bluuebunny/crossref_metadata_2025 huggingface dataset:
Comparison of three stool-processing methods for detection of Salmonella serogroups B, C2, and D by PCR
Authors: Kongmuang U , Luk J M , Lindberg A A
Published: January 2020

Three different stool sample-processing methods (centrifugation, immunomagnetic separation, and selective enrichment cultivation) for the identification of Salmonella serogroups by PCR were studied. The corresponding sensitivities in an ethidium bromide stained-agarose gel were 10(5), 10(3), and 10 bacteria, respectively. The PCR assay with overnight enrichment performed as well as, or even better than, the conventional culture technique. Of 485 clinical stool samples, PCR correctly identified all 230 culture-positive samples as well as mixed Salmonella infections in four cases.
Natural infections with Borrelia spirochetes in two dogs from Florida
Authors: Breitschwerdt E B , Nicholson W L , Kiehl A R , Steers C , Meuten D J , Levine J F
Published: January 2020

Spirochetemia is a rarely reported observation in dogs. We describe the clinical, hematologic, and immunodiagnostic features of two spirochetemic dogs from northern Florida and the subsequent isolation and preliminary characterization of a Borrelia species from one dog in which culture of a sample for spirochetes was attempted. Results of polyacrylamide gel electrophoresis, monoclonal antibody testing, and PCR analysis indicate that the Florida isolate is not Borrelia burgdorferi, the only other member of the genus that has been isolated in Florida. Our findings also indicate that a member of the genus Borrelia potentially causes disease in dogs in Florida and that serologic cross-reactivity of the Florida canine Borrelia isolate with B. burgdorferi probably contributes to the inaccurate diagnosis of canine Lyme disease in the region.
Comparative evaluation of alternative methods for broth dilution susceptibility testing of fluconazole against Candida albicans
Authors: Pfaller M A , Grant C , Morthland V , Rhine-Chalberg J
Published: January 2020

A comparative evaluation of methods for broth macro- and microdilution susceptibility testing of fluconazole was conducted with 119 clinical isolates of Candida albicans. Macro- and microdilution testing were performed according to National Committee for Clinical Laboratory Standards recommendations. For reference macrodilution testing, an 80% inhibition endpoint (MIC 80%) was determined after 48 h of incubation in accordance with National Committee for Clinical Laboratory Standards proposed standard M27-P. Microdilution endpoints were scored as the first tube or well in which a prominent reduction in turbidity (score 2 out of a possible 4) was observed compared with the growth control (Micro MIC-2). Alternative endpoint criteria were assessed independently of the reference MIC 80% and Micro MIC-2 values and included a colorimetric microdilution endpoint determined by using an oxidation-reduction indicator (Alamar Blue; Alamar Bio-sciences Inc., Sacramento, Calif.). The MICs for the two microdilution test systems were read after 24 and 48 h of incubation. The percentage of fluconazole MICs within 2 doubling dilutions of the macrodilution reference values was 94% for both microdilution tests read at 24 h. Agreement was slightly lower at 48 h and ranged from 91 to 93%. Comparison of Micro MIC-2 and colorimetric microdilution MICs resulted in agreements of 97 and 93% at 24 and 48 h, respectively. These results show excellent agreement among alternative methods for fluconazole susceptibility testing.
Microplate-based DNA hybridization assays for detection of human retroviral gene sequences
Authors: Dyster L M , Abbott L , Bryz-Gornia V , Poiesz B J , Papsidero L D
Published: January 2020

Nonisotopic, microwell-based DNA hybridization assays for the specific detection of human immunodeficiency virus type 1 (HIV-1) gag, human T-cell lymphotropic virus type I (HTLV-I) pol, and HTLV-II pol DNA sequences were evaluated. The performances of these detection kits (Gene Detective enzyme oligonucleotide assays; Cellular Products, Inc., Buffalo, N.Y.) were assessed by using clinical samples whose infection status were established by amplification by PCR and then liquid hybridization detection by using virus-specific probes. Peripheral blood mononuclear cell lysates from 59 HIV-1-, 35 HTLV-I-, and 19 HTLV-II-infected individuals and from 15 healthy blood donors were used as substrates for PCR amplification. The results of the study demonstrated a clinical sensitivity of 100%. In addition, the enzyme oligonucleotide assays were able to detect 1 to 10 proviral copies subsequent to PCR amplification, indicating an analytical sensitivity comparable to that of liquid hybridization.
Identification of a granulocytotropic Ehrlichia species as the etiologic agent of human disease
Authors: Chen S M , Dumler J S , Bakken J S , Walker D H
Published: January 2020

Six patients from northern Minnesota and Wisconsin with a febrile illness accompanied by granulocytic cytoplasmic morulae suggestive of ehrlichial infection were identified. Two patients died, and splenic granulocytes of one patient contained cytoplasmic vacuoles with organisms ultrastructurally characteristic of ehrlichiae. From one patient, a 1.5-kb DNA product was amplified by PCR with universal eubacterial primers of 16S rDNA. Analysis of the nucleotide sequence of the amplified product revealed 99.9 and 99.8% similarities with E. phagocytophila and E. equi, respectively, neither of which has previously been known to infect humans. From the variable regions of the determined sequence, a forward primer specific for three organisms (human granulocytic ehrlichia, E. phagocytophila, and E. equi) and a reverse primer for these ehrlichiae and E. platys were designed. By nested PCR with amplification by the universal primers and then reamplification with the specific primers described above, the expected 919-bp product was generated from the blood of the index patient and three additional patients. Blood from these four patients and two more patients with granulocytic morulae contained DNA which was amplified by nested PCR involving a combination of a universal primer and the human granulocytic ehrlichia-E. phagocytophila-E. equi-E. platys group-specific primer. This apparently vector-borne human granulocytic ehrlichia has only 92.5% 16S rDNA homology with E. chaffeensis. Nested PCR with group-specific primers did not amplify E. chaffeensis DNA, and E. chaffeensis-specific primers did not amplify DNAs of the human granulocytic ehrlichia. Thus, six patients were shown to be infected by an Ehrlichia species never previously reported to infect humans.
Application of PCR to detect Norwalk virus in fecal specimens from outbreaks of gastroenteritis
Authors: Moe C L , Gentsch J , Ando T , Grohmann G , Monroe S S , Jiang X , Wang J , Estes M K , Seto Y , Humphrey C
Published: January 2020

Norwalk virus (NV) and other small round-structured viruses (SRSVs) are frequent causes of gastroenteritis outbreaks. The recent cloning and sequencing of the NV genome has made it possible to detect NV and Norwalk-related viruses from fecal specimens by reverse transcription (RT)-PCR. We applied this technique to the examination of a total of 139 fecal specimens from 19 outbreaks characterized by NV serology, including 56 samples from 7 NV outbreaks, 36 from 6 Norwalk-related virus outbreaks, and 47 from 6 outbreaks with SRSVs visualized by electron microscopy that were serologically unrelated to NV. Three primer pairs were evaluated: two pairs in the polymerase region of NV and one pair near the 3' end of the genome. When one set of primers (primer pair 51-3) from the polymerase region was used, 40% of all samples were positive by RT-PCR and specimens from the NV outbreaks were more likely to be positive (64%) than those from outbreaks associated with Norwalk-related viruses (44%) or SRSVs (8%). To determine the relationship of the outbreak strains to NV, we compared the sequences of a 145-base portion of the polymerase gene from 10 specimens obtained from five different outbreaks characterized as NV by serology. No two outbreak strains had the same sequence in this 145-base portion of the polymerase gene, and the identities of the nucleotide and amino acid sequences of these products compared with the sequences of the corresponding region of NV ranged from 62 to 79% and 69 to 90%, respectively. Because of sequence diversity in the polymerase region, the successful application of RT-PCR to investigations of outbreaks of suspected NV-associated gastroenteritis will depend on the use of either multiple primer pairs or primers made against regions of the genome that are more conserved.
Isolation of Borrelia burgdorferi from biopsy specimens taken from healthy-looking skin of patients with Lyme borreliosis
Authors: Kuiper H , van Dam A P , Spanjaard L , de Jongh B M , Widjojokusumo A , Ramselaar T C , Cairo I , Vos K , Dankert J
Published: January 2020

Erythematous skin lesions due to infection with Borrelia burgdorferi will often disappear without antibiotic treatment. The aim of the study was to assess whether after disappearance of the erythematous skin lesion B. burgdorferi is still present in the healthy-looking skin of untreated patients. In six patients, a skin biopsy specimen was taken at the site of a previous erythematous skin lesion 1 to 6 months after disappearance of the lesion. Four of them presented with early disseminated Lyme borreliosis. In one additional patient with early disseminated Lyme borreliosis, the site of a previous tick bite was biopsied. None of these patients had been treated with antibiotics before presentation. The cultures of the skin biopsy specimens of the seven patients showed growth of Borrelia species. By rRNA gene restriction analysis and genospecies-specific PCR, six isolates were classified as Borrelia garinii and one as Borrelia group VS461. These results show that B. burgdorferi can still be cultured from the skin after disappearance of the erythematous skin lesion or at the site of a previous tick bite.
Detection of Borrelia burgdorferi in urine of Peromyscus leucopus by inhibition enzyme-linked immunosorbent assay
Authors: Magnarelli L A , Anderson J F , Stafford K C
Published: January 2020

An inhibition enzyme-linked immunosorbent assay was developed to detect Borrelia burgdorferi, the etiologic agent of Lyme borreliosis, in urine from white-footed mice (Peromyscus leucopus). Of the 87 urine specimens tested from 87 mice collected in widely separated tick-infested sites in Connecticut, 57 (65.5%) contained detectable concentrations of spirochetal antigens. Forty-seven (62.7%) of 75 serum samples analyzed contained antibodies to B. burgdorferi. In culture work with tissues from bladders, kidneys, spleens, or ears, 50 of 87 mice (57.5%) were infected with B. burgdorferi. Thirty-eight (76%) of 50 infected mice had antigens of this spirochete in urine, while 36 (72%) individuals had infected bladders. Of those with infected bladders, 24 (66.7%) mice excreted subunits or whole cells of B. burgdorferi into urine. Successful culturing of B. burgdorferi from mouse tissues, the presence of serum antibodies to this bacterium, and detection of antigens to this spirochete in urine provide further evidence that multiple assays can be performed to verify the presence of B. burgdorferi in P. leucopus.
Detection of bacteremia by Difco ESP blood culture system
Authors: Morello J A , Leitch C , Nitz S , Dyke J W , Andruszewski M , Maier G , Landau W , Beard M A
Published: January 2020

In a multicenter study, the Difco ESP blood culture system (Difco Laboratories, Detroit, Mich.) was compared with the BACTEC NR660 system (Becton Dickinson Diagnostic Instrument Systems, Sparks, Md.). The ESP system monitors each blood culture bottle every 12 to 24 min to detect changes in oxygen consumption and gas production by microbes. Equal volumes of blood were inoculated into aerobic ESP-80A and BACTEC 6A, 16A, or PEDS Plus broths and anaerobic ESP-80N and BACTEC 7A or 17A broths and were incubated for up to 7 days. ESP bottles contain supplemented tryptic soy broth without antimicrobial agent-adsorbing resins. From 7,532 aerobic compliant sets, the ESP system detected 356 clinically significant positive cultures and the BACTEC NR660 system detected 329. From 6,007 anaerobic cultures, the ESP system detected 234 clinically significant positive cultures and the BACTEC NR660 system detected 198. In aerobic broths, 292 organisms were isolated from both systems and 78 organisms were isolated from the ESP system alone, whereas 54 organisms were isolated from the BACTEC NR660 system alone (P < 0.05). Among individual organisms, pneumococci were isolated significantly more often in ESP aerobic broths. In anaerobic broths, 180 organisms were isolated from both systems and 68 organisms were isolated from the ESP system alone, whereas 35 organisms were isolated from the BACTEC NR660 system alone (P < 0.05). Aerobic gram-positive organisms as a group and Candida spp. were isolated significantly more often in ESP anaerobic broths. Both systems detected 207 clinically significant bacteremic episodes and the ESP system alone detected 63, whereas the BACTEC NR660 system alone detected 32 (P < 0.05). Significantly more episodes of bacteremia caused by Staphylococcus epidermidis and anaerobes were detected by the ESP system. The differences in the numbers of organisms detected >6h earlier in ESP broths compared with BACTNEC NR660 broths were significant, as were earlier times to detection. Although the total number of organisms detected was not significantly different, the ESP system alone detected more organisms in a shorter time than did the BACTEC NR660 system alone. The continuous monitoring capability of the ESP system makes it an attractive alternative to the BACTEC NR660 system.
Cokeromyces recurvatus, a mucoraceous zygomycete rarely isolated in clinical laboratories
Authors: Kemna M E , Neri R C , Ali R , Salkin I F
Published: January 2020

Cokeromyces recurvatus Poitras was isolated from an endocervical specimen obtained from a 37-year-old, insulin-dependent diabetic. The patient's diabetic condition had been well controlled for 10 years, and she had no other known medical problem. This is only the fourth time that this zygomycete has been recovered from a human source. While there was no evidence of tissue invasion in the present patient, the observation of fungus-like structures in two separate Papanicolaou-stained cervical smears prepared 1 year apart suggests that C. recurvatus may be capable of colonizing endocervical tissue.
Potential use of BacT/Alert automated blood culture system for antifungal susceptibility testing
Authors: Hazen K C , Chery M P , Han Y
Published: January 2020

A simple, rapid susceptibility test is needed to determine the possible resistance of yeasts to commonly used antifungal agents. The BacT/Alert automated blood culture system was evaluated as one technology for development of such a test. Yeast nitrogen base was used as the growth medium, and amphotericin B was the test antifungal agent. Isolates of various Candida species, Torulopsis glabrata, Saccharomyces cerevisiae, and Cryptococcus neoformans were evaluated. The results suggest that detection of amphotericin B resistance of yeast isolates within 12 to 14 h after inoculation of test medium is possible.
Inhibition of PCR-based assay for Bordetella pertussis by using calcium alginate fiber and aluminum shaft components of a nasopharyngeal swab
Authors: Wadowsky R M , Laus S , Libert T , States S J , Ehrlich G D
Published: January 2020

A PCR-based assay for Bordetella pertussis was inhibited by using a calcium alginate fiber-tipped swab with an aluminum shaft but not by using a Dacron fiber-tipped swab with a plastic shaft. The calcium alginate fiber component inhibited the assay following storage for less than 1 min in a suspension of 10(3) CFU of B. pertussis per ml, whereas the aluminum shaft component required storage for at least 48 h in order to cause inhibition. We recommend the Dacron swab over the calcium alginate swab for collecting specimens for testing in PCR-based assays.
Serologic responses in diagnostic tests for brucellosis in cattle vaccinated with Brucella abortus 19 or RB51
Authors: Stevens M G , Hennager S G , Olsen S C , Cheville N F
Published: January 2020

Serologic responses in the particle concentration fluorescence immunoassay and the card, complement fixation, and tube agglutination tests were measured for 10 weeks after vaccination of cattle with either Brucella abortus 19 or the lipopolysaccharide O-antigen-deficient mutant, strain RB51. The responses of strain 19-vaccinated cattle were positive, whereas those of strain RB51-vaccinated cattle were negative, in all of the tests. These results indicate that cattle vaccinated with strain RB51 fail to produce antibodies that can be detected by conventional serologic tests that are used to diagnose bovine brucellosis.
Antibiotic susceptibilities of AIDS-associated mycoplasmas
Authors: Poulin S A , Perkins R E , Kundsin R B
Published: January 2020

Because mycoplasmas may be a cofactor in the progression of human immunodeficiency virus infection to AIDS, their susceptibilities to antibiotics need to be known in the event that appropriate therapy is required. The mycoplasmas studied were a stock culture strain of Mycoplasma fermentans, two strains of M. fermentans isolated from patients with AIDS, M. fermentans var. incognitus, Mycoplasma penetrans, and Mycoplasma pirum. The antibiotics tested were doxycycline, tetracycline, clindamycin, ofloxacin, erythromycin, azithromycin, and clarithromycin at levels consistent with the attainable levels in serum. By the macrodilution metabolic inhibition method, all six mycoplasma strains were susceptible to doxycycline, tetracycline, clindamycin, ofloxacin, azithromycin, and clarithromycin. M. penetrans was susceptible to erythromycin. The M. fermentans strains and M. pirum were resistant to erythromycin. The macrodilution metabolic inhibition method results showed agreement with the Sensititre Gram Positive MIC Panel results for tetracycline, clindamycin, and erythromycin. MICs of clarithromycin for all six mycoplasma isolates tested were low, indicating susceptibility.
Early serodiagnosis of acute human cytomegalovirus infection by enzyme-linked immunosorbent assay using recombinant antigens
Authors: Vornhagen R , Plachter B , Hinderer W , The T H , Van Zanten J , Matter L , Schmidt C A , Sonneborn H H , Jahn G
Published: January 2020

DNA fragments from eight different reading frames of human cytomegalovirus (HCMV) were generated by PCR and subsequently cloned and expressed in Escherichia coli in fusion with glutathione S-transferase. The recombinant viral antigens were evaluated in immunoblot analyses. The most reactive antigens were purified and further evaluated in ELISAs. For this, sera from healthy blood donors and immunocompetent individuals with acute HCMV infection, and follow-up sera from transplant recipients with acute primary HCMV infection were used. The results of our experiments indicate that only three particular recombinant polypeptides from two viral proteins are necessary for serodiagnosis. While a fragment covering amino acids (aa) 495 to 691 of pp150 (150/1) was the most suitable antigen for the identification of infected individuals in general, immunoglobulin M antibodies against the C-terminal parts of pp150 (aa 862 to 1048; 150/7) and p52 (aa 297 to 433; 52/3) proved to be excellent serological markers to monitor acute HCMV infection. The selected recombinant antigens enable the improvement of serodiagnosis of HCMV-related diseases, especially during the early stages of infection.
Comparison of different strains of Borrelia burgdorferi sensu lato used as antigens in enzyme-linked immunosorbent assays
Authors: Magnarelli L A , Anderson J F , Johnson R C , Nadelman R B , Wormser G P
Published: January 2020

Eight strains of Borrelia burgdorferi sensu lato were tested with serum samples from persons who had Lyme borreliosis or syphilis in class-specific enzyme-linked immunosorbent assays (ELISAs). Antigens of B. burgdorferi sensu stricto, of Borrelia garinii, and of Borrelia spirochetes in group VS461 were prepared from cultured bacteria isolated from ticks, a white-footed mouse (Peromyscus leucopus), or human tissues in North America, the former Soviet Union, and Japan. Nearly all of the serum specimens that contained immunoglobulins to strain 2591, a Connecticut isolate, were also positive in antibody tests with the other seven strains. In general, all eight strains reacted similarly and were suitable as coating antigens in class-specific ELISAs. Assay sensitivities ranged from 82.6 to 100% in analyses for immunoglobulin M and G antibodies. Compared with reference antigen strain 2591, strains 231 (a tick isolate from Canada) and NCH-1 (a human skin isolate from Wisconsin) resulted in higher antibody titers in an ELISA. Syphilitic sera cross-reacted in all tests regardless of the antigen used. Key immunodominant proteins are shared among the closely related strains of B. burgdorferi sensu lato tested, but it is suspected that variations in antigen compositions among these spirochetes may sometimes affect assay performance for detecting serum antibodies.
Comparison of updated Vitek Yeast Biochemical Card and API 20C yeast identification systems
Authors: Fenn J P , Segal H , Barland B , Denton D , Whisenant J , Chun H , Christofferson K , Hamilton L , Carroll K
Published: January 2020

The updated Vitek Yeast Biochemical Card (YBC) was compared with the API 20C by using 409 germ tube-negative yeasts and Geotrichum spp. that were either clinical or proficiency sample isolates. The API 20C was the reference standard. The 409 isolates represented nine genera and 21 species. Morphology agars were inoculated and interpreted for each isolate. The API 20C identified 406 isolates (99.3%), while the Vitek YBC identified 367 (89.7%). Both systems identified the majority of yeasts after 24 h of incubation--73.4% were identified by the API 20C and 77.4% were identified by the Vitek YBC. The Vitek 24-h reading had some incorrect identifications. These included 14 isolates of Candida tropicalis that were identified as Candida parapsilosis (91 to 97% reliability) and 3 isolates of Candida krusei that were called Blastoschizomyces capitatus (Geotrichum capitatum), Candida rugosa, and Candida zeylanoides. In total, the Vitek YBC misidentified 30 isolates, while the API 20C misidentified 3 isolates. In addition, results for 14 isolates with the Vitek YBC were listed under the category "no identification." Morphology agars were required for identification with 89 isolates (21.9%) when the API 20C was used and with 50 isolates (12.6%) when the Vitek YBC was used. Apart from the price of the Vitek instrument, the API 20C costs $1.28 more per test than the Vitek YBC. Overall, the updated Vitek YBC compares favorably with the API 20C in the identification of common yeasts such as Torulopsis glabrata, C. parapsilosis, and Cryptococcus neoformans. However, problems were encountered with the Vitek system in the identification of C. tropicalis, C. krusei, Trichosporon spp., and some Cryptococcus spp. The routine use of morphology agars with either method is recommended.
A case of diarrhea, bacteremia, and fever caused by a novel strain of Escherichia coli
Authors: Bratoeva M P , Wolf M K , Marks J K , Cantey J R
Published: January 2020

A nonenteropathogenic strain of Escherichia coli from a patient with diarrhea and bacteremia possessed the attaching-effacing eae gene, was invasive in the gentamicin invasion assay, and expressed two types of pili and K1 antigen. This unique combination places the strain in a new category of attaching-effacing E. coli.
Identification and characterization of Serpulina hyodysenteriae by restriction enzyme analysis and Southern blot analysis
Authors: Sotiropoulos C , Coloe P J , Smith S C
Published: January 2020

Chromosomal DNA restriction enzyme analysis and Southern blot hybridization were used to characterize Serpulina hyodysenteriae strains. When chromosomal DNAs from selected strains (reference serotypes) of S. hyodysenteriae were digested with the restriction endonuclease Sau3A and hybridized with a 1.1-kb S. hyodysenteriae-specific DNA probe, a common 3-kb band was always detected in S. hyodysenteriae strains but was absent from Serpulina innocens strains. When the chromosomal DNA was digested with the restriction endonuclease Asp 700 and hybridized with two S. hyodysenteriae-specific DNA probes (0.75 and 1.1 kb of DNA), distinct hybridization patterns for each S. hyodysenteriae reference strain and the Australian isolate S. hyodysenteriae 5380 were detected. Neither the 1.1-kb nor the 0.75-kb DNA probe hybridized with Asp 700- or Sau3A-digested S. innocens chromosomal DNA. The presence of the 3-kb Sau3A DNA fragment in S. hyodysenteriae reference strains from diverse geographical locations shows that this fragment is conserved among S. hyodysenteriae strains and can be used as a species-specific marker. Restriction endonuclease analysis and Southern blot hybridization with these well-defined DNA probes are reliable and accurate methods for species-specific and strain-specific identification of S. hyodysenteriae.
Enzyme immunoassay using native envelope glycoprotein (gp160) for detection of human immunodeficiency virus type 1 antibodies
Authors: Nair B C , Ford G , Kalyanaraman V S , Zafari M , Fang C , Sarngadharan M G
Published: January 2020

An enzyme immunoassay using the purified native gp160 for the detection of human immunodeficiency virus type 1 (HIV-1) antibody was developed. This assay was determined to be highly specific, since (i) 157 serum samples that were confirmed negative by Western blot (immunoblot) (WB) were negative, (ii) 41 serum samples from populations with medical conditions that might cause nonspecific assay reactivity were all negative, and (iii) all 15 serum samples that showed false-positive reactions in one or more commercial HIV-1 screening tests were negative. The assay gave 100% specificity with a randomly selected and unlinked panel of 1,000 serum samples from healthy blood donors. The sensitivity of the assay was assessed by testing 238 samples confirmed as HIV-1 antibody positive by a standardized WB assay. All 238 serum samples (100%) were reactive in the native gp160 assay. In a dilution panel of 14 weakly WB-positive serum samples, 7 samples reacted two-to fivefold more strongly in the gp160 assay than in a virus lysate-based assay; the remaining 7 samples gave comparable reactivities in the two tests. The reactivities of 13 of these 14 serum samples in the gp160 assay were higher than in a commercial enzyme immunoassay that uses a recombinant envelope protein as the antigen. The native gp160 assay was more sensitive to identify seroconversion. In a well-characterized panel of sequential blood samples from a seroconverter, the new assay detected antibodies at least one sample ahead of the other commercial assays tested.
Detection of Bartonella (Rochalimaea) quintana by routine acridine orange staining of broth blood cultures
Authors: Larson A M , Dougherty M J , Nowowiejski D J , Welch D F , Matar G M , Swaminathan B , Coyle M B
Published: January 2020

Bartonella quintana was isolated from 34 BACTEC nonradiometric aerobic resin blood cultures for 10 adults. Nine patients were initially diagnosed by routine acridine orange staining of routine cultures that had been incubated for 8 days. All subcultures grew on chocolate agar within 3 to 12 days (median, 6 days). The PLUS 26 high-volume aerobic resin medium, combined with acridine orange stain and subculture, is an effective system for detection and isolation of B. quintana from blood.
Human infections caused by Brevibacterium casei, formerly CDC groups B-1 and B-3
Authors: Gruner E , Steigerwalt A G , Hollis D G , Weyant R S , Weaver R E , Moss C W , Daneshvar M , Brown J M , Brenner D J
Published: January 2020

Forty-one clinical strains of CDC coryneform groups B-1 and B-3 were compared biochemically, by analysis of cell wall sugars, amino acids, and cellular fatty acids, and by DNA relatedness to the type strains of Brevibacterium casei, Brevibacterium epidermidis, and Brevibacterium linens. Twenty-two strains were shown to be B. casei, while five other strains formed a phenotypically inseparable genomospecies in the same genus. The remaining isolates were genetically heterogeneous, and most are probably members of the genus Brevibacterium. They were not further identified, but they were biochemically distinguishable from B. casei. Eleven of the clinical strains of B. casei were isolated from blood, and two each were isolated from cerebrospinal fluid and from pleural fluid. At least five isolates were from multiple blood or cerebrospinal fluid cultures. To our knowledge, these strains are the first described clinical isolates identified as B. casei, which was previously considered to be a nonpathogenic species.
Controlled clinical evaluation of Isolator and ESP aerobic blood culture systems for detection of bloodstream infections
Authors: Kirkley B A , Easley K A , Washington J A
Published: January 2020

A controlled clinical evaluation comparing the Isolator system (Wampole Laboratories, Cranbury, N.J.) and the ESP 80A blood culture bottle in the automated ESP system (Difco Laboratories, Detroit, Mich.) was performed with 10,535 blood culture sets from patients with suspected septicemia. Of 1,150 positive cultures, 844 positive cultures from 285 patients with 394 septic episodes fulfilled the study criteria for minimum blood sample requirements in each system and clinical significance of isolates. The Isolator system detected statistically significantly more positive cultures of Staphylococcus aureus (P < 0.001), Enterococcus spp. (P = 0.007), Escherichia coli (P = 0.001), Alcaligenes xylosoxidans (P = 0.02), Xanthomonas maltophilia (P = 0.01), Candida albicans (P < 0.001), and Candida glabrata (P = 0.05). The Isolator system detected significantly more septic episodes due to S. aureus (P < 0.001), X. maltophilia (P = 0.02), and C. albicans (P = 0.004) than did the ESP 80A bottle; however, the two systems did not otherwise significantly differ in their abilities to detect septic episodes due to other organisms.
Rapid biochemical screening for Salmonella, Shigella, Yersinia, and Aeromonas isolates from stool specimens
Authors: De Ryck R , Struelens M J , Serruys E
Published: January 2020

Four screens for the rapid (4 to 6 h) biochemical detection of pathogens from enteric isolation media are described. The Salmonella screen consisted of Kligler iron agar (KIA), motility-indole-urea-tryptophan-deamination semisolid medium (MIU-TDA), and the o-nitrophenyl-beta-D-galactopyranoside (ONPG) test; the Shigella screen consisted of KIA, MIU-TDA, the ONPG test, and the lysine decarboxylation-indole test; the Yersinia screen consisted of a rhamnose broth; the Aeromonas screen consisted of a xylose agar plate. When tested on 2,102 fresh isolates and 71 stock strains, the screens correctly detected 212 enteric pathogens (sensitivity, 100%), with a specificity of 98.1%.
Comparison of E test with agar dilution for antimicrobial susceptibility testing of Neisseria gonorrhoeae
Authors: Van Dyck E , Smet H , Piot P
Published: January 2020

A collection of 150 Neisseria gonorrhoeae isolates from Africa, where various resistance mechanisms among N. gonorrhoeae isolates are common, was used to the compare E test (AB Biodisk, Solna, Sweden) with agar dilution susceptibility testing. MICs obtained by the E test agreed within 1 log2 concentration by the agar dilution method for 97.5, 97.3, 96.6, 94, and 84.7% of the tested isolates for penicillin, ciprofloxacin, chloramphenicol, tetracycline, and trimethoprim-sulfamethoxazole, respectively. No significant difference in susceptibility categorization was observed between either method. The E test is an attractive alternative to the agar dilution technique and is a more appropriate method for N. gonorrhoeae susceptibility testing in developing countries.
Biotypes of Arcanobacterium haemolyticum
Authors: Carlson P , Lounatmaa K , Kontiainen S
Published: January 2020

Colony morphology, beta hemolysis on horse blood agar, beta-glucuronidase activity, and ability to ferment sucrose and/or trehalose defined two biotypes of Arcanobacterium haemolyticum. One, the smooth type, grew as smooth, beta-hemolytic colonies and was beta-glucuronidase negative but often fermented sucrose and/or trehalose, while the other, the rough type, grew as rough colonies and was nonhemolytic, beta-glucuronidase positive, and negative for sucrose and trehalose fermentation. About 75% of the A. haemolyticum strains studied (n = 138) were of the smooth type. The smooth type predominated in wound infections, while the rough type was isolated almost exclusively from respiratory tract specimens; thus, 84% of the smooth-type strains were derived from wounds and 91% of the rough-type strains were isolated from respiratory tracts.
Development of a standardized screening method for detection of vancomycin-resistant enterococci
Authors: Swenson J M , Clark N C , Ferraro M J , Sahm D F , Doern G , Pfaller M A , Reller L B , Weinstein M P , Zabransky R J , Tenover F C
Published: January 2020

The incidence of vancomycin resistance among enterococci is increasing in the United States and elsewhere in the world, but automated susceptibility testing methods have difficulty detecting resistance expressed by certain strains. The agar screening method described by Willey et al. (B. M. Willey, B. N. Kreiswirth, A. E. Simor, G. Williams, S. R. Scriver, A. Phillips, and D. E. Low, J. Clin. Microbiol. 30:1621-1624, 1992) has been proposed as a reliable method for confirming vancomycin resistance. In this study, we investigated various parameters associated with the agar screening method and, on the basis of the findings, established optimum testing conditions for the method. First, to evaluate media and vancomycin concentrations, one laboratory used Mueller-Hinton and brain heart infusion agars supplemented with 4, 6, and 8 micrograms of vancomycin per ml to test 100 genetically characterized enterococcal strains. On the basis of the results obtained, brain heart infusion agar supplemented with 6 micrograms of vancomycin per ml was selected for further study. Subsequently, eight laboratories used the medium to test both reference and clinical isolates. There was very good performance with the reference strains and, among 158 clinical isolates tested, the method demonstrated sensitivity and specificity of 100% and from 96 to 99%, respectively.
Characterization of epidemic Neisseria meningitidis serogroup C strains in several Brazilian states
Authors: Sacchi C T , Tondella M L , de Lemos A P , Gorla M C , Berto D B , Kumiochi N H , Melles C E
Published: January 2020

Epidemic strains of the Neisseria meningitidis C:2b:P1.3 electrophoretic type 11 complex were responsible for an outbreak in Curitiba, Parana State, Brazil, from 1990 to 1991. Strains of this complex were also isolated in other Brazilian states and were responsible for a meningococcal disease epidemic in São Paulo State in 1990. Serotyping both with monoclonal antibodies and by multilocus enzyme electrophoresis was useful for typing these epidemic strains related to the increased incidence of meningococcal disease. The genetic similarity of members of the electrophoretic type 11 complex was confirmed by the ribotyping method by using EcoRI or ClaI endonuclease restriction enzymes.
Oligopeptide-based enzyme immunoassay for ovine lentivirus antibody detection
Authors: Kwang J , Torres J V
Published: January 2020

Ovine progressive pneumonia virus (OPPV) is a lentivirus which causes a progressive disease in sheep. Immunodominant epitopes have been identified in the envelope gp40 glycoprotein. Synthetic peptides representing these regions are able to detect the presence of OPPV antibodies in 96% of infected sheep.
Rapid detection of methicillin-resistant Staphylococcus aureus by Crystal MRSA ID System
Authors: Qadri S M , Ueno Y , Imambaccus H , Almodovar E
Published: January 2020

A commercially available method for the rapid detection of methicillin-resistant Staphylococcus aureus (BBL Crystal MRSA ID System) was evaluated and compared with conventional methods. All 52 isolates of methicillin-susceptible and 142 isolates of intrinsic methicillin-resistant S. aureus were correctly identified in 4 h by the test method, whereas correct identification took 11 to 24 h by conventional methods. The test is simple, rapid, and easy to perform and the results are easy to interpret.
Comparison of cord blood and peripheral blood mononuclear cells as targets for viral isolation and drug sensitivity studies involving human immunodeficiency virus type 1
Authors: Salomon H , Belmonte A , Nguyen K , Gu Z , Gelfand M , Wainberg M A
Published: January 2020

We have shown that umbilical cord blood mononuclear cells (CBMC) are at least as sensitive as peripheral blood mononuclear cells (PBMC) for isolation of human immunodeficiency virus type 1 from the PBMC of infected individuals. Viral replication was more efficiently monitored by a p24 antigen capture assay than by a viral reverse transcriptase test, regardless of whether CBMC or PBMC were employed. We also found that CBMC and PBMC yielded similar results with regard to the susceptibility profiles of both wild-type and drug-resistant variants of human immunodeficiency virus type 1 for 3'-azido-3'-deoxythymidine, 2',3'-dideoxycytidine, and the (-) enantiomer of 2',3'-dideoxy-3'-thiacytidine. Finally, viruses isolated on CBMC could be routinely grown on PBMC and vice versa.
Quantification of human immunodeficiency virus type 1-infected mononuclear cells in peripheral blood of seropositive subjects by newly developed flow cytometry analysis of the product of an in situ PCR assay
Authors: Re M C , Furlini G , Gibellini D , Vignoli M , Ramazzotti E , Lolli E , Ranieri S , La Placa M
Published: January 2020

The presence of human immunodeficiency virus type 1 (HIV-1) proviral DNA in peripheral blood mononuclear cells (PBMC) of three groups (group 1, more than 500 CD4+ T cells per microliter; group 2, between 200 and 499 CD4+ T cells per microliter; group 3, fewer than 200 CD4+ T cells per microliter) of HIV-1-infected patients, in different stages of the disease, was determined by using a newly developed flow cytometry analysis of the product of in situ PCR assay and compared with other markers of viral replication (HIV-1 p24 antigenemia and viral isolation). Results showed varied percentages of HIV-1-infected PBMC, ranging from 0.6 to 20%. Patients with more than 500 CD4+ T cells per microliter showed the lowest percentage of HIV-1-infected PBMC (2.1 +/- 1.7), compared with patients with CD4+ T-cell counts of between 200 and 499 per microliter (6.5% +/- 4.1%; P < 0.001) and patients with fewer than 200 CD4+ T cells per microliter (4.9% +/- 4.7%; P < 0.05). The difference in the percentage of HIV-1-infected PBMC between group 2 and group 3 patients may in part reflect the loss of CD4+ T lymphocytes in more advanced stages of the disease. However, the results clearly indicate a striking coincidence between the fall of the CD4+ T-cell count below 400/microliter and the sharp increase in PBMC virus loading and p24 antigenemia. Since the procedure is relatively easy to perform, it could be used to monitor the evolution of HIV-1 infection and may prove a useful adjunct in tailoring therapeutic strategies.
Evaluation of new monoclonal antibody-based latex agglutination test for detection of cryptococcal polysaccharide antigen in serum and cerebrospinal fluid
Authors: Kiska D L , Orkiszewski D R , Howell D , Gilligan P H
Published: January 2020

We evaluated the performance of CRYPTO-LEX (Trinity Laboratories, Inc., Raleigh, N. C.), a new mouse immunoglobulin M monoclonal antibody latex agglutination reagent which reacts with the capsular polysaccharide of the four serogroups of Cryptococcus neoformans. This test was compared with CALAS (Meridian Diagnostics, Cincinnati, Ohio) for the ability to detect cryptococcal antigen in serum and cerebrospinal fluid (CSF). A total of 580 clinical specimens (327 serum and 253 CSF samples), primarily from human immunodeficiency virus-infected patients, were tested in this study. Sixty-seven specimens (44 serum and 23 CSF samples) were positive for cryptococcal antigen with both tests, and 511 (282 serum and 229 CSF samples) were negative. The two latex reagents agreed for 326 of 327 serum specimens (44 positives and 282 negatives). One serum specimen with a titer of 1:2 was CALAS positive but CRYPTO-LEX negative. The titer correlation coefficient for the two tests was 0.884 when two highly discordant serum specimens were eliminated from analysis of the data. The two latex tests agreed for 252 of 253 CSF specimens (23 positives and 229 negatives). One specimen with a titer of 1:2 was positive with CALAS and negative by CRYPTO-LEX. The correlation coefficient of the two tests for CSF titers was 0.886. The sensitivity and specificity of CRYPTO-LEX were 97 and 100%, respectively, with a 99.6% correlation with CALAS. These data show that the performance of CRYPTO-LEX is comparable to that of CALAS for detection of cryptococcal antigen in serum and CSF.
Endocarditis in a dog due to infection with a novel Bartonella subspecies
Authors: Breitschwerdt E B , Kordick D L , Malarkey D E , Keene B , Hadfield T L , Wilson K
Published: January 2020

Vegetative valvular endocarditis involving the aortic and, to a lesser extent, mitral valves was diagnosed echocardiographically in a 3-year-old spayed female Labrador retriever. Historically, the dog had been treated with tetracycline hydrochloride and prednisolone for positive seroreactivity to Ehrlichia canis and antinuclear antigens. Although three aerobic and anaerobic blood cultures failed to grow bacteria, blood cultured simultaneously by the lysis centrifugation technique grew a fastidious, gram-negative organism. Despite an initial therapeutic response, the owner elected euthanasia 17 days later. Necropsy confirmed aortic and mitral valvular endocarditis. Bacteria phenotypically similar to Bartonella species were visualized in the heart valve by light and electron microscopy, and Bartonella DNA from a frozen heart valve was amplified by PCR. Subsequent phenotypic and genotypic characterization of the isolate, including biochemical testing, cellular fatty acid analysis, DNA hybridization, and sequencing of the 16S rRNA gene indicated that this organism, which can induce endocarditis in dogs, is a novel Bartonella subspecies containing an insertion sequence unique among currently recognized Bartonella species. The name Bartonella vinsonii subsp. berkoffii subsp. nov. will be proposed for this organism.
Auxotrophic variants of Pseudomonas aeruginosa are selected from prototrophic wild-type strains in respiratory infections in patients with cystic fibrosis
Authors: Barth A L , Pitt T L
Published: January 2020

Twenty-four nutritionally dependent (auxotrophic) Pseudomonas aeruginosa strains were isolated from 20 cystic fibrosis (CF) patients and tested for their amino acid requirements. Two different methods were necessary to identify the nutritional status of all isolates. Methionine was the most common single amino acid required (9 of 24 isolates), followed by leucine and arginine or ornithine. In total, a requirement for 12 different compounds or combination of compounds was demonstrated. Auxotrophic and prototrophic pairs of isolates from the same patient were compared by macrorestriction analysis of DNA in pulsed-field gel electrophoresis. Thirteen of 18 pairs analyzed presented identical restriction fragment length polymorphism profiles following digestion of DNA with XbaI. Three of the remaining pairs showed percentage similarities of 77, 91, and 98%, and the profiles of two pairs could not be compared because of the excessive degradation of their DNA. These results suggest that auxotrophic and prototrophic P. aeruginosa isolates colonizing the same CF patient constitute an isogenic group and raise the possibility that auxotrophs are selected from the prototrophic population during the course of pulmonary infection in CF patients.
Four-year prospective study of STAPH-IDENT system and conventional method for reference identification of Staphylococcus, Stomatococcus, and Micrococcus spp.
Authors: Rhoden D L , Miller J M
Published: January 2020

A 4-year prospective study compared the accuracy of the STAPH-IDENT system (bioMérieux Vitek, Inc., Hazelwood, Mo.) with that of the reference procedure of the Centers for Disease Control and Prevention for the identification of Staphylococcus species, Stomatococcus mucilaginosus, and Micrococcus species. The study compared the results from 1,106 cultures (500 eye cultures, 217 strains submitted for reference identification, and 389 known stock strains) representing 21 species of the family Micrococcaceae. The overall agreement of genus and species identifications was 81.1%. The percent agreement for the five most common clinical isolates was as follows: Staphylococcus epidermidis, 97.1% (517 isolates); Staphylococcus hominis, 82.5% (57 isolates); Staphylococcus aureus, 77.2% (162 isolates); Staphylococcus haemolyticus, 75.8% (61 isolates); and Staphylococcus warneri, 64.1% (39 isolates). The lowest percent agreement was with Staphylococcus cohnii (11.1%; (9 isolates). Of the 217 isolates sent to the Centers for Disease Control and Prevention for identification, 60.4% (131) were correctly identified by the STAPH-IDENT system. Of these, S. epidermidis accounted for 23.9%, S. aureus accounted for 15.6%, S. warneri accounted for 6.9%, Staphylococcus lugdunensis accounted for 6.5%, S. haemolyticus accounted for 5.5%, and S. hominis accounted for 4.1%. The STAPH-IDENT system did not perform adequately when dealing with commonly encountered organisms and is unsuitable for identifying uncommon isolates.
Evaluation of nucleic acid-based test (PACE 2C) for simultaneous detection of Chlamydia trachomatis and Neisseria gonorrhoeae in endocervical specimens
Authors: Iwen P C , Walker R A , Warren K L , Kelly D M , Hinrichs S H , Linder J
Published: January 2020

A nucleic acid-based test (Gen-Probe PACE 2C System) was evaluated for the ability to detect Chlamydia trachomatis and Neisseria gonorrhoeae from endocervical specimens in a single assay. Three swab samples, randomized for collection order, were obtained from each patient and tested by N. gonorrhoeae and C. trachomatis culture and by the PACE 2C probe assay. Fifty of 395 specimens were culture positive for N. gonorrhoeae (17 specimens), C. trachomatis (26 specimens), or both (7 specimens), of which PACE 2C testing detected 48 specimens. The PACE 2C assay was positive for 56 specimens, including 8 specimens not positive by culture. Of the total of 10 discrepancies between culture and PACE 2C results, resolution testing yielded four false-negative culture, four false-positive PACE 2C, and two false-negative PACE 2C results. The sensitivity, specificity, and positive and negative predictive values for PACE 2C after reevaluation were 96.3, 98.8, 92.9 and 99.4%, respectively. The overall sensitivities for C. trachomatis and N. gonorrhoeae culture were 89.2 and 88.9%, respectively. The prevalence rate for C. trachomatis was 9.4%, and that for N. gonorrhoeae was 6.8%. The Gen-Probe PACE 2C System is a reliable alternative for screening endocervical specimens for both C. trachomatis and N. gonorrhoeae in a single assay.