bluuebunny/crossref_metadata_2025 huggingface dataset:
Molecular typing of Salmonella typhi strains from Dhaka (Bangladesh) and development of DNA probes identifying plasmid-encoded multidrug-resistant isolates
Authors: Hermans P W , Saha S K , van Leeuwen W J , Verbrugh H A , van Belkum A , Goessens W H
Published: January 2020

Seventy-eight Salmonella typhi strains isolated in 1994 and 1995 from patients living in Dhaka, Bangladesh, were subjected to phage typing, ribotyping, IS200 fingerprinting, and PCR fingerprinting. The collection displayed a high degree of genetic homogeneity, because restricted numbers of phage types and DNA fingerprints were observed. A significant number of the S. typhi strains (67%) were demonstrated to be multiple drug resistant (MDR). The vast majority of the MDR strains were resistant to chloramphenicol, ampicillin, trimethoprim, streptomycin, sulfamethoxazole, and tetracycline (R type CATmSSuT), a resistance phenotype that has also frequently been observed in India. Only two strains displayed a distinct MDR phenotype, R type AT-mSSuT. Pulsed-field gel electrophoresis demonstrated the presence of large plasmids exclusively in the MDR strains of both R types. The plasmids present in the S. typhi strains of R type CATmSSuT could be conjugated to Escherichia coli and resulted in the complete transfer of the MDR phenotype. PCR fingerprinting allowed discrimination of MDR and susceptible strains. The DNA fragments enabling discrimination of MDR and susceptible S. typhi strains by PCR were useful genetic markers for identifying MDR encoded by large plasmids of the H1 incompatibility group.
Serological diagnosis of bovine neosporosis by Neospora caninum monoclonal antibody-based competitive inhibition enzyme-linked immunosorbent assay
Authors: Baszler T V , Knowles D P , Dubey J P , Gay J M , Mathison B A , McElwain T F
Published: January 2020

Neospora caninum, a protozoan parasite closely related to Toxoplasma gondii, causes abortion and congenital infection in cattle. To investigate specific methods of antemortem diagnosis, the antibody responses of infected cows were evaluated by immunoblot assay and competitive inhibition enzyme-linked immunosorbent assay (CI-ELISA) by using a monoclonal antibody (MAb), MAb 4A4-2, against N. caninum tachyzoites. MAb 4A4-2 bound diffusely to the exterior surface of N. caninum tachyzoites and recognized a single 65-kDa band in immunoblots. MAb 4A4-2 was unreactive to antigens of two closely related apicomplexan protozoa, Toxoplasma gondii and Sarcocystis cruzi. Binding of MAb 4A4-2 was inhibited by mild periodate treatment of N. caninum antigen, demonstrating the carbohydrate nature of the epitope. Immunoblot analysis of N. caninum tachyzoite antigens with sera from cows with confirmed Neospora-induced abortion revealed at minimum 14 major antigens ranging from 11 to 175 kDa. Although the recognized antigens varied from cow to cow, antigens of 116, 65, and 25 kDa were detected in all cows with abortion confirmed to be caused by N. caninum. The binding of MAb 4A4-2 to N. caninum tachyzoite antigen was consistently inhibited by sera from Neospora-infected cows in a CI-ELISA format and was not inhibited by sera from Neospora antibody-negative cows. Furthermore, sera from cattle experimentally infected with T. gondii, S. cruzi, Sarcocystis hominis, or Sarcocystis hirsuta, which had cross-reactive antibodies recognizing multiple N. caninum antigens by immunoblot assay, did not inhibit binding of MAb 4A4-2 in the CI-ELISA. Thus, MAb 4A4-2 binds a carbohydrate epitope on a single N. caninum tachyzoite surface antigen that is recognized consistently and specifically by Neospora-infected cattle.
Murine model of invasive pulmonary aspergillosis following an earlier stage, noninvasive Aspergillus infection
Authors: Nawada R , Amitani R , Tanaka E , Niimi A , Suzuki K , Murayama T , Kuze F
Published: January 2020

Aspergillus spp. occasionally cause invasive pulmonary aspergillosis following noninvasive infection in patients with underlying bronchopulmonary disorders regardless of their systemic immunological conditions. We developed a murine model of invasive pulmonary aspergillosis following an earlier stage, noninvasive Aspergillus infection. BALB/c mice were inoculated intratracheally with agarose beads containing Aspergillus fumigatus conidia. Two weeks after inoculation, half of the mice were immunosuppressed with cortisone acetate. During a 4-week observation period, the survival rate of infected immunosuppressed mice was significantly lower (P < 0.01) than that of infected nonimmunosuppressed mice. The number of CFU in the lungs gradually decreased in the nonimmunosuppressed mice, whereas a time-related significant increase (P < 0.05) of CFU was demonstrated in the immunosuppressed mice. In the lungs of the nonimmunosuppressed mice, there was marked accumulation of neutrophils, lymphocytes, and macrophages (in this order) around the agarose beads in the bronchi. Aspergillus hyphae were surrounded by the inflammatory cells and did not invade the lung parenchyma. In contrast, in the immunosuppressed mice, Aspergillus hyphae proliferated markedly and invaded the lung parenchyma after immunosuppression. In this model, the two-dimensional extents of the lesions were also evaluated with an image-processing system. Time-related increase of the area of peribronchial necrotic lesions was significant (P < 0.05) after immunosuppression. This model should therefore be useful for investigating the pathophysiology of noninvasive Aspergillus infection and invasive pulmonary aspergillosis and also for clarifying the mechanism of conversion to the invasive disease from the noninvasive stage.
Serological diagnosis of ovine enzootic abortion by comparative inclusion immunofluorescence assay, recombinant lipopolysaccharide enzyme-linked immunosorbent assay, and complement fixation test
Authors: Griffiths P C , Plater J M , Horigan M W , Rose M P , Venables C , Dawson M
Published: January 2020

Since the 1950s, serological diagnosis of ovine enzootic abortion (OEA), caused by strains of Chlamydia psittaci, has been based mainly on the complement fixation test (CFT), which is neither particularly sensitive nor specific since antibodies to other chlamydial and enterobacterial pathogens may be detected. In this study. a recombinant enzyme-linked immunosorbent assay (rELISA) (medac, Hamburg, Germany), based on a unique chlamydial genus-specific epitope of Chlamydia trachomatis L2 lipopolysaccharide, was evaluated for sensitivity and specificity as a primary screening assay for OEA by comparison with the CFT. A comparative inclusion immunofluorescence assay (IFA), in which antibody titers to C. psittaci and Chlamydia pecorum were examined, was used as the reference test for 573 serum samples from four flocks. Reactivity to C. pecorum was measured since inapparent intestinal infections by C. pecorum are believed to be common in British flocks. In detecting positive sera from an abortion-affected flock, in which a C. pecorum infection was also suggested by IFA, the rELISA outperformed the CFT with significant evidence for increased sensitivity (P = 0.003). In two flocks in which C. pecorum infections alone were suggested by IFA, the rELISA and CFT were prone to detect low levels of false-positive results, but the values were not significant. The rELISA provided results in one flock in which sera that were anticomplementary could not be resolved by the CFT. In another flock in which abortion had not occurred but infection by both chlamydial species was suspected, no significant difference was found between the sensitivities of the rELISA and CFT. The rELISA could not differentiate ovine C. psittaci and C. pecorum infections but was shown to be a more sensitive primary screening test for OEA than was the CFT, particularly where abortion had occurred and even when antibodies due to additional inapparent infection(s) by C. pecorum were present.
Subspecies typing of Vibrio parahaemolyticus by pulsed-field gel electrophoresis
Authors: Wong H C , Lu K T , Pan T M , Lee C L , Shih D Y
Published: January 2020

Vibrio parahaemolyticus is one of the most important food-borne pathogens in Taiwan, Japan, and other costal regions. We report on the development of a pulsed-field gel electrophoresis (PFGE) method for the molecular typing of this pathogen. Genomic DNA was digested with SfiI, and the fragments were resolved on 1% agarose with a contour-clamped homogeneous electric field apparatus set at 190 V and a pulse time of 3 to 80 s. A total of 130 selected isolates obtained from outbreaks during 1993 and 1994 on Taiwan were also characterized by this PFGE method. These isolates were grouped into 14 PFGE types which consisted on one to six patterns, and a total of 39 patterns were identified. Most of these domestic clinical isolates could be clustered into several major types (types A, B, C, and G). These major types showed relatively low degrees of similarity to several foreign strains and other domestic but environmental strains. Strain CCRC12863, which originated from Japan, was close to the group consisting of F, G, and H PFGE types, suggesting a clonal relationship between this Japanese strain and other domestic isolates.
Bicentric evaluation of Access Toxo immunoglobulin M (IgM) and IgG assays and IMx toxo IgM and IgG assays and comparison with Platelia Toxo IgM and IgG assays
Authors: Decoster A , Lecolier B
Published: January 2020

The recent Access immunoanalysis system (Sanofi Diagnostics Pasteur) for the serological diagnosis of toxoplasmosis was compared with the Abbott Toxo IMx EIA system, taking the Platelia Toxo immunoglobulin G (IgG) and Platelia Toxo IgM systems as references and using as confirmation methods an indirect fluorescence assay or a dye test for IgG and an immunosorbent agglutination assay (ISAGA) for IgM. A total of 1,461 serum samples were studied, of which 128 were collected from 42 recently seroconverted patients. Sensitivity and specificity rates of the Access system were 97.7 and 99.5%, respectively, for IgM and 98.6 and 100%, respectively, for IgG. Sensitivity and specificity rates of the Abbott IMx EIA system were 91 and 100%, respectively, for IgM and 92.5 and 100%, respectively, for IgG. The Access Toxo IgG and IgM EIA systems were found to be more sensitive than the Abbott Toxo IgG and IgM IMx EIA systems.
Molecular microbiological investigation of an outbreak of hemolytic-uremic syndrome caused by dry fermented sausage contaminated with Shiga-like toxin-producing Escherichia coli
Authors: Paton A W , Ratcliff R M , Doyle R M , Seymour-Murray J , Davos D , Lanser J A , Paton J C
Published: January 2020

Shiga-like toxin-producing Escherichia coli (SLTEC) strains are a diverse group of organisms which are known to cause diarrhea and hemorrhagic colitis in humans. This can lead to potentially fatal systemic sequelae, such as hemolytic-uremic syndrome (HUS). Strains belonging to more than 100 different O:H serotypes have been associated with severe SLTEC disease in humans, of which only O157 strains (which are uncommon in Australia) have a distinguishable cultural characteristic (sorbitol negative). During an outbreak of HUS in Adelaide, South Australia, a sensitive PCR assay specific for Shiga-like toxin genes (slt) was used to test cultures of feces and suspected foods. This enabled rapid confirmation of infection and identified a locally produced dry fermented sausage (mettwurst) as the source of infection. Cultures of feces from 19 of 21 HUS patients and 7 of 8 mettwurst samples collected from their homes were PCR positive for slt-I and slt-II genes. SLTEC isolates belonging to serotype O111:H- was subsequently isolated from 16 patients and 4 mettwurst samples. Subsequent restriction fragment length polymorphism analysis of chromosomal DNA from these isolates with slt-specific probes indicated that at least three different O111:H- genotypes were associated with the outbreak. Pulsed-field gel electrophoresis of genomic DNA restricted with XbaI showed that two of these restriction fragment length polymorphism types were closely related, but the third was quite distinct. However, SLTEC strains of other serotypes, including O157:H-, were also isolated from some of the HUS patients.
Brain abscess caused by Schizophyllum commune: an emerging basidiomycete pathogen
Authors: Rihs J D , Padhye A A , Good C B
Published: January 2020

Despite the worldwide distribution and prevalence of Schizophyllum commune, an emerging basidiomycetous pathogen, human infections occur only rarely. We describe the first well-documented pulmonary infection caused by S. commune which disseminated to the brain of a 58-year-old patient undergoing empiric corticosteroid therapy. Magnetic resonance imaging scans revealed ring-enhancing masses. Histologic examination of biopsy tissue from lungs and brain showed hyaline, septate, branched hyphae with clamp connections. Cultures of the lung tissue grew S. commune, which produced numerous, characteristic flabelliform and medusoid fruiting bodies on Czapek's agar. The isolate was susceptible to amphotericin B (MIC, < 0.03 microgram/ml) and fluconazole (MIC, 8 micrograms/ml). Despite treatment with antifungal and antibacterial agents, the patient developed progressive pulmonary failure and bacterial sepsis and died.
Testing of Mycobacterium tuberculosis susceptibility to ethambutol, isoniazid, rifampin, and streptomycin by using Etest
Authors: Wanger A , Mills K
Published: January 2020

Etest (AB BIODISK, Solna, Sweden) is a precise MIC method and the practical method of choice for the susceptibility testing of many fastidious organisms, including rapidly growing mycobacteria. Methods recommended by the National Committee for Clinical Laboratory Standards for the susceptibility testing of Mycobacterium tuberculosis include the Bactec (Becton Dickinson, Sparks, Md.) broth and agar proportion methods. A comparison of Etest with the Bactec broth method for testing the susceptibility of M. tuberculosis to four first-line antituberculous agents demonstrated equivalent interpretive results for 100% of the isolates tested. Agreements with agar proportion MICs, within +/-2 log2 dilutions, were 90, 93, 100, and 94% for ethambutol, isoniazid, rifampin, and streptomycin, respectively. Etest MICs were easily read within 5 to 10 days of inoculation. Preparation of the inoculum with a turbidity equivalent to a McFarland 3.0 standard prepared from growth on an agar surface and with a broth with a Bactec growth index of > 999 yielded equivalent results. Clinical isolates for which the MICs were reproducible were also identified as possible quality control strains. The Etest method appears to be an alternative method for testing the susceptibility of M. tuberculosis isolates to the four most commonly used therapeutic agents.
Species-specific identification of Candida krusei by hybridization with the CkF1,2 DNA probe
Authors: Carlotti A , Couble A , Domingo J , Miroy K , Villard J
Published: January 2020

The species specificity of the Candida krusei DNA fingerprinting probe CkF1,2 has been investigated. A total of 149 pathogenic and nonpathogenic fungal and bacterial DNAs were screened with CkF1,2. The probe was cold labeled with peroxidase, and its specificity was assessed by using Southern blot, dot blot, and colony blot hybridization. Its sensitivity was determined by dot blot hybridization. The CkF1,2 probe proved to be species specific. It hybridized with DNA for the 112 C. krusei strains studied, whereas it failed to hybridize under low-stringency conditions to 37 DNAs from 27 different yeast species, including Candida albicans, Candida glabrata, Candida norvegensis, Candida inconspicua, Candida tropicalis, Candida valida, Candida zeylanoides, and Yarrowia lipolytica, as well as DNAs from the filamentous fungi and bacteria tested. However, CkF1,2 hybridized strongly with DNA of the yeast species Issatchenkia orientalis, the putative ascogenous perfect state of C. krusei. Amounts as small as 60 to 120 ng of C. krusei target DNA were detected by dot blot hybridization with CkF1,2. It permitted the direct screening of colony blots for early identification. The CkF1,2 probe has potential value as a diagnostic reagent for identifying C. krusei.
Persistence, replacement, and microevolution of Cryptococcus neoformans strains in recurrent meningitis in AIDS patients
Authors: Sullivan D , Haynes K , Moran G , Shanley D , Coleman D
Published: January 2020

Six separate human immunodeficiency virus-positive patients with cryptococcal meningitis were each found to have been infected with a unique strain of Cryptococcus neoformans on the basis of genomic DNA finger-printing analysis with the microsatellite sequence-containing oligonucleotide probe (GGAT)4 and by random amplification of polymorphic DNA. Two patients (A and B) experienced a recurrent episode of infection. Between 12 and 16 single-colony isolates recovered from primary isolation media (> 50% of C. neoformans colonies recovered) from cerebrospinal fluid specimens were fingerprinted from both patients during each episode. The fingerprints of both isolate collections from patient B were very similar, although minor polymorphisms were evident in both sets of profiles. The fingerprints of the isolate collection from the initial episode of infection in patient A were also identical to each other, apart from minor polymorphisms, but they were clearly different from the corresponding profiles of the isolate collection from the recurrent episode, the latter of which were completely identical, apart from minor polymorphisms in a single isolate. Furthermore, prolonged storage and in vitro subculture of the isolates did not alter the fingerprint profiles. These results provided convincing evidence that patients A and B were each infected with a single C. neoformans strain during each episode of infection and that in patient B, the same strain persisted and caused both episodes, while in patient A, a different strain was responsible for each episode. The prevalence of polymorphisms in multiple single-colony isolates from both patients also suggested that C. neoformans populations may undergo microevolution.
Emergence of teicoplanin-resistant coagulase-negative staphylococci
Authors: Cercenado E , García-Leoni M E , Díaz M D , Sánchez-Carrillo C , Catalán P , De Quirós J C , Bouza E
Published: January 2020

Over a period of 5 years we have recovered 32 clinical isolates of coagulase-negative staphylococci (CoNS) exhibiting either decreased levels of susceptibility or true resistance to teicoplanin (MICs, 16 to 128 micrograms/ml); these isolates make up 0.55% of the total CoNS isolated by us. Twenty-nine of the strains were also methicillin resistant, and all were susceptible to vancomycin. Fourteen of the strains were Staphylococcus epidermidis, fourteen were Staphylococcus haemolyticus, and four were Staphylococcus hominis. In one case, a strain of S. haemolyticus was isolated with a vancomycin-resistant, teicoplanin-resistant Enterococcus faecalis strain. All strains were nosocomially acquired and were isolated from 17 different wards. Teicoplanin resistance occurred as a sporadic phenomenon, and none of the isolates were epidemiologically related. The isolates were from 30 patients, 13 of whom presented with true infections (43%). Five (38%) of the 13 patients with true infections had been previously treated with vancomycin. None of the infected patients were previously treated with teicoplanin. The in vivo development of resistance to teicoplanin among CoNS strains limits the therapy of infections by these microorganisms. There is a need for surveillance of nosocomial isolates of CoNS to determine resistance to glycopeptides.
Contemporary testing for enteric pathogens: the potential for cost, time, and health care savings
Authors: Morris A J , Murray P R , Reller L B
Published: January 2020

We sent a questionnaire to 79 clinical microbiology laboratories seeking information on contemporary practices when investigating for bacterial and protozoan enteric pathogens. Data from the 67 respondents (response rate of 85%) showed that a minority of laboratories (40% for stool culture and 45% for ova and parasite [O&P] examinations) had restrictions for testing in place and that fewer laboratories (24% for stool culture and 19% for O&P examinations) rejected specimens from patients who had been in the hospital for > 3 days. Using two estimates, 15 and 40%, for the proportion of all specimens received from patients in the hospital for > 3 days, we calculated savings for the average hospital in this survey. Reagent savings of $4,000 to $10,000 and time savings of 274 to 731 h per year might have been realized. Moreover, between $26,000 and $71,000 in patient charges could have been prevented. On the basis of this survey, wider application of rejection criteria when testing for enteric pathogens appears possible. If implemented, savings to the nation's health care system could be between $27 and $73 million a year.
Evaluation of L-pyrrolidonyl peptidase paper strip test for differentiation of members of the family Enterobacteriaceae, particularly Salmonella spp
Authors: Inoue K , Miki K , Tamura K , Sakazaki R
Published: January 2020

The L-pyrrolidonyl peptidase activities of 1,033 strains of the family Enterobacteriaceae were investigated by the paper strip method to evaluate their usefulness for screening those organisms, especially Salmonella cultures. We also evaluated the usefulness of indole and tryptophan deaminase paper strip tests as supplements to the L-pyrrolidonyl peptidase test for the rapid identification of Salmonella cultures. The paper strip tests are simple, and the results are obtainable within 10 min.
Quantitative assay of PCR-amplified hepatitis B virus DNA using a peroxidase-labelled DNA probe and enhanced chemiluminescence
Authors: Erhardt A , Schaefer S , Athanassiou N , Kann M , Gerlich W H
Published: January 2020

We have developed a sensitive and quantitative assay for hepatitis B virus (HBV) DNA in serum or plasma in which PCR and then microtiter hybridization analysis are used. Assay of HBV DNA in serum or plasma is important for demonstrating viral replication, indicating and monitoring antiviral therapy, determining the infectivities of virus carriers, and ensuring the safety of blood products. Under optimum conditions PCR can amplify one HBV DNA molecule to 10(8) copies, but detection of this amount of DNA still requires hybridization with labelled probes or a nested PCR. We labelled one strand of the PCR product with a biotinylated primer. The double-stranded amplicon was incubated in streptavidin-coated microplate wells. The nonlabelled strand was removed after denaturation of the double-stranded DNA with alkali, and the bound strand was hybridized with a peroxidase-coupled single-stranded probe. The amount of bound peroxidase was measured in a luminometer. Four picograms of amplicon was detectable in this system, whereas conventional ethidium bromide staining requires a 1,000 times higher amplicon concentration. The performance of the new assay was compared with those of nested PCR and a PCR system that uses a digoxigenin label, hybridization to a solid-phase adsorbed probe, and colorimetric detection. The chemiluminescence assay was found to be almost as sensitive as nested PCR and approximately five times more sensitive than the colorimetric test.
Chromosomal DNA restriction endonuclease digestion patterns of beta-lactamase-producing Enterococcus faecalis isolates collected from a single hospital over a 7-year period
Authors: Seetulsingh P S , Tomayko J F , Coudron P E , Markowitz S M , Skinner C , Singh K V , Murray B E
Published: January 2020

Twenty-three beta-lactamase (beta-lac)-producing, highly gentamicin-resistant Enterococcus faecalis isolates collected over a 7-year period from the same hospital were examined by pulsed-field gel electrophoresis of SmaI-digested genomic DNA. The beta-lac+ isolates appeared to form a single clonal group, which had been previously designated the mid-Atlantic pattern. Eleven variations of the mid-Atlantic clone, differing by one to six bands, were identified; some of the changes were likely due to plasmid bands. However, a number of isolates had indistinguishable patterns, including some recovered over a 4-year period. There was a surprising lack of movement of the beta-lac determinant to other strains, although this trait was transferable in vitro by conjugation. We conclude that a single clone (the mid-Atlantic clone) of beta-lac+ E. faecalis has remained endemic in this hospital for at least 7 years. The reason(s) for the apparent lack of spread to other strains of E. faecalis is unknown.
Differentiation of Chlamydia spp. by sequence determination and restriction endonuclease cleavage of RNase P RNA genes
Authors: Herrmann B , Winqvist O , Mattsson J G , Kirsebom L A
Published: January 2020

The amplification of DNA from Chlamydia trachomatis by PCR with degenerated primers yielded a 345-bp fragment of the putative RNase P RNA gene. From the deduced DNA sequence of this gene in C. trachomatis, a modified primer pair was designed. The primer pair was subsequently used to obtain the corresponding gene products from Chlamydia pneumoniae and Chlamydia psittaci. Sequence comparisons revealed similarities of 76.6% between C. trachomatis and C. pneumoniae, 79.5% between C. trachomatis and C. psittaci, and 84.7% between C. pneumoniae and C. psittaci. Furthermore, the three species were differentiated by fragment length polymorphism analysis after restriction enzyme cleavage of the PCR products. Sequence variations among 14 serotypes of C. trachomatis were confined to one purine base substitution in the putative RNase P RNA gene of lymphogranuloma venereum strains L1 to L3. Complete sequence similarity was found for nine strains of C. pneumoniae of different geographic origins. Taken together, our results indicate a possibility of the general application of this method in clinical bacteriology. Analysis of the secondary structures of the putative RNase P RNA genes from the different Chlamydia species suggested that a novel structural element in the domain of RNase P RNA is involved in base pairing with the 3'-terminal CCA motif of a tRNA precursor. This structure has not previously been found among RNase P RNAs of members of the division Bacteria.
Experimental transmission of Bartonella henselae by the cat flea
Authors: Chomel B B , Kasten R W , Floyd-Hawkins K , Chi B , Yamamoto K , Roberts-Wilson J , Gurfield A N , Abbott R C , Pedersen N C , Koehler J E
Published: January 2020

Bartonella henselae is an emerging bacterial pathogen, causing cat scratch disease and bacillary angiomatosis. Cats bacteremic with B. henselae constitute a large reservoir from which humans become infected. Prevention of human infection depends on elucidation of the natural history and means of feline infection. We studied 47 cattery cats in a private home for 12 months to determine the longitudinal prevalence of B. henselae bacteremia, the prevalence of B. henselae in the fleas infesting these cats, and whether B. henselae is transmitted experimentally to cats via fleas. Vector-mediated transmission of B.henselae isolates was evaluated by removing fleas from the naturally bacteremic, flea-infested cattery cats and transferring these fleas to specific-pathogen-free (SPF) kittens housed in a controlled, arthropod-free University Animal Facility. B. henselae bacteremia was detected in 89% of the 47 naturally infected cattery cats. A total of 132 fleas were removed from cats whose blood was simultaneously cultured during different seasons and were tested individually for the presence of B. henselae DNA by PCR. B. henselae DNA was detected in 34% of 132 fleas, with seasonal variation, but without an association between the presence or the level of bacteremia in the corresponding cat. Cat fleas removed from bacteremic cattery cats transmitted B. henselae to five SPF kittens in two separate experiments; however, control SPF kittens housed with highly bacteremic kittens in the absence of fleas did not become infected. These data demonstrate that the cat flea readily transmits B. henselae to cats. Control of feline infestation with this arthropod vector may provide an important strategy for the prevention of infection of both humans and cats.
Preparation of mycobacterial DNA from blood culture fluids by simple alkali wash and heat lysis method for PCR detection
Authors: Kulski J K , Pryce T
Published: January 2020

A sodium iodide-isopropanol (NI) method was compared with an alkali wash and heat lysis (AH) procedure for the preparation and extraction of DNA from BACTEC 13A blood culture fluid samples from AIDS patients for use in a PCR for the detection and identification of mycobacteria. The sensitivity and efficiency of the DNA extraction methods were assessed by a multiplex PCR which detected the members of the genus Mycobacterium and differentiated between M. intracellulare, M. tuberculosis, and M. avium isolates with a limit of detection of between 0.28 pg (67 cells) and 120 pg (28,571 cells) of standard mycobacterial DNA. The PCR amplified mycobacterial DNA prepared by the AH procedure from 40 acid-fast bacillus-positive blood cultures with growth index values of > 20 U but not from 48 blood cultures with growth index values of < 21 U. The AH method was about 10 times more sensitive than the NI method for extracting DNA from 13 acid-fast bacillus-positive BACTEC fluid samples for PCR analysis. The study shows that the AH procedure in combination with the multiplex PCR is a simple, specific, and sensitive method which can be used in the routine diagnostic laboratory to detect and identify different members of the genus Mycobacterium in blood culture fluid samples from AIDS patients.
Molecular epidemiology of Mycobacterium bovis in Texas and Mexico
Authors: Perumaalla V S , Adams L G , Payeur J B , Jarnagin J L , Baca D R , Suárez Güemes F , Ficht T A
Published: January 2020

Seventy-nine Mycobacterium bovis isolates recovered from Mexican and Texas cattle were categorized into 16 and 25 distinct types on the basis of IS6110 and direct-repeat fingerprint patterns, respectively. By using a combination of both fingerprint patterns, 30 distinct restriction fragment length polymorphism types were defined. Fifty-eight of 79 isolates (73%) were distributed among nine clusters. Clustered isolates were identified within herds, as well as in geographically disperse herds in Texas and Mexico. This observation is consistent with active transmission within herds and among herds, presumably as a result of active or historical cattle movements. The majority of bovine isolates (64 of 79) exhibited a single copy of IS6110. Interestingly, in contrast to previous studies, a high percentage of bovine isolates (15 of 79) exhibited multiple IS6110 copies (two to five) distributed among 11 different restriction fragment length polymorphism types. It is speculated that transmission from noncattle sources may be responsible. Continued fingerprinting of isolates originating from nonbovine sources and herd surveys is expected to provide useful information regarding the epidemiology of tuberculosis in this region.
Ehrlichia canis-like agent isolated from a man in Venezuela: antigenic and genetic characterization
Authors: Perez M , Rikihisa Y , Wen B
Published: January 2020

We report the first isolation and molecular and antigenic characterization of a human ehrlichial species in South America. A retrospective study was performed with serum specimens from 6 children with clinical signs suggestive of human ehrlichiosis and 43 apparently healthy adults who had a close contact with dogs exhibiting clinical signs compatible with canine ehrlichiosis. The evaluation was performed by the indirect fluorescent-antibody assay with Ehrlichia chaffeensis Arkansas, Ehrlichia canis Oklahoma, and Ehrlichia muris antigens. The sera from two apparently healthy humans were positive by the indirect fluorescent-antibody assay for all three antigens. Of the three antigens, samples from humans 1 and 2 showed the highest antibody titers against E. chaffeensis and E. muris, respectively. The remaining serum samples were negative for all three antigens. One year later examination of a blood sample from subject 1 revealed morulae morphologically resembling either E. canis, E. chaffeensis, or E. muris in monocytes in the blood smear. The microorganism, referred to here as Venezuelan human ehrlichia (VHE), was isolated from the blood of this person at 4 days after coculturing isolated blood leukocytes with a dog macrophage cell line (DH82). The organism was also isolated from mice 10 days after intraperitoneal inoculation of blood leukocytes from subject 1. Analysis by electron microscopy showed that the human isolate was ultrastructurally similar to E. canis, E. chaffeensis, and E. muris. When the virulence of VHE in mice was compared with those of E. chaffeensis, E. canis, and E. muris, only VHE and E. muris induced clinical signs in BALB/c mice at 4 and 10 days, respectively, after intraperitoneal inoculation. VHE was reisolated from peritoneal exudate cells of the mice. Only E. chaffeensis- and E. muris-infected mice developed significant splenomegaly. Western immunoblot analysis showed that serum from subject 1 reacted with major proteins of the VHE antigen of 110, 80, 76, 58, 43, 35, and 34 kDa. Human serum against E. chaffeensis reacted strongly with 58-, 54-, 52-, and 40-kDa proteins of the VHE antigen. Anti-E. canis dog serum reacted strongly with 26- and 24-kDa proteins of VHE. In contrast, anti-E. sennetsu rabbit and anti-E. muris mouse sera did not react with the VHE antigen. Serum from subject 1 reacted with major proteins of 90, 64, or 47 kDa of the E. chaffeensis, E. canis, and E. muris antigens. This reaction pattern suggests that this serum sample was similar to serum samples from E. chaffeensis-infected human patients in Oklahoma. The base sequence of the 16S rRNA gene of VHE was most closely related to that of E. canis Oklahoma. On the basis of these observations, we suggest that VHE is a new strain or a subspecies of E. canis which may cause asymptomatic persistent infection in humans.
Frequency, intensity, species, and strains of oral Candida vary as a function of host age
Authors: Kleinegger C L , Lockhart S R , Vargas K , Soll D R
Published: January 2020

While the age of the host has been suggested as a determining factor in yeast carriage, no studies in which the genetic relatedness of isolates has been assessed in combination with the frequency and intensity of carriage as a function of host age have been performed in a single geographical locale and over a short time window. Therefore, by using a simple plating protocol to determine the frequency and intensity of carriage, sugar assimilation patterns to determine species, and Southern blot hybridization with the DNA fingerprinting probe Ca3 combined with computer-assisted analysis to determine the genetic relatedness of strains of Candida albicans, yeast carriage was analyzed as a function of age. All test individuals lived in the Iowa City, Iowa, locale and, except for some of the 0.5- to 1.5-year-olds, were dentate. The results demonstrate that for this test population, the frequency, average intensity, predominant species, and genetic relatedness of C. albicans strains varied as a function of host age. In addition, comparison with oral commensal organisms from the Ann Arbor, Mich., locale confirms the geographical specificity of C. albicans strains and the existence of an Iowa City-enriched strain which is most prevalent in elderly individuals.
Factors influencing Cryptosporidium testing in Connecticut
Authors: Roberts C L , Morin C , Addiss D G , Wahlquist S P , Mshar P A , Hadler J L
Published: January 2020

To describe patterns of testing for Cryptosporidium oocysts in stool samples, Connecticut laboratories were surveyed. Different detection methods were used. Most laboratories examined stools specifically for Cryptosporidium only on physician request. The rate of positive tests varted widely (0 to 28%). Higher rates of positivity were associated with the use of monoclonal antibody methods, the use of two or more staining procedures, and testing of stool specimens in addition to those requested by physicians.
Molecular epidemiology of an outbreak of multidrug-resistant Enterobacter aerogenes infections and in vivo emergence of imipenem resistance
Authors: De Gheldre Y , Maes N , Rost F , De Ryck R , Clevenbergh P , Vincent J L , Struelens M J
Published: January 2020

Molecular typing was used to investigate an outbreak of infection caused by multidrug-resistant Enterobacter aerogenes (MREA) susceptible only to gentamicin and imipenem in an intensive care unit (ICU). Over a 9-month period, ciprofloxacin-resistant E. aerogenes isolates were isolated from 34 patients, or 4.1% of ICU admissions, compared with a baseline rate of 0.1% in the previous period (P < 0.001). Infection developed in 15 (44%) patients. In vivo emergence of imipenem resistance (MIC, 32 micrograms/ml) of organisms causing deep-seated infection was observed in two (13%) of these patients following prolonged therapy with imipenem and gentamicin. Arbitrarily primed PCR (AP-PCR) analysis with ERIC1R and ERIC2 primers and pulsed-field gel electrophoresis (PFGE) analysis of XbaI macrorestriction patterns concordantly showed that outbreak-associated MREA isolates were clonally related and distinct from epidemiologically unrelated strains. AP-PCR and PFGE showed discrimination indices of 0.88 and 0.98, respectively. Space-time clustering of cases within units suggests that the epidemic-related MREA isolates were transmitted on the hands of the health care personnel. A case-control study and repeated environmental culture surveys failed to identify a common source or procedure associated with transmission. In spite of the early implementation of isolation measures, the incidence of MREA colonization remained stable until all colonized patients were discharged. This study confirms the usefulness of AP-PCR and PFGE analyses for the epidemiological study of E. aerogenes and underscores the difficulty of controlling the spread of multiresistant clones of this organism in the ICU setting. The emergence of imipenem resistance represents a threat because virtually no therapeutic option is available for such strains.
Subtype 2c of hepatitis C virus is highly prevalent in Italy and is heterogeneous in the NS5A region
Authors: Maggi F , Vatteroni M L , Fornai C , Morrica A , Giorgi M , Bendinelli M , Pistello M
Published: January 2020

Hepatitis C virus (HCV) isolates, obtained from 50 Italian patients with community-acquired infection, that had previously been classified as subtype 2a or 2b by current rapid genotyping methods were further characterized by partial sequence analysis. All the isolates were reclassified: 45 within subtype 2c and the other 5 as subtype 1b, 3a, or 4d. Thus, subtype 2c is much more prevalent than previously recognized, with about 30% of all HCV strains detected in Italy being subtype 2c. In contrast, isolates of subtypes 2a and 2b appear to be infrequent, if not absent. Further studies showed that subtype 2c isolates are heterogeneous in the NS5A region, in that they may or may not contain a 57-nucleotide (nt) segment spanning from nt 7533 to nt 7589 of the viral genome. Partial nucleotide sequencing of the NS5B region of four 2c subtypes excluded the possibility that the isolates possessing or not possessing the 57-nt segment in the NS5A region may have resulted from recombination phenomena.
Capture-S, a nontreponemal solid-phase erythrocyte adherence assay for serological detection of syphilis
Authors: Stone D L , Moheng M C , Rolih S , Sinor L T
Published: January 2020

A solid-phase erythrocyte adherence assay has been developed for the serological detection of reagin antibodies in syphilis. Capture-S (Immucor, Inc., Norcross, Ga.) is a nontreponemal, qualitative screening test for the detection of immunoglobulin G (IgG) and IgM antilipid antibodies in serum or plasma samples from blood donors. The Capture-S assay utilizes a modified Venereal Disease Research Laboratory antigen bound to microtitration wells and anti-IgG- plus anti-IgM-coated indicator erythrocytes as the detection system. The Capture-S assay was evaluated at six separate sites on 10,942 specimens. For patient samples of clinically diagnosed syphilis categories (n = 366), the Capture-S assay yielded a sensitivity of 80.7% versus 80.3% for the rapid plasma reagin (RPR) card test (Becton Dickinson Microbiology Systems, Cockeysville, Md.). In comparative experiments on patient and donor samples (n = 10,222), the Capture-S assay demonstrated a sensitivity of 94% compared to 91.2% for the RPR card test. The Capture-S and RPR card tests produced essentially equivalent specificities of 99.2% and 99.3%, respectively, for this sample population. For five test sites, the Capture-S and RPR card test demonstrated a 98.3% agreement (10,085 of 10,264) of test results. These evaluations indicate that the Capture-S compares favorably to the RPR card test in assay sensitivity and specificity, with the added benefits of ease of use, accommodation of high-volume testing, and potential for automation.
Genotypic characterization of five subspecies of Mycobacterium kansasii
Authors: Picardeau M , Prod'Hom G , Raskine L , LePennec M P , Vincent V
Published: January 2020

Different molecular typing methods including restriction fragment length polymorphism (RFLP) analysis with the major polymorphic tandem repeat (MPTR) probe and the IS1652 probe, pulsed-field gel electrophoresis (PFGE), amplified fragment length polymorphism (AFLP) analysis, and PCR restriction analysis of the hsp-65 gene (PRA) were applied to clinical and water isolates of Mycobacterium kansasii. RFLP with the MPTR probe, PRA, PFGE, and AFLP analysis revealed five homogeneous clusters which appeared to be subspecies. RFLP with the MPTR probe and PRA gave patterns specific for each cluster, whereas PFGE and AFLP analysis gave polymorphic patterns. IS1652 was present in two of the five clusters and provided polymorphic patterns for one cluster only. The two IS1652-positive clusters were Accuprobe negative (Accuprobe test; Gen-Probe Inc.), and only two other clusters were Accuprobe positive. A PCR test based on the detection of a species-specific fragment (M. Yang, B.C. Ross, and B. Dwyer, J. Clin. Microbiol. 31:2769-2772, 1993) was positive for all M. kansasii strains. This PCR test is an accurate, rapid, and specific M. kansasii identification test. No subspecies was particularly more virulent, because all clusters contained clinical strains, from AIDS patients and non-AIDS patients, and environmental strains.
Candida and Torulopsis: a blinded evaluation of use of pseudohypha formation as basis for identification of medically important yeasts
Authors: Odds F C , Rinaldi M G , Cooper C R , Fothergill A , Pasarell L , McGinnis M R
Published: January 2020

Seventy yeast isolates representing species in the genera Candida and Torulopsis but excluding Candida albicans were examined in three laboratories for production of pseudohyphae in Dalmau cultures. The microscopic morphology of the isolates was scrutinized by four individuals experienced in yeast identification and three inexperienced persons, all of whom were blinded as to the putative identification of the yeasts. For 49 (70%) of the 70 isolates, the seven observers recorded comparable scores for morphology, but 5 (7%) of the isolates showed extreme variation in recorded morphologies, from true hyphae formed to no pseudohyphae formed. Isolates of Candida parapsilosis and Torulopsis glabrata consistently did and did not form pseudohyphae, respectively: however, other Candida and Torulopsis spp. did not always express their expected morphologies. In 48 (19%) of 252 readings (seven observers), 36 isolates of Candida spp. were scored as forming no pseudohyphae, and in 22 (9.2%) of 238 readings, 34 isolates of Torulopsis spp. were recorded as forming true hyphae or pseudohyphae. These results show that pseudohypha formation is not a reliable characteristic for identification of yeasts at the genus level; we suggest that the merger of Torulopsis spp. into the genus Candida should be finally accepted.
Rapid and accurate identification of Staphylococcus species by tRNA intergenic spacer length polymorphism analysis
Authors: Maes N , De Gheldre Y , De Ryck R , Vaneechoutte M , Meugnier H , Etienne J , Struelens M J
Published: January 2020

PCR-amplified tRNA gene (tDNA) intergenic spacer length polymorphism (tDNA-ILP) was analyzed for its ability to identify to the species level type strains (n = 18) and clinical isolates (n = 163) of staphylococci. Amplified products obtained by PCR with outwardly directed consensus tDNA primers were separated by agarose and polyacrylamide gel electrophoreses. The results were compared with those obtained by biochemical identification and ribotyping. Each type strain presented a specific tDNA-ILP pattern. PCR with fluorescent primers allowed for the detection of labelled DNA fragments on polyacrylamide gels by using an automated laser fluorescence sequencer and provided enhanced pattern resolution in comparison with that by analysis on agarose gels. tDNA patterns indistinguishable from those of the type strains were produced by clinical isolates of all tested species except for some isolates of S. aureus (n = 3) and S. haemolyticus (n = 1), which showed variant patterns. Strains of S. saprophyticus and S. xylosus showed very closely related profiles, and S. cohnii subspecies were indistinguishable. The identities obtained by tDNA-ILP analysis agreed with those obtained by the biochemical method to the species level for 99% (162 of 163) of the strains tested and to the subspecies level for 96% (156 of 163) of the strains tested. These results indicate that fluorescence-labelled PCR analysis of tDNA-ILP provides an accurate and rapid molecular method for the identification of human staphylococci.
Molecular epidemiology of acquisition of ceftazidime-resistant gram-negative bacilli in a nonoutbreak setting
Authors: D'Agata E , Venkataraman L , DeGirolami P , Samore M
Published: January 2020

We prospectively studied the acquisition of ceftazidime-resistant gram-negative bacilli (CAZ-RGN) in two surgical intensive care units (SICU) during a nonoutbreak period. Surveillance cultures were obtained from patients at the time of admission and serially thereafter. CAZ-RGN isolates were typed by pulsed-field gel electrophoresis (PFGE). Three hundred and forty-three patients were enrolled from whom 1,621 baseline and follow-up cultures were obtained. The most common species isolated from patients were Pseudomonas aeruginosa (22), Enterobacter cloacae (21), Acinetobacter spp. (13), Enterobacter aerogenes (11), Citrobacter spp. (10), Pseudomonas spp. (non P. aeruginosa) (9), and Stenotrophomonas spp. (7). For each species, PFGE strain types were highly diverse; no single type was recovered from more than four patients. Twenty-eight patients acquired a CAZ-RGN during the SICU stay; in six (21%), emergence of resistance from a previously susceptible strain was documented on the basis of matching serial strain types. Transmission of CAZ-RGN between patients occurred but was infrequent, as judged by analyzing strain types of epidemiologically linked patients. In conclusion, colonization with CAZ-RGN in SICU was associated with diverse species and strains, as determined by molecular typing. Emergence of resistance from previously susceptible strains appeared to be more important than horizontal transmission in acquisition of CAZ-RGN in a nonoutbreak period.
Prospective study of human herpesvirus 6, human herpesvirus 7, and cytomegalovirus infections in human immunodeficiency virus-positive patients
Authors: Fabio G , Knight S N , Kidd I M , Noibi S M , Johnson M A , Emery V C , Griffiths P D , Clark D A
Published: January 2020

Blood samples from human immunodeficiency virus (HIV)-positive patients were monitored for cytomegalovirus (CMV), human herpesvirus 6 (HHV-6), and HHV-7 by PCR. We detected CMV in 17% of the patients, HHV-6 in 6%, and HHV-7 in 3%. The viral loads of CMV were significantly higher than those of HHV-6 (P = 0.007) or HHV-7 (P = 0.01). Detection of CMV and HHV-6 was associated with low and high CD4 counts, respectively.