts to adapt the RLB method for detection and differentiation of FLA species and strains. PCR seems to be more sensitive than RLB hybridization, though.     

Genetic characterization of highly pathogenic H5N1 avian influenza viruses isolated from poultry farms in Egypt

Twenty-four avian influenza viruses were collected from poultry farms in three different governorates in Egypt during the years 2006–2009 and genetically characterized. All the isolates were confirmed to be type A and subtype H5 influenza virus by chromatographic strip test and hemagglutination inhibition assay. The sequence and phylogenetic data revealed that all Egyptian isolates cluster together and belong to subclade 2.2.1 of the H5N1 virus of Eurasian origin. Within the clade, Egyptian isolates were classified into three major groups (A, B, and C) based on genetic similarity and chronology of the isolation. The majority of the recent isolates belonged to subgroup A. Interestingly, four strains, which were isolated from the same farm with two of the samples collected on the same day, were located in separate subgroups. In addition, the difference in topology between HA and NS phylogenetic trees, which indicates possible difference in dynamics of genetic evolution in two genes, was observed. Genetic characterization data of H5N1 isolates obtained from farms with different vaccination histories indicate the vaccines currently being used in Egypt do not provide adequate level of protection. Our study provides additional evidence for the need for updated vaccine and warrants continuous monitoring of H5N1 influenza virus in Egypt.     

Surveillance of acute hepatitis C in Cairo, Egypt

Surveillance of acute hepatitis has been set up in two fever hospitals in Cairo to diagnose acute hepatitis C. Patients were categorized as definite acute hepatitis C with positive hepatitis C virus (HCV) RNA and without anti-HCV antibody, or probable acute hepatitis C with positive HCV RNA, positive anti-HCV antibody, alanine aminotransferase >/=4 times the upper limit of normal (ULN), and high risk parenteral exposure in the 1--3 months prior to the beginning of symptoms. From May to November 2002, 315 patients were recruited in the study. Of these, 115 (36.5%) had acute hepatitis A, 89 (28.3%) had acute hepatitis B, and 111 (35.2%) had non-A non-B acute hepatitis. Of the total with complete data (n=309), 12 (3.9%, 95% CI=2.0%-6.7%) had definite acute hepatitis C, and 11 (3.6%, 95% CI=1.8%-6.3%) had probable acute hepatitis C. In patients with definite acute hepatitis C, dental exposure (n=5) and intravenous drug use (n=2), were the only high risk procedures found in the 6 months prior to diagnosis. Five patients had no identifiable parenteral exposure. In conclusion, results from this study suggest that acute hepatitis C can be diagnosed by surveillance of acute hepatitis in hospital settings in Cairo and that minor community exposures contribute substantially to local HCV transmission.     

Detection and molecular characterization of avipoxviruses isolated from different avian species in Egypt

Six clinical cases of avipoxvirus (APV) infection were investigated and molecular biologically studied. The samples were collected from different domesticated birds reared in the Egyptian backyard management system and were propagated on the chorioallantoic membrane of embryonated chicken eggs. The virus isolation was confirmed via PCR amplification of fpv167 (P4b) gene locus. All the studied isolates were characterized as Fowlpox-like viruses based on the amplicon length of fpv140 gene locus. The phylogenetic analysis of fpv167 (P4b) gene clustered Elsharqyia_FWPV1, Elsharqyia_FWPV2, Elsharqyia_FWPV3, Elsharqyia_FWPV4, and Elsharqyia_TKPV strains within subclade A1. Furthermore, Elsharqyia_PGPV strain was clustered within subclade A2 (Turkeypox virus) and showed 100 % nucleic acid identity with the wood pigeon Indian which was isolated in 2009. On the other hand, when the fpv140 gene was used for the phylogenetic analysis, Elsharqyia_PGPV was clustered within subclade A4 (Pigeonpox virus) with the other PGPVs. This study is considered the first molecular record for APVs circulating in the Egyptian birds. Further studies in a larger scale need to be developed to have a better understanding about the molecular characterization of the Egyptian APV strains.     

Some ecological and physiological studies on bacteria isolated from salt-affected soils of Egypt.

Members of Bacillaceae, Rhizobiaceae, actinomycetes and others were isolated from cultivated and non-cultivated saline soils. The high population of bacteria and actinomycetes were almost coincided with the relatively high levels of organic matter whatever the degree of soil salinity. Bacillus stearothermophilus and B. subtilis were more frequently isolated than other Bacillus species. Most of Rhizobium isolates were salt tolerant being able to grow in media containing 3% and 6% NaCl. The abilities of different bacterial isolates to attack citrus pectin, soluble and insoluble forms of cellulose were also tested.     

Genotypic characterization of symptomatic hepatitis E virus (HEV) infections in Egypt

BackgroundHepatitis E virus (HEV) is a common cause of acute viral hepatitis (AVH) in many developing countries. In Egypt, HEV seroprevalence is among the highest in the world; however, only a very limited number of Egyptian HEV sequences are currently available.ObjectivesThe objectives were to determine the HEV genotype(s) currently circulating in Egypt.Study designAVH patients without serologic evidence of hepatitis A, B, and C viruses were evaluated for possible HEV infection using serologic assays for anti-HEV IgM and anti-HEV IgG and real-time PCR for HEV RNA. Stool suspensions from suspected cases were inoculated into rhesus macaques to confirm the presence of HEV. Sequence analysis was utilized to determine HEV genotype.ResultsOf 287 subjects with AVH enrolled, 58 had serologic evidence of acute HEV infection. Stool samples for two of these patients were repeatedly positive for HEV RNA by real-time PCR. Macaques experimentally inoculated with these human stools also developed viremia. Sequence analysis of open reading frame (ORF) 1 demonstrated that these isolates belonged to HEV genotype 1 and were 3.9–9.5% divergent from other genotype 1 isolates. ORF2 was 5.3–8.7% divergent from previously reported Egyptian isolates.ConclusionsThis study strongly suggests that genotype 1 HEV related to other North African isolates is circulating in acute symptomatic patients in Egypt. Further evaluation of genotypic variability is underway in this highly endemic cohort and is considered an important component of our increased understanding of HEV pathogenesis.     

Purification and characterization of urease isolated from the pathogenic fungus Coccidioides immitis.

Coccidioides immitis, the causative agent of San Joaquin Valley fever (coccidioidomycosis), produces a urease which has been suggested to contribute to the virulence of this fungal pathogen. Urease catalyzes the hydrolysis of urea and has been proposed to at least partly account for alkalinity of the microenvironment in which C. immitis grows due to the release of ammonia and ammonium ions. The C. immitis urease was purified to homogeneity (1048-fold) from the mycelial cytosol by chromatographic fractionation. The sequence of 12 N-terminal amino-acid residues of the purified, native polypeptide was identical to that predicted by the translated urease gene sequence which has been reported. The isolated enzyme exhibited a specific activity in the presence of urea of 1750 micromol min(-1) mg(-1) protein, has a native molecular mass of 450 kDa, revealed a Km for urea of 4.1 mM, had a pH optimum of 8.0 and is heat stable. Hydroxyurea, acetohydroxamic acid (AHA) and boric acid each inhibited activity of the purified enzyme. Urease activity was enhanced by the presence of 5-10 mM concentrations of Mg2+ or Mn2+, but inhibited by Li+, Ni2+, Cu2+ or Zn2+. The reversible urease inhibitor, AHA, blocked enzyme activity in the crude mycelial cytosolic fraction when added at a concentration of 10 mM. On the other hand, 10 mM AHA added to 4-day-old mycelial cultures only partially decreased the amount of ammonium detected in the culture medium. It is evident, therefore, that C. immitis urease activity does not account for the total amount of ammonia secreted during in vitro growth of the pathogen. Other metabolic sources of ammonia, which may also contribute to the virulence of C. immitis, are under investigation.     

Urease from a potentially pathogenic coccoid isolate: purification, characterization, and comparison to other microbial ureases.

Strain SL100 is a gram-positive coccoid isolate prototype with an adhesin specific for gastric mucin and is representative of potentially pathogenic organisms obtained at biopsy from patients with gastric disorders. The urease of this isolate constitutes a significant fraction of the total cell protein, and the outcome of the purification strategy described herein suggests that it is associated with a cell wall fraction. The urease was purified 138-fold to apparent homogeneity, as indicated by gel electrophoresis, to a specific activity of 1,120 U/mg. The urease was unstable during purification in the absence of nickel, which is present in a metallocenter in other microbial ureases. When nickel sulfate was present during growth (5 microM) and in buffers during sonication and purification (100 microM), the urease was completely stable at room temperature during the purification procedure. The native urease was approximately 260 kDa and was composed of three subunits of 65 kDa and three subunits of 21 kDa. The purified urease was relatively stable in acid and retained most of its activity after incubation for 30 min at pH 1.3. The K(m)s for urease measured from whole cells and for the purified enzyme were 0.56 and 1.7 mM, respectively, indicating that some cell wall component(s) affects the affinity of the enzyme for urea. The V(max)s for urea hydrolysis measured from whole cells and for the purified enzyme were 8.1 and 1,120 mol/min/mg of protein, respectively. The kinetic parameters, relative abundance, and subunit composition are more similar to those of the ureases of Helicobacter than to those of the ureases of other microbial species. These similarities are consistent with an adaptation of this organism to colonization of the stomach and indicate that the urease may be a virulence factor during colonization.     

Genotypic and phenotypic characterization of Borrelia burgdorferi isolated from ticks and small animals in Illinois.

We have characterized 33 isolates of Borrelia burgdorferi from northern Illinois (32 isolates) and Wisconsin (1 isolate) representing the largest series of midwestern isolates investigated to date. The techniques used for molecular analysis of strains included (i) genospecies typing with species-specific PCR primers, (ii) plasmid profiling by pulsed-field gel electrophoresis of total genomic DNA, (iii) large-restriction-fragment pattern (LRFP) analysis by pulsed-field gel electrophoresis of MluI-digested genomic DNA (J. Belfaiza, D. Postic, E. Bellenger, G. Baranton, and I. Saint Girons, J. Clin. Microbiol. 31:2873-2877, 1993), (iv) sodium dodecyl sulfate-polyacrylamide gel electrophoresis of total proteins, (v) microsequencing of high-performance liquid chromatography-purified peptides derived from proteins showing high levels of expression, (vi) amino acid composition analysis of proteins, and (vii) immunological analysis of proteins with a polyclonal antiserum of human origin. Five reference strains as well as two atypical tick isolates from California (DN127) and New York (25015) were included for comparison. All of the Illinois and Wisconsin isolates were typed as B. burgdorferi sensu stricto with genospecies-specific PCR primers. The isolates were found to be heterogeneous with regard to their plasmid and protein profiles. One isolate from Illinois possessed two large-molecular-size plasmids instead of the usual 49-kb plasmid. Fragment patterns resulting from MluI digestion of genomic DNA from the 33 isolates and strains DN127 and 25015 were separable into six distinct LRFPs, five of which have not previously been described. Strain 25015 and an isolate from Illinois (CT39) shared an unusual LRFP that is not typical of other B. burgdorferi sensu stricto strains, suggesting that they may represent a fifth species of B. burgdorferi sensu lato. Five of the 33 isolates and strains DN127 and 25015 showed high-level expression of proteins with molecular masses of approximately 22 kDa. Investigation of these proteins by microsequencing of individual peptides and total amino acid composition analysis indicated that the 22-kDa proteins expressed by the seven strains were polymorphic OspC proteins. By using a polyclonal serum of human origin, expression of OspC could be detected in all 33 Illinois and Wisconsin isolates.     

OXA-163-producing Klebsiella pneumoniae in Cairo, Egypt, in 2009 and 2010

Two genetically unrelated OXA-163-carrying Klebsiella pneumoniae strains were identified from two infection cases in June 2009 and May 2010 in Cairo, Egypt. OXA-163-producing Enterobacteriaceae had been previously reported in Argentina only. Both patients had no history of travel abroad. The emergence of this newly recognized OXA-48-related β-lactamase able to hydrolyze cephalosporins and carbapenems is especially worrying in a geographic area where OXA-48 is endemic and effective surveillance for antibiotic resistance is largely unaffordable.     

Genotypic characterization of enteropathogenic Escherichia coli (EPEC) isolated in Belgium from dogs and cats

Enteropathogenic Escherichia coli (EPEC) are isolated from man and farm animals but also from dogs and cats. They produce typical histological lesions called 'attaching and effacing' lesions. Both plasmid and chromosomal elements are involved in the pathogenesis of EPEC infection. The presence of these genetic elements was investigated in 14 dog and three cat EPEC isolates. A bfpA-related gene was detected in five of the 17 isolates in association with high molecular weight plasmids, and a locus of enterocyte effacement (LEE) was present in all isolates. The LEE was inserted in the selC region in only 12% of the isolates. The eae, tir, espA and espB genes were analyzed by multiplex PCR. The results indicated the presence of those genes in the tested isolates with heterogeneity in the gene subtypes present: eae gamma-tir alpha-espA alpha-espB alpha (65%), eae beta-tir beta-espA beta-espB beta (29%), eae alpha-tir alpha-espA alpha-espB alpha (6%). Moreover, the espD gene was also present in dog and cat EPEC. The DEPEC and CEPEC form a heterogeneous group and five of them are closely related to human EPEC.     

Genotypic and phenotypic characterization of Lactobacillus plantarum strains isolated from Thai fermented fruits and vegetables

Ten Lactobacillus strains originally isolated from Thai fruits and vegetables fermentation were characterized by various phenotypic and genotypic methods. The phenotypic analysis using the method of carbohydrate fermentation patterns (API50CHL) revealed that the isolates belonged to the L. plantarum species. This was further confirmed by 16S rRNA gene sequencing. Multilocus sequence typing (MLST) revealed a strongly clonal population structure and a low genotypic diversity in this collection. However, the analyzed L. plantarum population demonstrated a higher level of diversification after API50CHL that reflects the role of available carbohydrate sources in bacterial evolution. Our results support the postulate that a combination of conventional biochemical and genotyping methods allows a thorough characterization and identification of isolates. We propose that genotypic characterization could be complemented by biochemical characterization to discriminate L. plantarum strains. (© 2009 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Genotypic characterization of carbapenem-nonsusceptible Acinetobacter spp. isolated in Latin America

The principal aim of this study was to evaluate the genomic diversity among the imipenem-nonsusceptible Acinetobacter spp. (INSA) collected from the Latin American medical centers within the SENTRY Antimicrobial Surveillance Program. The INSA isolates were collected from patients with bloodstream infections, who were hospitalized in seven Latin American countries between 1997 and 1999. For epidemiologic comparison, 20 carbapenem-susceptible Acinetobacter spp. (CSA) isolates were collected in the same period of time from the respective medical centers. A total of 23 Acinetobacter spp. isolates exhibiting imipenem MIC values of >/=8 microg/ml were typed by ribotyping, an automated molecular method. The isolates showing an identical ribogroup were also typed by pulsed-field gel electrophoresis (PFGE). The antimicrobial susceptibility to various antimicrobial agents was evaluated using a reference broth microdilution technique. Among the INSA isolates, 13 distinct ribogroups were observed, whereas 16 ribogroups were detected among the CSA. Nearly 57% of the INSA belonged to only four ribogroups. Identical ribogroups and PFGE patterns were observed among INSA and CSA isolates collected from medical centers located in different countries (Brazil and Argentina). Our results showed: (1) a higher genomic variability among the CSA; (2) presence of epidemic clones among INSA isolates encountered in Latin American medical centers; and (3) spread of INSA and CSA epidemic clones between Latin American countries.     

汕头地区医院感染多重耐药铜绿假单胞菌的基因型特征分析

目的了解汕头地区ICU和非ICU医院感染多重耐药铜绿假单胞菌分离株的基因型特征。方法收集汕头地区两家三级甲等医院的医院感染铜绿假单胞菌菌株,对这些菌株的耐药性和基因型进行分析。采用纸片扩散法进行药敏试验,脉冲场凝胶电泳法对多重耐药菌株进行基因分型,电泳结果用BionumericsV4.0软件进行聚类分析。结果共分离到132株铜绿假单胞菌,其中45株分离自ICU,87株来自非ICU病房。132株菌株中,有46株多重耐药菌（耐三种抗菌素或以上）。对46株多重耐药菌（25株来自ICU,21株来自非ICU病房）进行脉冲场凝胶电泳,结果显示来自ICU病房的25株菌株具有较高程度的同源性,而来自非ICU病房的菌株则具有明显的基因多态性。结论交叉感染是ICU多重耐药铜绿假单胞菌医院感染的重要传播途径。     

Isolation and identification of pathogenic Acanthamoeba strains in Tenerife, Canary Islands, Spain from water sources

A comprehensive survey to document the presence of free-living amoebae of the genus Acanthamoeba was conducted in tap water and sea water sources related to human environments in Tenerife, Canary Islands, Spain. Acanthamoeba identification was based on the morphology of cyst and trophozoite forms and PCR amplification with a genus-specific primer pair. The pathogenic potential of Acanthamoeba isolates was characterized by temperature and osmotolerance assays and PCR reactions with two primer pairs related to Acanthamoeba pathogenesis. The results demonstrate the presence of potentially pathogenic strains in both sources. Thus, some of the amoebae in these aquatic habitats can act as opportunistic pathogens, could play a role in the diseases of aquatic organisms, and may present a risk to human health.