bluuebunny/crossref_metadata_2025 huggingface dataset:
Partial characterization of Streptococcus suis type 2 hemolysin
Authors: Feder I , Chengappa M M , Fenwick B , Rider M , Staats J
Published: January 2020

Streptococcus suis type 2 was evaluated for hemolysin production. Supernatants of S. suis type 2 grown in Todd-Hewitt broth were assayed for hemolytic activity by a photometric assay. Twenty-two additional serotypes of S. suis (1,3 to 22, and 1/2) were evaluated for hemolysin production; nine of them (1/2, 1, 4, 5, 14, 15, 17, 19, and 20) were positive. The effects of temperature, atmosphere, centrifugation, sonication, chemicals, bovine serum albumin, fetal calf serum, and enzymes on S. suis type 2 hemolysin activity were studied. Maximum hemolysis occurred after incubation in RPMI 1640 medium at 40 degrees C in 6% CO2 and after growth in Todd-Hewitt broth at 37 degrees C under anaerobic conditions. Hemolytic activity was absent after the addition of fetal calf serum and decreased after the addition of trypsin or amylase. However, treatment of erythrocytes with amylase or trypsin prior to incubation with supernatant also resulted in a decrease in hemolytic activity. The addition of bovine serum albumin caused increased hemolytic activity. Dipyridyl and EDTA had negligible effects on hemolysis. Hemolytic S. suis type 2 culture supernatant injected intraperitoneally failed to cause death in BALB/c mice. Data from our study indicate that S. suis type 2 hemolysin is a secreted or loosely cell bound, thermolabile molecule whose activity is growth condition dependent.
Detection of Streptococcus pneumoniae in sputum samples by PCR
Authors: Gillespie S H , Ullman C , Smith M D , Emery V
Published: January 2020

A method for the detection of Streptococcus pneumoniae in sputum samples by PCR has been developed. The assay employs oligonucleotide primers specific for a portion of the autolysin gene lytA of S. pneumoniae. Other closely related streptococci, Haemophilus influenzae, and Moraxella catarrhalis do not give a positive result in the assay. The assay was capable of detecting between 10 and 100 CFU of S. pneumoniae in distilled water and 1.4 x 10(4) CFU/ml in simulated sputum samples. Sputum samples from 33 patients with acute pneumonia were collected and subjected to culture, PCR, and C-polysaccharide antigen detection by enzyme-linked immunosorbent assay (ELISA). A significant isolate of S. pneumoniae was isolated from 14 patients, of which 13 were positive by PCR and C-polysaccharide antigen ELISA. No positive results were obtained for the 19 patients in whom other pathogens or upper respiratory tract floras only were isolated. The sensitivity of the autolysin PCR is 92.8%, the specificity is 100%, the predictive value of a positive result is 100%, and the predictive value of a negative result is 95%. This suggests that autolysin PCR is suitable for the detection of S. pneumoniae in clinical samples.
Detection of Pseudomonas pseudomallei by PCR and hybridization
Authors: Lew A E , Desmarchelier P M
Published: January 2020

A molecular method for the detection of Pseudomonas pseudomallei was developed on the basis of the differences in the 23S rRNA sequences of related species of the genus Pseudomonas. An 18-base oligonucleotide probe, designed following partial sequencing of 23s ribosomal DNA (rDNA), was used for the identification and detection of P. pseudomallei either by hybridization or by direct PCR. Optimal detection was obtained by hybridization of the probe with PCR-amplified rDNA rather than with total genomic DNA or colony blots. One nanogram of template DNA amplified in a PCR mixture containing 14% glycerol could be detected in slot blots hybridized with the digoxigenin-labelled probe and the lumigen PPD detection system. Amplified rDNA sequences from 41 P. pseudomallei strains of various origins hybridized with the probe. The probe also hybridized with three Pseudomonas mallei reference strains under conditions of high stringency but failed to hybridize with amplified rDNA sequences from other closely related Pseudomonas spp. PCR with a conserved primer and the 18-base oligonucleotide probe (direct PCR) specifically amplified P. pseudomallei and P. mallei. By using these methods, approximately 10(4) P. pseudomallei cells per ml could be detected in artificially inoculated blood samples and in blood dried on filter paper following Chelex extraction. The detection limit in blood was increased to 10(2) cells per ml by concentration of bacteria from 0.5 ml of blood or by a 24-h blood culture enrichment prior to PCR. Approximately 10(3) cells per ml were detected in seeded sputum samples. The detection times by direct PCR and indirect PCR and then probe hybridization were approximately 5 h and 24 h, respectively. These results indicate that amplification of conserved rDNA sequences by PCR directly or by hybridization with a probe to PCR fragments offers promise for the detection of P. pseudomallei and P. mallei.
Effects of manganese on growth of Mycoplasma salivarium and Mycoplasma orale
Authors: Watanabe T
Published: January 2020

The growth of Mycoplasma salivarium ATCC 23064 and Mycoplasma orale ATCC 15539 was inhibited by MnCl2. The growth-inhibitory effect was much more remarkable on M. orale than M. salivarium and was much more remarkable in medium supplemented with 10% (vol/vol) horse serum (HS) than 20% (vol/vol) HS. It was suggested that isolates of Mycoplasma from the oral cavity could be roughly identified as either M. salivarium and M. orale by examination of the growth (color changes) in PPLO broth supplemented with 10% (vol/vol) HS and 0.2 mM MnCl2.
Comparison of three methods for determination of a single MIC of an antimicrobial agent
Authors: Miller L A , Rittenhouse S F , Utrup L J , Poupard J A
Published: January 2020

Determining a single MIC of an antimicrobial agent that is not included on a routine susceptibility panel can be significantly time-consuming and costly. The UniScept MICRO-MIC, the JustOne, and the E test are single-MIC systems. These systems as well as disk diffusion were compared on the bases of time, cost, and ease of use.
Prevalence of Corynebacterium urealyticum in urine specimens collected at a university-affiliated medical center
Authors: Ryan M , Murray P R
Published: January 2020

Corynebacterium urealyticum (formerly Corynebacterium group D2) has been implicated as a cause of alkaline-encrusted cystitis and urinary tract struvite calculi. Despite preselecting urine specimens with neutral and alkaline pHs and using prolonged incubation on a selective medium, isolation of this organism was rarely observed in a population of hospitalized patients. We do not recommend routine cultures for this organism unless the urine is alkaline and struvite crystals, leukocytes, and erythrocytes are present.
Genetic variability and molecular typing of Shigella sonnei strains isolated in Canada
Authors: Preston M A , Borczyk A A
Published: January 2020

Restriction fragment length polymorphisms (RFLPs) of genomic DNAs from 49 clinical isolates of Shigella sonnei were analyzed by using a modified restriction endonuclease analysis procedure to investigate the genetic variability of this species. After cleavage with the restriction enzyme HaeIII or RsaI, DNA samples were electrophoresed in polyacrylamide gels and the RFLP patterns were visualized by silver staining. The results showed that among 20 strains associated with sporadic cases of infection in three Canadian provinces, 15 distinct RFLP patterns were revealed by HaeIII digestion and 12 distinct patterns were revealed by RsaI digestion. In contrast, the RFLP patterns of individual isolates within six groups of epidemiologically related isolates were identical to each other but distinct from those of unrelated isolates, and these patterns could be used to determine the genetic relationships between isolates associated with separate outbreaks of shigellosis. Our results indicate that the modified restriction endonuclease analysis technique represents a rapid, reproducible, and highly discriminatory method for the molecular typing of this species.
Enzyme capture assay for rapid identification of Escherichia coli in blood cultures
Authors: Huang S W , Wu J J , Chang T C
Published: January 2020

An enzyme capture assay (ECA) for rapid identification of Escherichia coli in blood cultures by using beta-D-glucuronidase as a marker was developed. Microdilution plates coated with antiglucuronidase were used to capture this enzyme from the cell lysates of blood cultures which showed growth of gram-negative bacteria. The assay, using 4-methylumbelliferyl-beta-D-glucuronide as a fluorogenic substrate, had a detection limit of 0.1 ng/ml (3 x 10(-13) M) for the enzyme; this was approximately equal to a cell concentration of 10(6) CFU of E. coli per ml. Among 212 blood cultures showing growth of gram-negative bacteria, 77 specimens were found to contain E. coli by conventional culture procedures and 73 samples were positive by ECA. Among the 135 blood cultures from which E. coli was not isolated, ECA gave one false-positive (Salmonella enteritidis) reaction. Thus, the sensitivity and specificity for the identification of E. coli in blood cultures by ECA were 94.8% (73/77) and 99.3% (134/135), respectively. From the finding of positive growth in the culture bottle, the assay can be completed within 4 h. In view of the high rate of isolation of E. coli from bacteremic patients, the test can be performed in parallel with conventional culture protocols; this may shorten the identification time for E. coli, and proper antimicrobial treatments may be started 24 h earlier than when results of conventional identification systems are used.
Two-year study of endemic enteric pathogens associated with acute diarrhea in New Caledonia
Authors: Germani Y , Morillon M , Begaud E , Dubourdieu H , Costa R , Thevenon J
Published: January 2020

A longitudinal study of diarrheal disease among patients of all ages with acute diarrhea was carried out in New Caledonia from January 1990 to December 1991. Stool samples from 2,088 diarrheal patients were examined for parasites, rotavirus, and bacterial pathogens. Potential sources of contamination (drinking water, seawater and bovine and porcine feces) were investigated. One or more enteric pathogens were identified in 41.8 and 40.6% of the persons with diarrhea, in 1990 and 1991, respectively. Salmonella spp., Shigella spp., HEp-2 cell adherent Escherichia coli (diffuse adherent and enteroaggregative), enteropathogenic E. coli (EPEC) (EPEC adherence factor-positive strains belonging to classical serotypes), localized adherent E. coli (non-EPEC), and enterotoxigenic E. coli were the frequently identified enteropathogenic bacteria. Other major enteropathogens were Entamoeba histolytica and Giardia lamblia. Campylobacter jejuni, Clostridium difficile, Clostridium perfringens, Yersinia enterocolitica, and rotavirus were isolated from only a few patients. No Vibrio spp., Aeromonas spp., Plesiomonas spp., Shiga-like-toxin-producing E. coli, enterohemorrhagic E. coli, or enteroinvasive E. coli were identified. Shiga-like toxin I-producing E. coli were present in adult bovines and calves, and heat-stable enterotoxin II-producing enterotoxigenic E. coli were found in pigs.
Usefulness of gram staining of blood collected from total parenteral nutrition catheter for rapid diagnosis of catheter-related sepsis
Authors: Moonens F , el Alami S , Van Gossum A , Struelens M J , Serruys E
Published: January 2020

The accuracy of Gram staining of blood drawn from catheters used to administer total parenteral nutrition was compared with paired quantitative blood cultures for the diagnosis of catheter-related sepsis. Gram staining was positive in 11 of 18 episodes of catheter-related sepsis documented by quantitative culture (sensitivity, 61%) but in none of the 5 episodes of fever unrelated to catheter infection. Thus, this procedure enabled the rapid presumptive diagnosis and guidance of antimicrobial therapy for total parenteral nutrition catheter sepsis, with a positive predictive value of 100% and a negative predictive value of 42%.
Evidence of pestivirus RNA in human virus vaccines
Authors: Harasawa R , Tomiyama T
Published: January 2020

We examined live virus vaccines against measles, mumps, and rubella for the presence of pestivirus RNA or of pestiviruses by reverse transcription PCR. Pestivirus RNA was detected in two measles-mumps-rubella combined vaccines and in two monovalent vaccines against mumps and rubella. Nucleotide sequence analysis of the PCR products indicated that a modified live vaccine strain used for immunization of cattle against bovine viral diarrhea is not responsible for the contamination of the vaccines.
Comparison of techniques and evaluation of three commercial monoclonal antibodies for laboratory diagnosis of varicella-zoster virus in mucocutaneous specimens
Authors: Pérez J L , García A , Niubò J , Salvà J , Podzamczer D , Martín R
Published: January 2020

A comparison of direct antigen detection in cell scrapings with culture techniques (tube culture and shell vial method) for diagnosis of varicella-zoster virus (VZV) mucocutaneous infections was done in parallel in two groups of specimens. A total of 100 specimens were from patients with clinical diagnosis of VZV infection (group 1), and 69 were from patients with no suspicion of VZV infection (group 2) but mainly with herpes simplex virus infections. In addition, three commercially available monoclonal antibodies (Whittaker, Biosoft Clone 2013, and Ortho 3B3) directed against VZV antigens were evaluated in parallel in the last 87 group 1 specimens. Overall, 80% of the group 1 specimens were confirmed positive by direct detection, in comparison with 56% positive by tube culture and/or shell vial. None of the group 2 specimens were positive for VZV by any of the methods, and none of the monoclonal antibodies assayed reacted with any herpes simplex virus stock strains. Antiviral therapy and the length of evolution time of lesions affected negatively the performance of all laboratory methods, but to a lesser extent in direct detection techniques than in culture techniques. The Whittaker and Biosoft reagents (indirect immunofluorescence assay) showed statistically significant differences in sensitivity with respect to the Ortho antibody (P = 0.002 and P = 0.039, respectively; two-tailed binomial test). Direct antigen detection is a rapid, easy-to-perform, sensitive, and specific technique and appears to be the method of choice for laboratory confirmation of VZV mucocutaneous infections.
Comparison of PCR with other tests for early diagnosis of canine ehrlichiosis
Authors: Iqbal Z , Chaichanasiriwithaya W , Rikihisa Y
Published: January 2020

The purpose of the study was to compare the sensitivity of PCR with those of cell culture reisolation of Ehrlichia canis, the indirect fluorescent antibody test (IFA), and Western immunoblotting (WI) in the early diagnosis of canine ehrlichiosis. Five German shepherd dogs were intravenously inoculated with 10(7) E. canis-infected DH82 cells. Blood was collected on alternate days during a 2-week postinoculation period. Mononuclear cell fractions were harvested and used for E. canis reisolation and DNA extraction for PCR. The plasma was used for assaying antibodies against E. canis. By PCR, the 16S rRNA gene of E. canis was detected in the mononuclear cell specimens collected as early as day 4 to 10 postexposure (PE). E. canis was reisolated from the blood starting on day 2 PE from all five dogs. The indirect fluorescent antibody test and Western immunoblotting could detect E. canis antibodies as early as 2 to 8 days PE. Cell culture reisolation proved to be the most sensitive and definitive for early diagnosis of ehrlichiosis, but it is not very convenient, since it takes a long time (14 to 34 days) to show up positive. The sensitivity of PCR is comparable to or slightly less than that of other established methods; however, the convenience, quickness, and direct nature of detecting E. canis DNA is expected to make PCR more useful for clinical diagnosis.
Typing of Candida krusei clinical isolates by restriction endonuclease analysis and hybridization with CkF1,2 DNA probe
Authors: Carlotti A , Grillot R , Couble A , Villard J
Published: January 2020

The use of restriction endonuclease analysis and Southern hybridization with our new CkF1,2 DNA probe, cold labeled with peroxidase, for the typing of Candida krusei isolates has been investigated. Fifty-five clinical samples isolated from forty-five patients hospitalized in eight centers, one environmental strain, and two reference strains were evaluated. Patterns were analyzed by a computer-assisted method and compared by numerical analysis. Clearer and less ambiguous patterns were obtained by restriction with endonuclease HinfI. It generated 9 to 14 (average, 11) well-separated fragments in the range of 6.5 to 2.0 kb. Both their numbers and sizes varied greatly among the strains studied. The CkF1,2 probe hybridized with one to seven fragments of HinfI patterns. A total of 48 distinct types were distinguished among the 58 strains studied. HinfI and CkF1,2 patterns showed similarities of less than 83 and 75% for unrelated strains and more than 91 and 100% for related strains, respectively. The methods showed 100% typeability, 98% reproducibility, and a discriminatory power of 1. C. krusei isolates from each patient were distinct, whether from one hospital or from different hospitals. Multiple isolates from the same patient were identical, both over time and at different anatomic sites. An endogenous origin is suggested for the colonizing and infecting isolates among the 45 patients. The CkF1,2 probe enhanced discrimination of the strains and provided a definitive comparison for strain identity. Genetic linkages between isolates were assessed at the subspecies level, and 12 clusters were delineated. A typing scheme is proposed for epidemiological studies of C. krusei.
Development of o.a.s.i.s., a new automated blood culture system in which detection is based on measurement of bottle headspace pressure changes
Authors: Stevens C M , Swaine D , Butler C , Carr A H , Weightman A , Catchpole C R , Healing D E , Elliott T S
Published: January 2020

o.a.s.i.s. (Unipath Ltd., Basingstoke, United Kingdom) is a new automated blood culture system. The metabolism of microorganisms is detected by measuring changes in the pressure of the headspace of blood culture bottles. These changes are measured by monitoring the position of a flexible sealing septum, every 5 min, with a scanning laser sensor. This noninvasive system can detect both gas absorption and production and does not rely solely on measuring increasing carbon dioxide levels. A research prototype instrument was used to carry out an evaluation of the media, the detection system, and its associated detection algorithm. In simulated blood cultures, o.a.s.i.s. supported growth and detected a range of clinical isolates. Times to positivity were significantly shorter in o.a.s.i.s. than in the BACTEC 460 system. Results of a clinical feasibility study, with a manual blood culture system as a control, confirmed that o.a.s.i.s. was able to support the growth and detection of a variety of clinically significant organisms. On the basis of these findings, full-scale comparative clinical trials of o.a.s.i.s. with other automated blood culture systems are warranted.
Identification of Vibrio hollisae associated with severe gastroenteritis after consumption of raw oysters
Authors: Carnahan A M , Harding J , Watsky D , Hansman S
Published: January 2020

Vibrio hollisae was recovered from the stool culture of a 40-year-old female hospitalized for severe abdominal cramping, vomiting, fever, and watery diarrhea. She had consumed two dozen raw oysters 5 days prior. There was only a single colony on thiosulfate-citrate-bile salts-sucrose-agar, and definitive identification required conventional test media with 1% NaCl.
CHROMagar Candida, a new differential isolation medium for presumptive identification of clinically important Candida species
Authors: Odds F C , Bernaerts R
Published: January 2020

CHROMagar Candida is a novel, differential culture medium that is claimed to facilitate the isolation and presumptive identification of some clinically important yeast species. We evaluated the use of this medium with 726 yeast isolates, including 82 isolated directly on the medium from clinical material. After 2 days of incubation at 37 degrees C, 285 C. albicans isolates gave distinctive green colonies that were not seen with any of 441 other yeast isolates representing 21 different species. A total of 54 C. tropicalis isolates also developed distinctive dark blue-gray colonies with a halo of dark brownish purple in the surrounding agar. C. krusei isolates (n = 43) also formed highly characteristic rough, spreading colonies with pale pink centers and a white edge that was otherwise encountered only rarely with isolates of C. norvegensis. Trichosporon spp. (n = 34) formed small, pale colonies that became larger and characteristically rough with prolonged incubation. Most of the other 310 yeasts studied formed colonies with a color that ranged from white to pink to purple with a brownish tint. The only exceptions were found among isolates identified as Geotrichum sp. or Pichia sp., some of which formed colonies with a gray to blue color and which in two instances formed a green pigment or a dark halo in the agar. The specificity and sensitivity of the new medium for the presumptive identification of C. albicans, C. krusei, and C. tropicalis exceeded 99% for all three species. A blinded reading test involving four personnel and 57 yeast isolates representing nine clinically important species confirmed that colonial appearance after 48 h of incubation on CHROMagar Candida afforded the correct presumptive recognition of C. albicans, C. tropicalis, C, krusei, and Trichosporon spp. None of nine bacterial isolates grew on CHROMagar Candida within 72 h, and bacteria (Escherichia coli) grew from only 4 of 104 vaginal, 100 oral, and 99 anorectal swabs. The new medium supported the growth of 19 of 23 dermatophyte fungi tested and 41 of 43 other molds representing a broad range of fungal pathogens and contaminants. In parallel cultures of 348 clinical specimens set up on Sabourand agar and CHROMagar Candida, both media grew yeasts in the same 78 instances. CHROMagar Candida is recommended as a useful isolation medium capable of the presumptive identification of the yeast species most commonly isolated from clinical material and facilitating recognition of mixed yeast cultures.
Quantitation of hepatitis C virus genome molecules in plasma samples
Authors: Manzin A , Bagnarelli P , Menzo S , Giostra F , Brugia M , Francesconi R , Bianchi F B , Clementi M
Published: January 2020

A competitive reverse transcription PCR (cRT-PCR)-based assay for the quantitative detection of hepatitis C virus (HCV) viremia was developed, optimized, and applied to the direct molecular analysis of clinical samples from nine patients with persistent HCV infection. As for other competitive PCR-based applications, this method consists of the reverse transcription and subsequent amplification of two RNA species in the same tube: the wild-type template (to be quantified) and a known amount of a modified synthetic template. These templates have identical primer recognition sites and very similar (but not identical) sizes, thus allowing direct detection of both template species after gel electrophoresis and ethidium bromide staining. The results obtained by this cRT-PCR application for testing clinical samples from HCV-infected patients mainly indicate that the competitive approach reaches the degree of sensitivity (fewer than 5 HCV RNA molecules per 100 microliters) necessary to evaluate viral load in all HCV-infected patients, independently of clinical conditions, and that this technique is flexible enough to quantify highly divergent levels of cell-free HCV genome copy numbers in biological samples. Interestingly, we observed a sample-to-sample variation in the loss of detectable HCV genome molecules in serum in comparison with that in plasma from the same patient, thus indicating that serum specimens, although widely used in the past few years for qualitative molecular investigation of HCV-infected patients, cannot be used to obtain reliable quantitative data on HCV viremia from these patients.
Surveillance of susceptibility testing methodologies for Haemophilus influenzae in Canada, including evaluation of disk diffusion test
Authors: Scriver S R , Hoban D J , McGeer A , Moore T C , Walmsley S L , Low D E
Published: January 2020

We surveyed 75 clinical laboratories to determine if National Committee for Clinical Laboratory Standards (NCCLS) were being used for the susceptibility testing of Haemophilus influenzae. Of the 66 laboratories that performed susceptibility testing, all claimed to follow current NCCLS guidelines. However, upon further questioning, only 23, all of which used disk diffusion testing, accurately interpreted and followed the guidelines. Proficiency testing of 22 of these laboratories found that an unacceptable number of interpretive errors (> 10%) occurred. These results query the merit of routine disk diffusion susceptibility testing of H. influenzae to beta-lactam agents.
Genotyping of Canadian hepatitis C virus isolates by PCR
Authors: Andonov A , Chaudhary R K
Published: January 2020

We used PCR for hepatitis C virus (HCV) genotyping with type-specific primers from the core and NS5 genes. Type I was predominant in the general population (58% in blood donors) as well as in different risk groups, such as intravenous drug abusers (58%), blood transfusion recipients (64%), hemophiliacs (62%), and patients with HCV chronic liver disease (76%). Types II, III, and IV were less prevalent in Canada, being found in 10.92, 6.72, and 5.88% of the population, respectively. The type II core primer was not type specific and reacted with the majority of our type I HCV samples, suggesting a false-positive dual infection with two different genotypes (I and II). Digestion of these amplified type I and type II products with restriction endonuclease AccI proved to be very useful in the exclusion of false-positive dual type I and type II infections.
Evaluation of two commercial human T-cell lymphotropic virus western blot (immunoblot) kits with problem specimens
Authors: Gallo D , Diggs J L , Hanson C V
Published: January 2020

We evaluated two commercial human T-cell lymphotropic virus (HTLV) Western blot (WB; immunoblot) kits, Cambridge Biotech Corp. (CBC) and Diagnostic Biotechnology Ltd. (DBL). Both methods employ HTLV type I (HTLV-I) viral lysate and rgp21. The DBL WB kit also distinguishes between HTLV-I and HTLV-II antibodies, using an HTLV-I-specific and an HTLV-II-specific recombinant. Fifty weakly reactive HTLV-II-positive plasma specimens which were falsely negative with the Abbott enzyme immunoassay (EIA) and 50 Ortho EIA false-positive samples were selected to determine sensitivity and specificity. The sensitivities of the CBC and the DBL WB kits were 90 and 68%, respectively. All positive samples reacted with rgp21 in both kits, but some did not display core bands. Five samples were typed as HTLV-I and four were typed as dual infection by the DBL WB kit. The specificities of the CBC and DBL kits were 48 and 70%, respectively. The most prevalent WB reaction with the negative samples was with the core protein, p19, followed by p24 and p28 for CBC and rgp21 and p28 for DBL. DBL had two false-positive interpretations, and CBC had none, rgp21 was the most sensitive antigen in both kits for the weakly reactive HTLV-II samples. If all samples not reacting with this protein were interpreted as WB negative, regardless of other bands, the specificity would improve to 90% for CBC and 86% for DBL.
Spread and maintenance of a dominant methicillin-resistant Staphylococcus aureus (MRSA) clone during an outbreak of MRSA disease in a Spanish hospital
Authors: Dominguez M A , de Lencastre H , Linares J , Tomasz A
Published: January 2020

It was not until November 1989 that the 1,000-bed University-affiliated Hospital de Bellvitge "Princeps d'Espanya" in Barcelona first acquired methicillin-resistant Staphylococcus aureus (MRSA). Since that time, the outbreak of MRSA disease has continued. We have analyzed by genomic DNA fingerprinting 189 MRSA isolates collected between late 1989 and the end of 1993. The isolates include both invasive and colonizing strains as well as isolates from health-care workers and environmental sources. In addition, 52 clinical isolates of methicillin-susceptible S. aureus (MSSA) collected in the same hospital were also analyzed. Isolates were classified into clonal types on the basis of molecular typing techniques. A single MRSA clone (I::B::a) belonging to ClaI type I, pulsed-field gel electrophoretic pattern B, and Tn554 pattern a was responsible for the great majority of infections (73% of blood cultures and 79% of specimens from other clinical sources). This clone appeared at the very beginning of the outbreak, spread throughout the hospital wards, and was also carried by inpatients and health-care workers and on environmental surfaces. In contrast, no dominant lineage was apparent among MSSA isolates (33 distinct pulsed-field gel electrophoretic patterns among 52 isolates). Two MSSA isolates seem to have originated from the dominant clone by deletion of the mecA gene and some additional DNA. In several isolates, different mecA polymorphs were present in identical chromosomal backgrounds or cells with distinct chromosomal backgrounds carried the same mecA polymorph, suggesting horizontal transfer of the mecA gene.
Emergence of fluconazole-resistant strains of Candida albicans in patients with recurrent oropharyngeal candidosis and human immunodeficiency virus infection
Authors: Ruhnke M , Eigler A , Tennagen I , Geiseler B , Engelmann E , Trautmann M
Published: January 2020

After repeated use of fluconazole for therapy of oropharyngeal candidosis, the emergence of in vitro fluconazole-resistant Candida albicans isolates (MIC, > or = 25 micrograms/ml) together with oral candidosis unresponsive to oral dosages of up to 400 mg of fluconazole were observed in patients with human immunodeficiency virus (HIV) infection. Antifungal susceptibility testing was done by broth microdilution and agar dilution techniques on C. albicans isolates recovered from a cohort of patients with symptomatic HIV infection who were treated repeatedly with fluconazole for oropharyngeal candidosis. In vitro findings did show a gradual increase in the MICs for C. albicans isolates recovered from selected patients with repeated episodes of oropharyngeal candidosis. Primary resistance of C. albicans to fluconazole was not seen. Cross-resistance in vitro occurred between fluconazole and other azoles (ketoconazole, itraconazole), but to a lesser extent. The results of the study suggest that the development of clinical resistance to fluconazole could be clearly correlated to in vitro resistance to fluconazole. Itraconazole may still serve as an effective antifungal agent in patients with HIV infection and oropharyngeal candidosis nonresponsive to fluconazole.
Comparison of broth macrodilution, broth microdilution, and E test antifungal susceptibility tests for fluconazole
Authors: Sewell D L , Pfaller M A , Barry A L
Published: January 2020

A comparison of the E test, the broth microdilution test, and the reference broth macrodilution susceptibility test of the National Committee for Clinical Laboratory Standards for fluconazole susceptibility testing was performed with 238 clinical isolates of Candida species and Torulopsis (Candida) glabrata. An 80% inhibition endpoint MIC was determined by the reference broth macrodilution method after 48 h of incubation. The MICs obtained by the two study methods were read after 24 and 48 h of incubation. Overall, excellent agreement within 2 doubling dilutions was obtained between the broth microdilution and the broth macrodilution methods for the combined results for all species at both 24 h (93%) and 48 h (94%). The correlation of 24-h MIC endpoints between the E test and the broth macrodilution methods was 37% for T. glabrata, 56% for Candida tropicalis, 93% for Candida albicans, and 90% for other Candida species. The percent agreement at 48 h ranged from 34% for T. glabrata to 97% for Candida species other than C. albicans and C. tropicalis. These initial results support the further evaluation of the E test as an alternative method for fluconazole susceptibility testing of Candida species.
Differentiation of Salmonella serovar infantis isolates from human and animal sources by fingerprinting IS200 and 16S rrn loci
Authors: Pelkonen S , Romppanen E L , Siitonen A , Pelkonen J
Published: January 2020

We genotyped Salmonella serovar infantis (referred to as S. infantis), which is the most widespread serovar among animals and the third most common cause of human salmonellosis in Finland. Molecular fingerprinting of the 16S rrn locus and the Salmonella-specific insertion sequence IS200 was used to type the 131 isolates originating from the main sources of S. infantis infection. The number of IS200 elements in S. infantis varied from zero to seven; three or more copies were present in 97% of the isolates, and 71% had four copies. There were four conserved chromosomal positions of IS200, which allowed us to group the isolates into three major clonal groups. We defined 11 unique IS200 profiles and five different ribotypes which, in combination, generated 15 genotypes highly restricted to the infection sources: 8 genotypes were typical of isolates from broiler chickens and cattle and seven genotypes were typical of isolates from humans. The eight genotypes of isolates from chickens represented two clonal groups which were differentially associated with chicken-producing companies. The typing scheme allows efficient discrimination between isolates from various infection sources and within sources and, therefore, provides a unique molecular tool for use in the study of the epidemiology of S. infantis infection.
Comparison of four enzyme immunoassays for detection of immunoglobulin M antibodies against rubella virus
Authors: Matter L , Gorgievski-Hrisoho M , Germann D
Published: January 2020

We evaluated four tests for the detection of rubella virus-specific immunoglobulin M antibodies. Primarily, consecutive serum samples were tested by two different assays. Selected panels of sera from patients with proven or likely recent rubella and false-positive and true-negative results in the two primary assays were further tested with two recently developed, fully automated techniques. The four tests were comparable in overall accuracy, but their dynamic ranges may differ considerably. Ways to optimize the predictive values are discussed. We conclude that automated assays may be used without causing significant changes in diagnostic accuracy or distortions in notifications of the incidence of rubella compared with the use of established tools.
Comparison of peptide enzyme-linked immunosorbent assay and radioimmunoprecipitation assay with in vitro-translated proteins for detection of serum antibodies to human papillomavirus type 16 E6 and E7 proteins
Authors: Sun Y , Shah K V , Müller M , Muñoz N , Bosch X F , Viscidi R P
Published: January 2020

Antibodies to human papilloma virus (HPV) type 16 (HPV-16) E6 and E7 proteins in serum are markers for HPV-associated invasive cervical carcinoma. We compared two assays, a radioimmunoprecipitation assay with in vitro-translated HPV-16 E6 and E7 proteins and an enzyme-linked immunosorbent assay (ELISA) with E6 and E7 synthetic peptides, for their abilities to discriminate serologically between patients with invasive cervical cancer and controls. Among the patients, antibody prevalences were higher by the E6 radioimmunoprecipitation assay (55.7%) than by the E6 peptide ELISA (15.5%), but among the controls, they were lower by the radioimmunoprecipitation assay (1.7%) than by the E6 peptide ELISA (5%). For E7, antibody prevalences among the patients were comparable by the radioimmunoprecipitation assay (43%) and the peptide ELISA (41%), but among the controls they were higher by the E7 peptide ELISA (17.4%) than by the radioimmunoprecipitation assay (4.1%). There was good agreement between the E7 radioimmunoprecipitation assay and the E7 peptide ELISA among patients but not among controls. In tests with representative sera, heat denaturation of the translated proteins resulted in a complete loss of reactivity to the E6 protein and a marked decrease in reactivity to the E7 protein. Our study showed that the radioimmunoprecipitation assay discriminates better than the peptide ELISA between patients with invasive cervical cancer and controls and that this is related to the ability of the radioimmunoprecipitation assay to detect conformational epitopes.
Comparison of three commercial MIC systems, E test, fastidious antimicrobial susceptibility panel, and FOX fastidious panel, for confirmation of penicillin and cephalosporin resistance in Streptococcus pneumoniae
Authors: Krisher K K , Linscott A
Published: January 2020

The performances of three commercial broth microdilution MIC assays adapted for use with fastidious organisms--the E test (ET), Fastidious Antimicrobial Susceptibility panel (FAS), and FOX Fastidious panel (FOX)--were compared with a MIC using Mueller-Hinton broth with 5% lysed horse blood (MHLHB) to confirm penicillin and cephalosporin resistance in clinical isolates of Streptococcus pneumoniae. Of the isolates screened for penicillin resistance, 5 (12.8%) were categorized as susceptible, 16 (41.0%) were categorized as intermediate, and 18 (46.2%) were categorized as resistant by MHLHB. Only the isolates exhibiting intermediate-to-resistant MICs were included in the comparison. Agreement within +/- 1 log2 dilution was found in 91, 21, and 76% of the ET, FAS, and FOX MICs, respectively, compared with the MHLHB MIC. No very major or major discrepancies occurred with the ET or FOX; however, two very major interpretive errors occurred with the FAS. Agreement between the ET and MHLHB for cefotaxime, ceftriaxone, and cefuroxime was 88, 85, and 100%, respectively. Less than 50% of cephalosporin MICs categorized as > 0.5 microgram/ml by MHLHB were detected by FAS or FOX. Of the methods compared, the ET was the most reliable alternative for susceptibility testing of pneumococci.
Standardized microtiter assay for determination of syncytium-inducing phenotypes of clinical human immunodeficiency virus type 1 isolates
Authors: Japour A J , Fiscus S A , Arduino J M , Mayers D L , Reichelderfer P S , Kuritzkes D R
Published: January 2020

A standardized assay in 96-well microtiter plates for syncytium-inducing (SI) human immunodeficiency virus type 1 phenotype detection using MT-2 cells has been developed. SI variants were found in 67% of the patients with advanced human immunodeficiency virus disease. The occurrence of the SI phenotype increased with lower CD4+ counts. There was no association between p24 antigenemia and the SI phenotype.
Evaluation of E test as a rapid method for determining MICs for nutritionally variant streptococci
Authors: Douglas C P , Siarakas S , Gottlieb T
Published: January 2020

E test was evaluated as an alternative rapid and simple method of MIC estimation for nutritionally variant streptococci. E test with various media was compared with conventional broth and plate dilution techniques supplemented with 0.001% (wt/vol) pyridoxal hydrochloride (vitamin B6). Of the 14 strains tested with E test, isosensitest agar supplemented with 5% defibrinated horse blood and 0.001% pyridoxal HCl, with and without 0.01% cysteine, gave complete agreement within one twofold-dilution titer of the agar reference method and between 93 and 86% agreement within one twofold-dilution titer of the broth reference method. E test MICs with other media were comparable; however, these were considerably more difficult to interpret. Use of Mueller-Hinton and Columbia-based supplemented agar showed hazy growth and double zoning around the endpoint, respectively. The addition of 0.01% (wt/vol) cysteine to media exhibited no significant effect, and incubation in 5% carbon dioxide (CO2) did not affect MICs.
Development and evaluation of PCR assay for detection of low levels of Cowdria ruminantium infection in Amblyomma ticks not detected by DNA probe
Authors: Peter T F , Deem S L , Barbet A F , Norval R A , Simbi B H , Kelly P J , Mahan S M
Published: January 2020

The sensitivities of a PCR assay and a DNA probe assay were compared for the detection of Cowdria ruminantium in Amblyomma ticks that were fed on C. ruminantium-infected, clinically reacting, and recovered carrier animals. The PCR assay and DNA probe detected infection in 86.0 and 37.0%, respectively, of 100 ticks fed on a febrile animal. In 75 ticks fed on carrier animals, PCR and the DNA probe detected infection in 28.0 and 1.33% of ticks, respectively. This demonstrates that the DNA probe has poor sensitivity for the detection of low levels of infection in ticks and that PCR is necessary for this purpose. The PCR assay had a detection limit of between 1 and 10 C. ruminantium organisms and did not amplify DNA from Ehrlichia canis, which is phylogenetically closely related to C. ruminantium, Theileria parva, or uninfected Amblyomma hebraeum or A. variegatum. PCR detected infection in A. hebraeum and A. variegatum adult ticks infected with one of six geographically different C. ruminantium strains. Amplification was also possible from desiccated ticks and ticks fixed in 70% ethanol, 10% buffered formalin, or 2% glutaraldehyde. The PCR assay supersedes the DNA probe and older detection methods for the detection of C. ruminantium in ticks, particularly those fed on carrier animals, and is suitable for both prospective and retrospective studies which require accurate detection of C. ruminantium in individual ticks. Application of the PCR assay should significantly improve the understanding of heartwater epidemiology, particularly through the determination of field tick infection rates.
PCR amplification of rRNA intergenic spacer regions as a method for epidemiologic typing of Clostridium difficile
Authors: Cartwright C P , Stock F , Beekmann S E , Williams E C , Gill V J
Published: January 2020

From January to March 1993, a suspected outbreak of antibiotic-associated diarrhea occurred on a pediatric oncology ward of the Clinical Center Hospital at the National Institutes of Health. Isolates of Clostridium difficile obtained from six patients implicated in this outbreak were typed by both PCR amplification of rRNA intergenic spacer regions (PCR ribotyping) and restriction endonuclease analysis of genomic DNA. Comparable results were obtained with both methods; five of the six patients were infected with the same strain of C. difficile. Subsequent analysis of 102 C. difficile isolates obtained from symptomatic patients throughout the Clinical Center revealed the existence of 41 distinct and reproducible PCR ribotypes. These data suggest that PCR ribotyping provides a discriminatory, reproducible, and simple alternative to conventional molecular approaches for typing strains of C. difficile.
Restriction endonuclease analysis of field isolates of feline herpesvirus type 1 and identification of heterogeneous regions
Authors: Maeda K , Kawaguchi Y , Ono M , Tajima T , Mikami T
Published: January 2020

The genomic heterogeneity of 78 isolates of feline herpesvirus type 1 (FHV-1) recently isolated from cats suspected to have feline viral rhinotracheitis was analyzed by examining the digestion patterns found with restriction endonuclease MluI. The FHV-1 field isolates were classified into at least three genotypes, namely, the C7301, F2 (an attenuated vaccine strain), and C7805 types. The C7301 type seems to be a major type, since 64 of the 78 isolates belonged to this type. Eight and six isolates belonged to the F2 and C7805 types, respectively. Compared with the C7301 type, the heterogeneous region of the F2 type was localized to a 4.3-kbp EcoRI fragment within the US segment and the heterogeneous region of the C7805 type was localized to a 5.5-kbp XbaI fragment within the UL segment. Northern (RNA) blot analysis revealed no differences in the products transcribed from these regions. In addition, nucleotide sequence analysis showed that the MluI sites not found in the F2 and C7805 types were located in the regions homologous to the herpes simplex virus type 1 gI and UL5 genes, respectively.
Comparison of antimicrobial susceptibility methods for detection of penicillin-resistant Streptococcus pneumoniae
Authors: Kiska D L , Kerr A , Jones M C , Chazotte N N , Eskridge B , Miller S , Jordan M , Sheaffer C , Gilligan P H
Published: January 2020

We sought to determine if commercially available susceptibility tests were accurate in detecting penicillin resistance and relative resistance in Streptococcus pneumoniae. We compared the reference MIC method with oxacillin disk screening and three commercial tests, E-test (AB Biodisk), JustOne (Radiometer America), and MicroScan Pos MIC Panel Type 6 (Baxter Diagnostics), with 80 selected clinical isolates. Thirty-three additional isolates were tested by the reference method and the E-test to further validate the latter method. Oxacillin screening was effective in detecting all penicillin-resistant and relatively resistant strains of S. pneumoniae. The MicroScan method was not effective in detecting penicillin resistance or relative resistance. The JustOne system classified only 6 (35%) of 17 resistant strains correctly, with 11 resistant strains classified as relatively resistant. The E-test correctly classified 30 (83%) of 36 resistant isolates, with 6 resistant isolates interpreted as relatively resistant. For determining penicillin MICs for S. pneumoniae, the E-test was the most accurate of the commercial systems that we studied.
Monitoring of leukocyte cytomegalovirus DNA in bone marrow transplant recipients by nested PCR
Authors: Yuen K Y , Lo S K , Chiu E K , Wong S S , Lau Y L , Liang R , Chan T K , Ng M H
Published: January 2020

A nested PCR assay for the detection of human cytomegalovirus (CMV) DNA was evaluated by weekly monitoring of blood samples taken from 101 bone marrow transplant (BMT) recipients. When peripheral blood leukocytes were used as the source of CMV DNA, even a modified assay with stringent temperature-cycling conditions was as sensitive as the standard assay. The sensitivity, specificity, and positive predictive value of two consecutively positive leukocytic PCR results with this modified assay in predicting CMV disease of 101 patients submitting 1,441 peripheral blood leukocyte samples were found to be 92.1, 63.5, and 60.3%, respectively. The positive predictive value of patients' seropositivity for CMV was 40%, while that of viremia was 72%. However, viremia followed CMV disease by a median of 1.5 days, while the first leukocytic positive PCR assay preceded disease by a median of 14 days. By use of the criteria of two consecutively positive PCR results instead of recipient CMV seropositivity for starting preemptive ganciclovir treatment, 38 of the 43 recipients with isolated single positive or negative assays (groups I and II) would be spared unnecessary ganciclovir treatment. Moreover, two other findings support the use of antiviral prophylaxis before engraftment in high-risk cases and subsequent preemptive treatment of patients with two consecutively positive PCR assays. First, for 7.9% of 76 patients with positive assays (groups II and III), the first positive PCR assay occurred before engraftment, which implied the presence of viral DNA in the blood (DNAemia) soon after transplantation. Second, isolated single positive assays which were clustered around the second to sixth weeks after transplantation were found for 18 patients (group II) and could represent abortive episodes of CMV infection.