bluuebunny/crossref_metadata_2025 huggingface dataset:
Comparison of PCR assay with bacterial culture for detecting Streptococcus pneumoniae in middle ear fluid of children with acute otitis media
Authors: Virolainen A , Salo P , Jero J , Karma P , Eskola J , Leinonen M
Published: January 2020

We have studied etiological diagnosis of acute otitis media (AOM) by comparing a newly developed pneumococcal PCR for Streptococcus pneumoniae to bacterial culture with 180 middle ear fluid (MEF) samples of 125 children with 125 episodes of AOM. For pneumococcal PCR assay, DNA from MEF samples was extracted by phenol-chloroform. The outer primers used amplified a 348-bp region of the pneumolysin gene, and the inner primers amplified a 208-bp region. S. pneumoniae was cultured in 33 (18%) samples, and pneumolysin PCR was positive for 51 (28%) of 180 MEF samples. Only 2 of 21 PCR-positive, S. pneumoniae culture-negative samples were positive for other otitis pathogens. By combining MEF culture and PCR results, 54 (30%) of 180 MEF samples had evidence of pneumococcal etiology. In conclusion, pneumolysin PCR is a sensitive and specific new method to study pneumococcal involvement in MEF samples of children with AOM.
Comparative quantitation of human cytomegalovirus DNA in blood leukocytes and plasma of transplant and AIDS patients
Authors: Gerna G , Furione M , Baldanti F , Sarasini A
Published: January 2020

A new method for the quantitation of human cytomegalovirus (HCMV) DNA was used to determine the levels of viral DNA in parallel in 120 blood leukocyte (leukoDNAemia) and plasma (plasmaDNAemia) samples from 8 heart or heart-lung transplant patients and 17 AIDS patients with disseminated HCMV infection. PlasmaDNAemia was consistently associated with leukoDNAemia in both groups of patients. However, at least in the transplant patients, plasmaDNAemia was not necessarily associated with clinical symptoms, appearing later and disappearing earlier than leukoDNAemia during the course of infection. Quantitative mean levels of leukoDNAemia were mostly higher than those of plasmaDNAemia in both transplant and AIDS patients. However, in the absence of antiviral treatment, plasmaDNAemia levels were significantly higher in AIDS patients than in transplant recipients, whereas leukoDNAemia levels were not significantly different between the two groups of patients. A significant correlation was found between leukoDNAemia and plasmaDNAemia in AIDS patients, as well as in transplant recipients, although to a lesser degree. However, from a diagnostic standpoint, quantitative determination of plasmaDNAemia appears to represent a much less sensitive parameter than that of leukoDNAemia (or antigenemia) for monitoring HCMV infections and antiviral treatment.
RTX toxin genotypes and phenotypes in Actinobacillus pleuropneumoniae field strains
Authors: Beck M , van den Bosch J F , Jongenelen I M , Loeffen P L , Nielsen R , Nicolet J , Frey J
Published: January 2020

Actinobacillus pleuropneumoniae serotype reference strains and 204 A. pleuropneumoniae field strains representing all 12 serotypes and both biovars 1 and 2, obtained from laboratories from various countries worldwide, were analyzed for the presence of the toxin genes apxIC, apxIA, apxIB, apxID, apxIIC, apxIIA, apxIIIC, apxIIIA, apxIIIB, and apxIIID by DNA-DNA hybridization with specific gene probes. Expression of the toxins ApxI, ApxII, and ApxIII was assessed by immunoblot analysis with monoclonal antibodies. The results show that the patterns of apx genes and those of the expressed Apx toxins in biovar 1 field strains are the same as those of the genes and toxins of corresponding serotype reference strain. We found only three strains which had certain apx genes missing compared with the genes in their serotype reference strains. Analysis of the expression of the three toxins showed that nearly all strains expressed their apx genes and produced the same Apx toxins as their serotype reference strain. We found only one strain that did not produce ApxI, although it contained the apxICABD genes, and one strain which did not express ApxII but which contained apxIICA. Several field strains which initially showed that their serotype did not correspond to the apx gene profile of the reference strain and which had an unexpected virulence for the given serotype revealed that their initial serotyping was erroneous. We show that the apx gene profiles are inherent to a given serotype. The method cannot differentiate between all 12 serotypes. However, it allowed us to distinguish five groups of toxin gene patterns which showed pathological, toxicological, and epidemiological significance. None of the biovar 2 strains contained apxIII genes. The apxI and apxII genes in the biovar 2 strains, however, were the same as those found in the serotype reference strains of biovar 1.
Intestinal colonization of symptomatic and asymptomatic schoolchildren with Blastocystis hominis
Authors: Nimri L , Batchoun R
Published: January 2020

A study of single stool specimens was done to determine the prevalence of intestinal parasites among 1,000 primary school children. A questionnaire was completed by each child's parents. Specimens were examined by using wet-mount preparation, formaline-ether concentration, and Sheather's flotation technique. Trichrome and acid-fast stains were done. Blastocystis hominis was observed in 203 (20.3%) of the specimens examined, and 175 specimens contained this organism in the absence of other pathogenic parasites. Older children had fewer B. hominis infections (6 to 7 years old, 50% infection rate; 8 to 9 years, 27.5%; 10 to 12 years, 9.5%). The most common complaints reported by 75 children harboring the parasite were a mild recurrent diarrhea, abdominal pain, nausea, anorexia, and fatigue. Blastocystosis is quite common among schoolchildren. Contaminated drinking water is suspected to be the source of infection.
Serovars of Mycobacterium avium complex isolated from patients in Denmark
Authors: Askgaard D S , Giese S B , Thybo S , Lerche A , Bennedsen J
Published: January 2020

Danish isolates of Mycobacterium avium complex were serotyped by the use of seroagglutination. The most prevalent serovars among patients with AIDS (n = 89) were 4 and 6, while among non-AIDS patients the most prevalent serovars were 1, 6, and 4, with no major differences between those in patients with pulmonary disease (n = 65) and those in patients with lymph node infection (n = 58). The results suggest a Scandinavian distribution of serovars with a predominance of serovar 6 and fail to demonstrate any selective protection against different serovars by Mycobacterium bovis BCG vaccination.
Interactions with lectins and agglutination profiles of clinical, food, and environmental isolates of Listeria
Authors: Facinelli B , Giovanetti E , Casolari C , Varaldo P E
Published: January 2020

On the basis of preliminary trials with 14 collection strains of Listeria, five lectins (Canavalia ensiformis, concanavalin A; Griffonia simplicifolia lectin I; Helix pomatia agglutinin; Ricinus communis agglutinin; and Triticum vulgaris wheat germ agglutinin) were selected to set up a microtiter agglutination assay. The lectin agglutination profiles of 174 clinical, food, and environmental strains of Listeria monocytogenes, Listeria innocua, and Listeria seeligeri were investigated. Data on the standard determination of the antigenic structure were available for clinical strains; nonclinical isolates were assigned to serogroup 1 or 4 with commercial antisera. The listeria-lectin interaction was related to serological type rather than species; in particular, the strains assigned to serogroup 1 or belonging to serovars 1/2a, 1/2b, 1/2c, 3a, 3b, and 7 were never agglutinated by G. simplicifolia lectin I. The five-lectin set proved to be capable of detecting differences between serologically identical isolates of L. monocytogenes. Of the 150 isolates of this species, 144 were distributed over 15 different lectin agglutination profiles and 6 autoagglutinated, the overall typeability being 96%. However, the profiles encountered among L. monocytogenes isolates were not randomly distributed. With strains assigned to serogroup 1 or belonging to serovars 1/2a, 1/2b, 1/2c, and 3b, the clinical isolates fell into only two of the eight patterns recorded overall; with strains of serogroup 4 and serovar 4b, food and environmental isolates were distributed over eight of the nine patterns found in total, while clinical isolates were distributed over five patterns. In a comparative study of 15 epidemiologically relevant isolates of L. monocytogenes from five distinct outbreaks, strains with identical phage types and/or DNA fingerprints displayed identical lectin profiles. The heterogeneity of agglutination profiles may form the basis of a new approach to L. monocytogenes typing.
Use of cephalexin-aztreonam-arabinose agar for selective isolation of Enterococcus faecium
Authors: Ford M , Perry J D , Gould F K
Published: January 2020

Cephalexin-aztreonam-arabinose agar (CAA), a new selective agar, was examined in comparison with nalidixic acid-colistin agar for the differentiation of Enterococcus faecium from other enterococci and the ability to isolate the organism from feces. Two hundred sixteen enterococcus isolates and a variety of gram-positive and gram-negative control strains were inoculated onto both media. All control strains of E. faecium were easily differentiated from Enterococcus faecalis and Enterococcus durans on the basis of arabinose fermentation on CAA. Differentiation of E. faecium from other enterococci or Streptococcus bovis was not possible on nalidixic acid-colistin agar. Increased isolation of E. faecium was demonstrated on CAA when both media were compared for the isolation of the organism from feces. CAA has been shown to possess excellent differential and selective features allowing the simple and effective isolation of E. faecium from heavily contaminated sites.
Detection of mip gene by PCR for diagnosis of Legionnaires' disease
Authors: Lindsay D S , Abraham W H , Fallon R J
Published: January 2020

The mip gene of Legionella pneumophila was demonstrated by PCR and probing in paired acute-phase and convalescent-phase sera from five patients with Legionnaires' disease but not in the acute-phase sera of 100 patients with pneumonia that showed no serological evidence of Legionella infection. PCR may help in cases presenting diagnostic difficulty.
PCR amplification of variable sequence upstream of katG gene to subdivide strains of Mycobacterium tuberculosis complex
Authors: Goyal M , Young D , Zhang Y , Jenkins P A , Shaw R J
Published: January 2020

PCR amplification of a species-specific 2-kb KpnI fragment of variable size located 10 kb upstream of the katG gene was used to subdivide 130 clinical isolates of Mycobacterium tuberculosis. Seven subtypes were identified, and their frequencies were distributed normally with respect to the size of the amplified product.
PCR-based restriction fragment length polymorphism typing of Helicobacter pylori
Authors: Fujimoto S , Marshall B , Blaser M J
Published: January 2020

We applied a molecular typing approach for Helicobacter pylori that uses restriction fragment length polymorphism (RFLP) analyses of an 820-bp PCR-amplified portion of the ureC gene in H. pylori. The PCR products were digested with restriction enzyme HhaI, MboI, or MseI, and the fragments generated were analyzed by agarose electrophoresis. Among 25 independent clinical isolates, each showed a different pattern when a combination of the three RFLP patterns was used. Using this method, we studied isolates from the antrum or the body of the stomach of 14 patients before and after antibiotic therapy. Before treatment, successful isolation of H. pylori from the two sites of the stomach was possible for 12 of the 14 patients. For 10 of these 12 patients, each pair of isolates had identical RFLP profiles. For the other two patients (16.7%), however, isolates from the antrum and the body of the stomach had different RFLP profiles. Treatment was successful for 6 of the 14 patients; of the 8 patients with treatment failures, 5 had identical isolate pairs. In each case, the isolates found posttreatment were the same as the pretreatment isolates. For one of the patients who was colonized with two different isolates pretreatment, one of the isolates was identified at both sites after unsuccessful treatment. We also studied six long-term follow-up patients who had sequential biopsies at intervals of up to 5 months. Each follow-up isolate from each patient had the same RFLP profile as the initial isolate. This typing method provides a reliable and reproducible typing scheme for the study of H. pylori infections and indicates that infection with more than one H. pylori isolate is not rare.
Evaluation of Gen-Probe Amplified Mycobacterium Tuberculosis Direct Test and PCR for direct detection of Mycobacterium tuberculosis in clinical specimens
Authors: Miller N , Hernandez S G , Cleary T J
Published: January 2020

The Gen-Probe Amplified Mycobacterium Tuberculosis Direct Test (AMTD) is a direct specimen assay for the identification of Mycobacterium tuberculosis from respiratory samples. rRNA is amplified, and the product is detected with a specific chemiluminescent probe. We performed a retrospective evaluation of three separate respiratory specimens from each of 250 patients by using the AMTD and compared the results with those of microscopy, culturing, and a patient chart review. From the latter results, 198 patients (594 specimens) were found negative for M. tuberculosis by culturing and clinical criteria. The overall specificity of the AMTD after discrepancy resolution was 98.5% (585 of 594). There were 52 patients with culture-proven and/or clinically diagnosed tuberculosis. Of these 156 specimens, the organism was cultured from 142 (91%), and acid-fast microscopy was positive for 105 (67.3%). The AMTD was positive for 142 (91%) specimens from these patients. Tuberculosis patient samples were tested by a PCR assay which uses primers for amplification of the IS6110 insertion sequence of the M. tuberculosis complex. The PCR assay detected 144 of the 156 (92.3%) specimens. Overall, when three specimens per patient were examined, the AMTD found all 52 patients positive for tuberculosis, while the PCR assay found 51 patients positive by agarose gel analysis and all 52 patients positive by Southern blot hybridization.
Use of pulsed-field gel electrophoresis for epidemiological study of Bordetella pertussis in a whooping cough outbreak
Authors: de Moissac Y R , Ronald S L , Peppler M S
Published: January 2020

We used pulsed-field gel electrophoresis (PFGE) of chromosomal DNA digested with XbaI to determine the distribution of different Bordetella pertussis strains from clinical isolates obtained during a large whooping cough outbreak that occurred in Alberta, Canada, from December 1989 to May 1991. Our initial study analyzed 28 clinical isolates, 14 from the city of Edmonton and 1 from each of 14 northern Alberta towns. These clinical isolates were randomly chosen over the course of the 18-month outbreak. The DNA profiles were more heterogeneous than anticipated and caused concern that PFGE was too sensitive a technique to characterize strains. Further analysis showed that this was not the case, as clusters of similar PFGE patterns were observed in strains isolated from the same outlying town. Identical PFGE patterns were also seen in clinical strains obtained from different members of the same family. Two PFGE pattern types, a and b, predominated in the outbreak, accounting overall for 44 of 70 B. pertussis strains tested. Results from isolates from outlying towns, however, indicated involvement of local strains rather than a single, highly infectious strain in the whooping cough outbreak in Alberta.
Comparison of traditional and molecular methods of typing isolates of Staphylococcus aureus
Authors: Tenover F C , Arbeit R , Archer G , Biddle J , Byrne S , Goering R , Hancock G , Hébert G A , Hill B , Hollis R
Published: January 2020

Fifty-nine Staphylococcus aureus isolates and 1 isolate of Staphylococcus intermedius were typed by investigators at eight institutions by using either antibiograms, bacteriophage typing, biotyping, immunoblotting, insertion sequence typing with IS257/431, multilocus enzyme electrophoresis, restriction analysis of plasmid DNA, pulsed-field or field inversion gel electrophoresis, restriction analysis of PCR-amplified coagulase gene sequences, restriction fragment length polymorphism typing by using four staphylococcal genes as probes, or ribotyping. Isolates from four well-characterized outbreaks (n = 29) and a collection of organisms from two nursing homes were mixed with epidemiologically unrelated stock strains from the Centers for Disease Control and Prevention. Several isolates were included multiple times either within or between the sets of isolates to analyze the reproducibilities of the typing systems. Overall, the DNA-based techniques and immunoblotting were most effective in grouping outbreak-related strains, recognizing 27 to 29 of the 29 outbreak-related strains; however, they also tended to include 3 to 8 epidemiologically unrelated isolates in the same strain type. Restriction fragment length polymorphism methods with mec gene-associated loci were less useful than other techniques for typing oxacillin-susceptible isolates. Phage typing, plasmid DNA restriction analysis, and antibiogram analysis, the techniques most readily available to clinical laboratories, identified 23 to 26 of 29 outbreak-related isolates and assigned 0 to 6 unrelated isolates to outbreak strain types. No single technique was clearly superior to the others; however, biotyping, because it produced so many subtypes, did not effectively group outbreak-related strains of S. aureus.
Virulence of Rhodococcus equi isolates from patients with and without AIDS
Authors: Takai S , Sasaki Y , Ikeda T , Uchida Y , Tsubaki S , Sekizaki T
Published: January 2020

Rhodococcus equi is an emerging opportunistic pathogen of human immunodeficiency virus-infected patients. Thirty-nine isolates of R. equi from immunocompromised patients with and without AIDS were analyzed for the presence of virulence plasmid DNA, expression of 15- to 17-kDa antigens, and their pathogenicities in mice. Of the human isolates, eight contained an 85-kb virulence plasmid, expressed 15- to 17-kDa antigens, and were virulent in mice. Nineteen isolates carried cryptic plasmids of various sizes, and the remaining 12 isolates did not contain any plasmids. These 31 isolates did not express virulence-associated antigens and were not virulent in mice. The results suggested that opportunistic infections in immunocompromised patients could be caused by both virulent and avirulent R. equi strains and that the pathogenesis of R. equi infection in immunocompromised patients appears to be different from that which occurs in foals.
A 2-year study of Helicobacter pylori in children
Authors: Raymond J , Bergeret M , Benhamou P H , Mensah K , Dupont C
Published: January 2020

From September 1990 to October 1992, Helicobacter pylori was searched for in 426 children, 2 days to 16 years old, requiring upper fibroscopy for various symptoms. H. pylori was detected in 77 children (18.1%). Recurrent abdominal pain was present in 63.3% of the patients with H. pylori versus 48.6% of a control group of 74 age-matched children negative for H. pylori, weight loss was present in 6.5% of the patients versus 0% of the control subjects, and a family history of peptic ulcer was present in 14.2% of the patients versus 5.4% of the controls. Micronodular gastritis was observed in 31 children with H. pylori infection (40.2%). Among the 24 children (31.1%) with H. pylori infection and a normal mucosa at endoscopy, 18 (75%) complained of recurrent abdominal pain. H. pylori was also found in 21 of 38 children (55.2%) being examined because of short stature. These findings indicate that H. pylori should be looked for in children with recurrent abdominal pain with or without weight loss or a family history of peptic ulcer. Its relevance in short-stature syndrome requires further clarification.
Direct identification of Mycobacterium species in BACTEC 7H12B medium by high-performance liquid chromatography
Authors: Cage G D
Published: January 2020

Primary cultures of mycobacteria grown in BACTEC 7H12B medium (Becton Dickinson and Co., Paramus, N.J.), with and without the addition of oleic acid-albumin-dextrose-catalase (OADC) enrichment (Becton Dickinson and Co., Cockeysville, Md.), were analyzed for their mycolic acid patterns by high-performance liquid chromatography. Of the 126 isolates grown in medium to which OADC was added, 117 (93%) were successfully identified to the species level. The time to identification of Mycobacterium tuberculosis (n = 65) averaged 19 days, and the average time was 21 days for nontuberculosis mycobacteria (n = 52) from initial specimen processing. None of the 10 isolates cultured without OADC were identified. The mycolic acid patterns were considered reliable for identification if the height of the tallest peak in the chromatogram was at least 50% of the internal standard peak height.
Detection of microsporidia by indirect immunofluorescence antibody test using polyclonal and monoclonal antibodies
Authors: Aldras A M , Orenstein J M , Kotler D P , Shadduck J A , Didier E S
Published: January 2020

During a screening for monoclonal antibodies (MAbs) to the microsporidian Encephalitozoon hellem, three murine hybridoma cell lines producing strong enzyme-linked immunosorbent assay (ELISA) reactivities were cloned twice, were designated C12, E9, and E11, and were found to secrete MAbs to the immunoglobulin M isotype. On subsequent ELISAs, the three MAbs reacted most strongly to E. hellem, and they reacted somewhat less to Encephalitozoon cuniculi and least to Nosema corneum, two other microsporidian species. The MAbs produced values of absorbance against microsporidia that were at least three times greater than reactivities obtained with control hybridoma supernatants or with uninfected host cell proteins used as antigens. By Western blot immunodetection, the three MAbs detected three E. hellem antigens with relative molecular weights (M(r)s) of 62, 60, and 52 when assayed at the highest supernatant dilutions producing reactivity. At lower dilutions, the MAbs detected additional proteins with M(r)s of 55 and 53. By using indirect immunofluorescence antibody staining, the MAbs, as well as hyperimmune polyclonal murine antisera raised against E. cuniculi and E. hellem, were able to detect formalin-fixed, tissue culture-derived E. cuniculi and E. hellem and two other human microsporidia, Enterocytozoon bieneusi and Septata intestinalis, in formalin-fixed stool and urine, respectively. E. bieneusi, however, stained more intensely with the polyclonal antisera than with the MAbs. Neither the MAbs nor the hyperimmune murine polyclonal antibodies detected Cryptosporidium, Giardia, Trichomonas, or Isospora spp. At higher concentrations, the polyclonal antisera did stain N. corneum and yeast cells. The background staining could be absorbed with Candida albicans. These results demonstrate that polyclonal antisera to E. cuniculi and E. hellem, as well as MAbs raised against E. hellem, can be used for indirect immunofluorescence antibody staining to detect several species of microsporidia known to cause opportunistic infections in AIDS patients.
Evaluation of Staf-Sistem 18-R for identification of staphylococcal clinical isolates to the species level
Authors: Piccolomini R , Catamo G , Picciani C , D'Antonio D
Published: January 2020

The accuracy and efficiency of Staf-Sistem 18-R (Liofilchem s.r.l., Roseto degli Abruzzi, Teramo, Italy) were compared with those of conventional biochemical methods to identify 523 strains belonging to 16 different human Staphylococcus species. Overall, 491 strains (93.9%) were correctly identified (percentage of identification, > or = 90.0), with 28 (5.4%) requiring supplementary tests for complete identification. For 14 isolates (2.8%), the strains did not correspond to any key in the codebook and could not be identified by the manufacturer's computer service. Only 18 isolates (3.4%) were misidentified. The system is simple to use, is easy to handle, gives highly reproducible results, and is inexpensive. With the inclusion of more discriminating tests and adjustment in supplementary code numbers for some species, such as Staphylococcus lugdunensis and Staphylococcus schleiferi, Staf-Sistem 18-R is a suitable alternative for identification of human coagulase-positive and coagulase-negative Staphylococcus species in microbiological laboratories.
Detection of African horse sickness virus by reverse transcription-PCR
Authors: Stone-Marschat M , Carville A , Skowronek A , Laegreid W W
Published: January 2020

Reverse transcription-PCR (RT-PCR) was used to detect African horse sickness virus (AHSV). A single primer pair which amplified a 423-bp fragment of the S8 gene which encodes the NS2 protein of AHSV was identified. Amplification of this fragment from all nine serotypes of AHSV was achieved with these primers. Between 10(1) and 10(2) copies of AHSV genomic double-stranded RNA could be detected by RT-PCR followed by agarose gel electrophoresis and ethidium bromide staining. Application of RT-PCR to blood samples from AHSV-infected horses resulted in earlier detection of viremia than virus isolation did. Furthermore, viremia was detected by RT-PCR in blood samples from horses infected with an avirulent isolate of AHSV which were negative by virus isolation. AHSV was also detected by RT-PCR in spleen and lung samples from horses which died of AHSV infection. These results indicate that RT-PCR is a rapid and sensitive method for the identification of horses infected with AHSV.
Enzyme electrophoresis, sero- and subtyping, and outer membrane protein characterization of two Neisseria meningitidis strains involved in laboratory-acquired infections
Authors: Guibourdenche M , Darchis J P , Boisivon A , Collatz E , Riou J Y
Published: January 2020

Two cases of laboratory-acquired infections due to Neisseria meningitidis were suspected to have occurred in two French hospitals. The first case occurred shortly, i.e., 3 days, after one strain had been handled by a laboratory technician, and the link between this strain and the strain causing meningitis was easily established. In the second case, infection occurred 3 weeks after 10 strains had been handled by a technician. In this case, it was necessary to use high-resolution markers in order to establish the link between the infecting strain and 1 of the 10 strains handled. The antigenic formulae of the two infecting strains (serogroup:serotype:subtype) were, respectively, C:NT:P1.12 and B:2a:P1.2. Outer membrane protein profile analysis and multilocus enzyme electrophoresis unequivocally confirmed the identity of the respective strains.
Detection and nucleotide sequence analysis of the speC gene in Swedish clinical group A streptococcal isolates
Authors: Norrby-Teglund A , Holm S E , Norgren M
Published: January 2020

The production of pyrogenic exotoxins SpeA, SpeB, and SpeC by group A streptococci has been associated with streptococcal toxic shock syndrome. Several epidemiological studies using DNA hybridization and PCR analysis have been performed in attempts to correlate one or several of the toxins with streptococcal toxic shock syndrome. The results reveal great variation in the occurrence of the speA and speC genes among clinical isolates. In this study, we show that the speC gene could be detected by nested PCR in five Swedish T1M1 strains isolated from patients infected with group A streptococci as well as in three Norwegian T1M1 isolates, previously reported to lack speC as determined by dot blot hybridization. To verify the identities of the amplified products, the nucleotide sequences of the PCR fragments from one Swedish T1M1 strain and from the toxin reference strain NY5 were determined. The nucleotide sequences showed that the amplified products were speC and of allele type C2, on the basis of the nucleotides in positions 438 and 456. However, one additional base pair substitution was found in NY5 at position 147 and in the Swedish isolate at position 157, which resulted in nonsynonymous amino acid changes. Thus, these speC genes represent two new allelic variants.
Campylobacter fetus diarrhea in a Hutterite colony: epidemiological observations and typing of the causative organism
Authors: Rennie R P , Strong D , Taylor D E , Salama S M , Davidson C , Tabor H
Published: January 2020

Following a case of Campylobacter fetus sepsis and meningitis in a 4-month-old female member of a Hutterite colony, an epidemiological investigation revealed at least 18 cases of diarrhea in other members of the colony. C. fetus was isolated from 7 of 15 fecal samples submitted from affected persons. A case control study suggested that persons who worked in the abattoir were 2.03 times more likely to have had diarrhea, but none of the risk factors studied were significant. The epicurve of the outbreak was inconclusive as to the likely mode of spread of C. fetus. All of the C. fetus strains isolated from the blood of the infant and from the fecal samples were the same by biochemical and antibiotic susceptibility tests. Pulsed-field gel electrophoresis showed that all isolates produced identical restriction endonuclease patterns and differed from other nonepidemiologically related strains of C. fetus.
Comparative virulence of blood and stool isolates of Shigella sonnei
Authors: Seymour C , Crowe H M , Wilson M E
Published: January 2020

Shigellemia is rare in developed countries and might result from the emergence of unusually virulent strains. We compared systemic invasiveness markers of isolates from the blood of 3 temporally clustered patients with Shigella sonnei bacteremia in Boston with those of 11 unrelated contemporaneous strains from stools of people in New England. We found no difference between the two groups in O-chain length by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, mouse 50% lethal dose, in vivo response to iron, and susceptibility to serum, which varied from moderately susceptible to ultrasusceptible. Mean intraperitoneal 50% lethal doses of smooth form I colonies for mice were equally low (10(5.8) CFU) in both groups, and the 50% lethal doses were lowered equally further in the two groups by predosing with iron to levels useful in mouse model sepsis studies. S. sonnei bacteremia may reflect compromised host defenses, not bacterial virulence.
Involvement of human papillomavirus type 20 in epidermodysplasia verruciformis skin carcinogenesis
Authors: Yutsudo M , Tanigaki T , Kanda R , Sasagawa T , Inoue T , Jing P , Yong-Il H , Hakura A
Published: January 2020

The involvement of human papillomavirus (HPV) in skin carcinogenesis in a patient with epidermodysplasia verruciformis was studied. This patient had disseminated pityriasis versicolor-like lesions, flat warts, and a malignant skin carcinoma. HPV types 3, 17, 20 (HPV-20), and 38 were isolated and molecularly cloned from the benign skin lesions of this patient. Of these HPVs, only HPV-20 was detected in the malignant skin carcinoma. Transcripts of HPV-20 were also expressed in the carcinoma. These findings suggest that HPV-20 was involved in the skin carcinogenesis in this patient.
Ribotyping patterns and emergence of metronidazole resistance in paired clinical samples of Helicobacter pylori
Authors: Rautelin H , Tee W , Seppälä K , Kosunen T U
Published: January 2020

Metronidazole-susceptible pretreatment isolates and metronidazole-resistant posttreatment isolates of Helicobacter pylori from 11 patients before and after unsuccessful triple therapy consisting of metronidazole, amoxicillin, and colloidal bismuth subcitrate were studied. Ribotyping (rRNA gene restriction pattern analysis) of the isolates demonstrated that all patients except one had identical digest patterns for pre- and posttreatment isolates.
Epidemic spread of Shigella sonnei shigellosis and evidence for development of immunity among children attending day-care centers in a communal settlement (Kibbutz)
Authors: Lerman Y , Yavzori M , Ambar R , Sechter I , Wiener M , Cohen D
Published: January 2020

An investigation of two Shigella sonnei shigellosis outbreaks that occurred in a communal settlement indicated that the transmission of the pathogen was restricted to day-care classes and secondary infection of family members was minimal. Development of serotype-specific immunity following S. sonnei infection was observed among infected children.
Absence of hepatitis B virus (HBV) DNA in children born after exposure of their mothers to HBV during in vitro fertilization
Authors: Quint W G , Fetter W P , van Os H C , Heijtink R A
Published: January 2020

During in vitro fertilization, 22 human embryos were exposed to hepatitis B virus (HBV) in contaminated human serum present in the culture medium. All mothers experienced hepatitis B during the first trimester of pregnancy, and two had hepatitis B surface antigen and HBV DNA, as determined by PCR, at the time of delivery. No HBV DNA was found in serum or lymphocytes from the exposed 22 infants. HBV DNA was also absent from one infant at autopsy.
Comparative evaluation of chemiluminescent DNA probe assays and exoantigen tests for rapid identification of Blastomyces dermatitidis and Coccidioides immitis
Authors: Padhye A A , Smith G , Standard P G , McLaughlin D , Kaufman L
Published: January 2020

Chemiluminescent DNA probe (Accuprobe) assays developed by Gen-Probe, Inc. (San Diego, Calif.), for the rapid identification of Blastomyces dermatitidis and Coccidioides immitis were evaluated and compared with the exoantigen test by using 74 mycelial cultures of B. dermatitidis and 72 mycelial cultures of C. immitis. Seventeen isolates of the dimorphic pathogen Paracoccidioides brasiliensis were included because of their gross morphologic and antigenic relatedness to B. dermatitidis. The heterologous fungi, namely, species of Chrysosporium, which are often confused with B. dermatitidis, and species of Malbranchea, which morphologically resemble C. immitis, were tested. All 74 of the B. dermatitidis mycelial isolates were correctly identified by the Accuprobe assay for B. dermatitidis within 2 h. However, the B. dermatitidis probe cross-hybridized with rRNA extracts of 10 of the 17 P. brasiliensis isolates, misidentifying them as B. dermatitidis. All 72 of the C. immitis isolates were identified correctly with the C. immitis probe. None of the other heterologous fungi belonging to Chrysosporium spp., Malbranchea spp., Onychocola canadensis, and Geotrichum sp. were cross-reactive with the B. dermatitidis and C. immitis probes. The exoantigen tests specifically identified 74 B. dermatitidis, 72 C. immitis, and 17 P. brasiliensis isolates within 48 to 72 h and differentiated the related heterologous fungi from the three dimorphic fungal pathogens.
Mixed-effect models for predicting microbial interactions in the vaginal ecosystem
Authors: Ross R A , Lee M L , Delaney M L , Onderdonk A B
Published: January 2020

Three statistical models that predict microbial interactions within the vaginal environment are presented. A large data set was assembled from in vivo studies describing the healthy vaginal environment, and the data set was analyzed to determine whether statistical models which would accurately predict the interactions of the microflora in this environment could be formulated. During assembly of the data set, two new variables were defined and were added to the data set, that is, cycle (sequence of menstrual cycle) and flow stage (subdivision of cycle determined by day of menstrual cycle). Concentrations of total aerobic (includes facultative) bacteria, total anaerobic bacteria, and a Corynebacterium sp. were identified by correlation analysis as variables with significant predictors. By using a regression method with a backward elimination procedure, significant predictors of these outcome variables were identified as the concentrations of Lactobacillus spp., anaerobic Streptococcus spp., and Staphylococcus spp., respectively. For all three outcome variables, pH and flow stage were also identified as significant independent variables. Because some of the data in the data set are repeated measurements for a subject, a mixed-effect model that accounts for the random effects of repeated-measurement data fit best the data set for predicting interactions between various members of the vaginal microflora. The predictive accuracies of the three models were tested by a comparison of model-predicted outcome-variable values with actual mean in vivo outcome-variable values. From these results, we concluded that it is possible to accurately predict vaginal microflora interactions by using a mixed-effect modeling system. The application of this type of modeling strategy and its future use are discussed.
Hybridization of strains of Escherichia coli O157 with probes derived from the eaeA gene of enteropathogenic E. coli and the eaeA homolog from a Vero cytotoxin-producing strain of E. coli O157
Authors: Willshaw G A , Scotland S M , Smith H R , Cheasty T , Thomas A , Rowe B
Published: January 2020

A total of 375 Escherichia coli O157 strains were tested by colony hybridization with the eae probe from the central portion of the eaeA gene of the classical enteropathogenic E. coli strain E2348/69. They were also tested with a probe, eaeO157, from the C-terminal end of the eae gene homolog from a Vero cytotoxin (VT)-producing strain of E. coli (VTEC) of serotype O157:H7. Both probes hybridized with all 246 O157:H7 or H- VTEC strains tested. The majority were from human infections, and the remainder were from cattle. A further 10 strains (H7 or H-) hybridized with both eae and eaeO157 sequences but not with VT probes. They resembled O157 VTEC and were probably naturally occurring derivatives that had lost VT genes. The remaining 119 strains of O157 were from human, animal, and food sources and belonged to 16 H types other than H7 or were H-. They were VT negative and differed in their properties from O157 VTEC: generally they fermented sorbitol in 1 day, produced beta-glucuronidase, and could not be phage typed by the scheme for O157 VTEC. The eae probe but not the eaeO157 sequence hybridized with 18 H8 or H39 strains, predominantly from human diarrhea. The remaining 101 VT-negative strains hybridized with neither probe. However, 16 strains of O157:H45 hybridized with a probe for diffusely adherent E. coli and attached to HEp-2 cells in a diffuse pattern. Serogroup O157 comprises strains with heterogeneous properties. The eaeO157 probe is a valuable addition to the VT probes used to differentiate O157 strains.
Comparison of four commercially available rapid enzyme immunoassays with cytotoxin assay for detection of Clostridium difficile toxin(s) from stool specimens
Authors: Merz C S , Kramer C , Forman M , Gluck L , Mills K , Senft K , Steiman I , Wallace N , Charache P
Published: January 2020

Rapid (2.5- to 3.5-h) enzyme immunoassays (EIAs) for the detection of Clostridium difficile toxins have been developed. We report the results of simultaneous testing of 700 fresh stool specimens by the tissue culture cytotoxin assay and four EIAs (Bartels Prima System C. difficile Toxin A EIA, Cambridge Biotech Cytoclone A+B EIA, Meridian Diagnostics Premier C. difficile Toxin A EIA, and TechLab C. difficile Tox-A Test EIA). In cases of disagreement, culturing for toxigenic C. difficile was performed. A total of 61 (8.7%) specimens from 46 patients were positive for C. difficile toxin. The sensitivity of the cytotoxin assay was 87%, and that of culture was 93%. In comparison with the cytotoxin assay results, the sensitivity and specificity of the EIAs were as follows: Bartels, 87 and 96%; Cambridge, 89 and 99%; Meridian, 87 and 98%; and TechLab, 87 and 95%, respectively. In comparison with the cytotoxin assay plus toxigenic culture results, the sensitivity and specificity of the EIAs were as follows: Bartels, 84 and 97%; Cambridge, 85 and 99%; Meridian, 79 and 98%; and TechLab, 80 and 96%, respectively. The EIAs varied in positive predictive values (PPVs). A high PPV was seen with the Cambridge EIA (96%); lower PPVs were seen with the TechLab (64%), Bartels (72%), and Meridian (80%) EIAs because of high false-positive rates. The negative predictive values (98 to 99%) were excellent with all EIAs. Results were indeterminant with 0.3% of the samples by the Meridian EIA and 3% by all the other EIAs. Although the EIAs were less sensitive than the cytotoxin assay, they provide same-day results and may be useful in laboratories without tissue culture facilities.
Characterization of endemic Shigella flexneri strains in Somalia: antimicrobial resistance, plasmid profiles, and serotype correlation
Authors: Casalino M , Nicoletti M , Salvia A , Colonna B , Pazzani C , Calconi A , Mohamud K A , Maimone F
Published: January 2020

One hundred twelve Shigella flexneri strain isolated from children with diarrheal disease in Somalia in 1983, 1984, 1988, and 1989 were analyzed for serotype, plasmid profile, and genetic location of antimicrobial resistance determinants. The prevalent serotypes were 4 (46% of the isolates), 1b (16%), 2a (16%), 3a (12%), and 6 (8%). Each serotype was associated with a characteristic predominant plasmid profile, whereas no specific correlation between antimicrobial resistance patterns and single serotypes was found. All but three of the strains were resistant at least to ampicillin, chloramphenicol, spectinomycin, and tetracycline. Of these resistant strains, 41 were resistant to sulfonamide and streptomycin and 14 were resistant to trimethoprim or trimethoprim and kanamycin. The genes for resistance to ampicillin, chloramphenicol, spectinomycin, and tetracycline formed a linkage group located on the chromosome of the strains of all serotypes. The genes for resistance to sulfonamide and streptomycin were located on a 6.3-kb plasmid in strains of serotypes 1b, 2a, and 4. Conjugative trimethoprim or trimethoprim and kanamycin resistance plasmids with lengths of 80 to 110 kb were present in strains of serotypes 1b, 2a, 3a, and 4. The systematic presence of a chromosomal component in this uncommon genetic plasmid-chromosome configuration may play a role in the emergence of increased genetic stability of resistance patterns in S. flexneri.
Comparison of PCR-based restriction length polymorphism analysis of urease genes with rRNA gene profiling for monitoring Helicobacter pylori infections in patients on triple therapy
Authors: Owen R J , Bickley J , Hurtado A , Fraser A , Pounder R E
Published: January 2020

Multiple isolates of Helicobacter pylori from antral biopsies of nine patients were examined by DNA fingerprinting. Analysis of rRNA gene patterns and HaeIII restriction fragment length polymorphism of PCR-amplified urease genes were compared and used to study colonization before and after failed triple therapy. H. pylori isolates from a single biopsy shared the same HaeIII DNA fingerprint regardless of the isolation method (plate or broth). DNA pattern types of paired strains of H. pylori were distinct between patients and were not grossly affected by treatment except for one patient with an altered strain type. H. pylori infections were generally associated with several subpopulations of strains, evident from the subtypic variation before and after treatment, detectable by both DNA fingerprinting methods. The urease gene patterns also provided evidence that some cultures of H. pylori probably contained a mixture of genomic subtypes. The study suggests that triple therapy has the effect either of inducing minor genomic variations or of changing the proportions of different subtypes of H. pylori. It was concluded that urease gene profiling provides a simple yet reliable method of establishing whether treatment failures are attributable to incomplete eradication of H. pylori.
Development of species-specific enzyme-linked immunosorbent assay for diagnosis of Johne's disease in cattle
Authors: Vannuffel P , Gilot P , Limbourg B , Naerhuyzen B , Dieterich C , Coene M , Machtelinckx L , Cocito C
Published: January 2020

The previously described (M. De Kesel, P. Gilot, M.-C. Misonne, M. Coene, and C. Cocito, J. Clin. Microbiol., 31:947-954, 1993) a362 recombinant polypeptide of Mycobacterium paratuberculosis was used as reagent for an enzyme-linked immunosorbent assay (ELISA). This ELISA, which is endowed with species specificity with respect to the other mycobacteria, was applied to the analysis of bovine paratuberculosis (Johne's disease), an endemic mycobacteriosis of cattle caused by M. paratuberculosis. The distribution of anti-a362 antibodies in the cattle population was analyzed by a computer program (mixture population model) to determine a cutoff value for the test. The prevalence of a362 seropositivity in the Belgian bovine population was estimated to be 12%. The sensitivity of the a362 assay was 70%, as determined with reference sera from the U.S. National Repository of Paratuberculosis Specimens. Some 40% of the animals in the herds with paratuberculosis analyzed were found to be positive by the a362 assay. The latter proved to be 95% specific with respect to both healthy and tuberculous cattle.
Direct detection of Vibrio cholerae in stool samples
Authors: Varela P , Pollevick G D , Rivas M , Chinen I , Binsztein N , Frasch A C , Ugalde R A
Published: January 2020

A direct method to detect Vibrio cholerae in stool samples was developed by using a PCR procedure that did not require a DNA purification step. Dilution (1/100) of stool samples prevented inhibition of the reaction by contaminants, and two consecutive PCRs, the second one with a nested primer, achieved the desired sensitivity. Comparison of the results obtained from stool swab samples processed by the two-step PCR and by an enzyme-linked immunosorbent assay using GM1 as the capture molecule showed that the former is more sensitive and gave positive results even when V. cholerae was not culturable or dead.
Detection of antibodies to human immunodeficiency virus in vaginal secretions by immunoglobulin G antibody capture enzyme-linked immunosorbent assay: application to detection of seminal antibodies after sexual intercourse
Authors: Bélec L , Grésenguet G , Dragon M A , Meillet D , Pillot J
Published: January 2020

In order to evaluate a commercial immunoglobulin G (IgG) antibody capture enzyme-linked immunoassay (ELISA) (Wellcozyme HIV1 + 2 Gacelisa; Murex Diagnostics Limited, Dartford, United Kingdom) for the detection of antibodies to human immunodeficiency virus (HIV) in vaginal secretion samples (VS) from HIV-seropositive and -seronegative women, serum samples (S) and VS were obtained from 129 African women living in the Central African Republic, a country of high HIV prevalence. Sera were tested for HIV by routine second-generation ELISA with confirmatory Western blot (immunoblot) (WB). By the Gacelisa IgG immuno-capture assay, 45 VS were positive and 84 were negative, whereas by WB, 44 VS were confirmed positive and 85 were confirmed negative. Considering WB as a reference, the IgG immunocapture assay in VS was 97.7% sensitive (43 of 44 positive samples) and 97.6% specific (83 of 85 negative samples). Of 42 HIV-seropositive women, 41 (97.6%) had S and VS that both were HIV positive (S+ VS+), and of 87 HIV-seronegative women, 83 (95.4%) had S and VS that both were HIV negative (S- VS-). Five women had discordant results for S and VS. One (S+ VS-) possibly had a false-negative VS result. Two (S- VS+) had similar indeterminate patterns for S and VS in WB. Two (S- VS+) had a typical HIV-positive pattern on WB of VS, whereas S results in WB were indeterminate in one case and negative in the other case; for both women, detection of prostatic acid-phosphatase was positive in VS, strongly suggesting recent sexual intercourse with an HIV-positive man. Because all HIV-infected men have detectable IgG antibodies to HIV in the seminal fluid, an HIV-seronegative rape victim with HIV-positive VS (S- VS+) should receive short-term antiviral therapy to prevent possible HIV transmission.