bluuebunny/crossref_metadata_2025 huggingface dataset:
Diagnostic value of the strand displacement amplification method compared to those of Roche Amplicor PCR and culture for detecting mycobacteria in sputum samples
Authors: Ichiyama S , Ito Y , Sugiura F , Iinuma Y , Yamori S , Shimojima M , Hasegawa Y , Shimokata K , Nakashima N
Published: January 2020

We compared the ability of the semiautomated BDProbeTec-SDA system, which uses the strand displacement amplification (SDA) method, with that of the Roche Amplicor-PCR system and the Septi-Chek AFB culture system to directly detect Mycobacterium tuberculosis complex (MTB) and other mycobacteria in sputum samples. A total of 530 sputum samples from 299 patients were examined in this study. Of the 530 samples, 129 were culture positive for acid-fast bacilli with the Septi-Chek AFB system; 95 for MTB, 29 for M. avium-M. intracellulare complex (MAC), and 5 for other mycobacteria. The BDProbeTec-SDA system detected 90 of the 95 samples culture positive for MTB (sensitivity, 94.7%), and the Amplicor-PCR system detected 85 of the 95 samples culture positive for MTB (sensitivity, 89.5%). The specificity of each system, based on the clinical diagnosis, was 99.8% for SDA and 100% for PCR, respectively. Among the 29 samples culture positive for MAC, the BDProbeTec-SDA system detected MAC in 24 samples (sensitivity, 82.8%), whereas the Amplicor-PCR system detected MAC in 23 samples (sensitivity, 79.3%). The specificities of the systems were 98.3 and 100%, respectively. The high degrees of sensitivity and specificity of the BDProbeTec-SDA system suggest that it should be very useful in clinical laboratories for the rapid detection of mycobacteria in sputum samples.
Typing of Pneumocystis carinii f. sp. hominis by single-strand conformation polymorphism of four genomic regions
Authors: Hauser P M , Francioli P , Bille J , Telenti A , Blanc D S
Published: January 2020

To better investigate Pneumocystis carinii f. sp. hominis epidemiology, we have developed a molecular typing method. Because of the limited genetic variability of the P. carinii hominis genome, a multitarget approach was used. Four variable regions of the genome were amplified by PCR, polymorphism in each region was assessed by the single-strand conformation polymorphism (SSCP) technique, and the results for the four regions of each patient were combined. Bronchoalveolar lavage specimens collected from 11 patients were examined. Four patients were probably infected by a single strain, since their specimens yielded simple SSCP patterns (two bands corresponding to one allele). The combinations of these patterns were unique, suggesting that the strains which infected these patients were different. For the other seven patients, complex patterns were found (three or four bands corresponding to two alleles). The presence of more than one allele of a region in a patient is likely to be due to coinfection. Polymorphism was also assessed by sequencing, which revealed variations at nucleotide positions previously reported to vary. About half of the observed alleles had already been reported by laboratories in different countries. Multitarget typing of P. carinii hominis by PCR-SSCP should allow investigation of strain diversity and thus be useful for future epidemiological studies.
Emergence of a unique O3:K6 clone of Vibrio parahaemolyticus in Calcutta, India, and isolation of strains from the same clonal group from Southeast Asian travelers arriving in Japan
Authors: Okuda J , Ishibashi M , Hayakawa E , Nishino T , Takeda Y , Mukhopadhyay A K , Garg S , Bhattacharya S K , Nair G B , Nishibuchi M
Published: January 2020

Active surveillance of Vibrio parahaemolyticus infection among hospitalized patients in Calcutta, India, was initiated in January 1994. The incidence of cases of V. parahaemolyticus infection suddenly increased in February 1996 and has remained high since then. One hundred thirty-four strains of V. parahaemolyticus isolated from January 1994 to August 1996 were examined for serovar, the presence of the thermostable direct hemolysin gene (tdh) and tdh-related hemolysin genes (trh1 and trh2), production of urease, and antibiogram. Strains of the O3:K6 serovar appeared for the first time in February 1996. The O3:K6 serovar strains accounted for 50 to 80% of the strains isolated during the high-incidence period (February to August 1996). All of the serovar O3:K6 strains carried the tdh gene but not the trh genes and did not produce urease. All of the isolates except two were sensitive to all of the antibiotics tested. These and the results of analysis by an arbitrarily primed PCR method indicated that the O3:K6 serovar strains belong to a unique clone. When the O3:K6 serovar strains, isolated from travelers arriving in Japan from Southeast Asian countries, were compared by the arbitrarily primed PCR method, the strains isolated between 1982 and 1993 were distinct from Calcutta O3:K6 while the strains isolated in 1995 and 1996 were indistinguishable from the Calcutta O3:K6 strains. The results suggest that this unique O3:K6 clone may have become prevalent not only in Calcutta but also in Southeast Asian countries very recently. Not only the O3:K6 strains but also the non-O3:K6, tdh-bearing strains isolated in 1996 produced thermostable direct hemolysin at high levels, and thus the level of hemolysin produced does not appear to have influenced the high incidence of serovar O3:K6 strains.
Determination of hepatitis C virus genotypes in the United States by cleavase fragment length polymorphism analysis
Authors: Marshall D J , Heisler L M , Lyamichev V , Murvine C , Olive D M , Ehrlich G D , Neri B P , de Arruda M
Published: January 2020

We describe the application of a new DNA-scanning method, which has been termed Cleavase Fragment Length Polymorphism (CFLP; Third Wave Technologies, Inc., Madison, Wis.), for the determination of the genotype of hepatitis C virus (HCV). CFLP analysis results in the generation of structural fingerprints that allow discrimination of different DNA sequences. We analyzed 251-bp cDNA products generated by reverse transcription-PCR of the well-conserved 5'-noncoding region of HCV. We determined the genotypes of 87 samples by DNA sequencing and found isolates representing 98% of the types typically encountered in the United States, i.e., types 1a, 1b, 2a/c, 2b, 3a, and 4. Blinded CFLP analysis of these samples was 100% concordant with DNA sequencing results, such that closely related genotypes yielded patterns with strong familial resemblance whereas more divergent sequences yielded patterns with pronounced dissimilarities. In each case, the aggregate pattern was indicative of genotypic grouping, while finer changes suggested subgenotypic differences. We also assessed the reproducibility of CFLP analysis in HCV genotyping by analyzing three distinct isolates belonging to a single subtype. These three isolates yielded indistinguishable CFLP patterns, as did replicate analysis of a single isolate. This study demonstrates the suitability of this technology for HCV genotyping and suggests that it may provide a low-cost, high-throughput alternative to DNA sequencing or other, more costly or cumbersome genotyping approaches.
Evaluation of the BBL Crystal Anaerobe identification system
Authors: Cavallaro J J , Wiggs L S , Miller J M
Published: January 2020

The BBL Crystal Anaerobe (ANR) identification system was evaluated, and the results were compared with those from conventional anaerobic methods. We tested 322 clinically significant anaerobic bacteria according to the manufacturer's instructions. The system identified correctly 286 of 322 (88.8%) of the anaerobic bacteria tested. Of these, 263 of 322 (81.7%) were identified correctly on initial testing and 49 were identified correctly only to the genus level; on repeat testing, 23 of 49 (46.9%) were identified correctly to both the genus and the species levels. A total of 26 (8.5%) were misidentified at the species level, and 10 (3.1%) were not identified. Performance characteristics for individual strains varied. The system correctly identified all tested strains of Campylobacter, Desulfomonas, Desulfovibrio, Leptotrichia, Mobiluncus, Peptostreptococcus, Porphyromonas, Provetella, Propionibacterium, Tisierella, and Veillonella and 36 of 37 (97.3%) Actinomyces strains, 42 of 46 (91.3%) B. fragilis group strains, 79 of 103 (76.7%) Clostridium strains, (however, the system failed to identify any of the 7 C. innocuum and 9 C. tetani strains tested), and 8 of 15 (53.3%) Bacteroides strains. This system was easy to use, did not involve the addition of reagents, and was faster than conventional anaerobic procedures. It would be a useful addition to the anaerobe laboratory of most hospitals.
A new agent of mycobacterial lymphadenitis in children: Mycobacterium heidelbergense sp. nov
Authors: Haas W H , Butler W R , Kirschner P , Plikaytis B B , Coyle M B , Amthor B , Steigerwalt A G , Brenner D J , Salfinger M , Crawford J T , Böttger E C , Bremer H J
Published: January 2020

Nontuberculous mycobacterial lymphadenitis presents an increasing clinical problem in immunocompetent young children. A slowly growing, nonphotochromogenic mycobacterium was recovered twice (isolates 2553/91 and 2554/91) from the lymphatic tissue of a child with recurrent cervical lymphadenitis. It could be differentiated biochemically from described Mycobacterium species, although it most closely resembled Mycobacterium malmoense by thin-layer chromatography and high-performance liquid chromatography of mycolic acids. A striking characteristic of the isolate was its high degree of susceptibility to antituberculous drugs in vitro, including isoniazid. Direct determination of the 16S rRNA gene sequence revealed a unique sequence and positioned the strain phylogenetically on a branch separate from M. malmoense within a group of slowly growing mycobacteria that show a high degree of similarity to M. simiae at the 16S rRNA gene level. Despite 99.6% sequence identity with M. simiae at the 16S rRNA gene level, DNA-DNA hybridization studies (hydroxyapatite method) demonstrated DNA relatedness of less than 40%. We conclude that this organism is a new species for which we propose the name M. heidelbergense. A culture of the type strain, strain 2554/91, has been deposited in the American Type Culture Collection as strain ATCC 51253.
Characterization of group A Streptococcus strains recovered from Mexican children with pharyngitis by automated DNA sequencing of virulence-related genes: unexpectedly large variation in the gene (sic) encoding a complement-inhibiting protein
Authors: Mejia L M , Stockbauer K E , Pan X , Cravioto A , Musser J M
Published: January 2020

Sequence variation was studied in several target genes in 54 strains of group A Streptococcus (GAS) cultured from children with pharyngitis in Mexico City. Although 16 distinct emm alleles were identified, only 4 had not been previously described. Virtually all bacteria (31 of 33 [94%] with the streptococcal pyrogenic exotoxin gene (speA) had emm1-related, emm3, or emm6 alleles. The gene (sic) encoding an extracellular GAS protein that inhibits complement function was unusually variable among isolates with the emm1 family of alleles, with a total of seven variants identified. The data suggest that many GAS strains infecting Mexican children are genetically similar to organisms commonly encountered in the United States and western Europe. Sequence variation in the sic gene is useful for rapid differentiation among GAS isolates with the emm1 family of alleles.
Evaluation of Vitek GNI+ and Becton Dickinson Microbiology Systems Crystal E/NF identification systems for identification of members of the family Enterobacteriaceae and other gram-negative, glucose-fermenting and non-glucose-fermenting bacilli
Authors: O'Hara C M , Westbrook G L , Miller J M
Published: January 2020

We evaluated the Vitek GNI+ and Becton Dickinson Crystal E/NF identification systems for their ability to accurately identify 619 and 626 strains, respectively, of members of the family Enterobacteriaceae and other glucose-fermenting and non-glucose-fermenting gram-negative rods. All strains tested were taken from a stock collection and passed three times on 5% sheep blood agar prior to testing. These strains represented a more rigorous challenge to both systems than one resulting from the testing of consecutive clinical isolates. Testing with both systems was done according to the manufacturers' instructions, and tests were repeated in duplicate when errors occurred. Vitek version 5.01 and Crystal version 3.0 softwares were used for identifications. The identification results from each system were compared with identifications previously determined with reference biochemicals. At the completion of the appropriate incubation period, the GNI+ and Crystal systems correctly identified 80.1 and 71.1% of the total isolates, respectively. After additional tests suggested by the software programs were completed, the GNI+ had an accuracy of 87.6% and the Crystal system's accuracy had improved to 87.9%. The error rates for the GNI+ and Crystal systems were 6.5 and 5.3%, respectively. A report of "no identification" was given for 6.0 and 6.9% of the isolates, respectively, and was associated with no particular organism group. One isolate each of Acinetobacter lwoffii and Vibrio alginolyticus would not grow in the Vitek card. The average times to detection for correct enteric identifications in the GNI+ system were 4.1 and 6.8 h for nonenteric identifications, while the Crystal results were routinely read at 18 h. We conclude that there was no significant difference (P > 0.05) between the results of the GNI+ card and those of the Crystal E/NF system after additional testing was performed with the group of organisms tested, but the overall accuracy for both systems in this study was below 90%.
Comparison of the Amplicor HIV-1 monitor test and the nucleic acid sequence-based amplification assay for quantitation of human immunodeficiency virus RNA in plasma, serum, and plasma subjected to freeze-thaw cycles
Authors: Griffith B P , Rigsby M O , Garner R B , Gordon M M , Chacko T M
Published: January 2020

The Amplicor HIV-1 Monitor test was compared to the nucleic acid sequence-based amplification (Nasba) assay system for the quantitation of human immunodeficiency virus (HIV) RNA in three different types of clinical samples: plasma, serum, and plasma subjected to freeze-and-thaw cycles. Each assay detected HIV RNA in the same 73 (90%) of 81 samples tested, and the quantitative results obtained with the two assays were significantly correlated. Both assays detected higher RNA levels in patients with CD4+ cell counts lower than 200 cells/mm3 than in patients with CD4+ cell counts higher than 200 cells/mm3. In addition, RNA levels in plasma higher than 5 logs predicted higher numbers of clinical events than did RNA levels in plasma lower than 5 logs. Quantitation of HIV RNA in paired plasma and serum samples showed lower HIV RNA content in serum than in the paired plasma sample, with mean differences between HIV RNA contents of plasma and serum of 0.54 and 0.28 log RNA copy/ml by the Nasba assay and the Amplicor HIV-1 Monitor assay, respectively. No significant loss of HIV RNA was detected with either assay in plasma samples subjected to multiple freeze-and-thaw cycles. These studies demonstrate that the Nasba and Amplicor assays perform similarly with plasma and serum samples. Further, the results indicate that freeze-and-thaw cycles do not result in significant loss of detectable HIV RNA.
Algaemia due to Prototheca wickerhamii in a patient with myasthenia gravis
Authors: Mohabeer A J , Kaplan P J , Southern P M , Gander R M
Published: January 2020

Prototheca wickerhamii is a rare cause of systemic infection in humans. While some cases occur in previously healthy individuals, others are associated with a variety of preexisting diseases. Here we present, for the first time, a case of P. wickerhamii algaemia in a patient with myasthenia gravis. The patient was successfully treated with amphotericin B.
Serodiagnosis of American trypanosomosis by using nonpathogenic trypanosomatid antigen
Authors: Monteón V M , Guzmán-Rojas L , Negrete-García C , Rosales-Encina J L , Lopez P A
Published: January 2020

Crithidia luciliae, a nonpathogenic trypanosomatid, could provide a good alternative source of antigen for serodiagnosis of Chagas' disease. An enzyme-linked immunosorbent assay showed 100% sensitivity and 83% specificity when 91 human serum samples from Chagas' disease patients and 127 human serum samples from people suffering from toxoplasmosis (21 samples), leishmaniasis (32 samples), systemic rheumatic diseases (33 samples), and heart diseases (41 samples) were tested simultaneously with Trypanosoma cruzi and C. luciliae crude extracts. By Western blotting, an immunodominant band (30 kDa) was recognized by chagasic sera on the C. luciliae crude extract; specificity reached 97% with respect to this protein band. The carbohydrate moieties were not antigenic.
Peritonitis associated with a CDC group EO-3 organism
Authors: Daley D , Neville S , Kociuba K
Published: January 2020

A 63-year-old female with chronic renal failure on continuous ambulatory peritoneal dialysis developed chronic peritonitis. A CDC group EO-3 organism was isolated from the peritoneal dialysis fluid on five occasions over a period of 4 months. This is the first reported isolation of this organism in which it is associated with a patient on continuous ambulatory peritoneal dialysis.
Characterization of saccharolytic Bacteroides and Prevotella isolates from infected dog and cat bite wounds in humans
Authors: Alexander C J , Citron D M , Hunt Gerardo S , Claros M C , Talan D , Goldstein E J
Published: January 2020

Saccharolytic, nonpigmented, anaerobic gram-negative rods isolated from infected dog and cat bite wounds in humans have been poorly characterized, and most are not included in the databases of kits used for anaerobic identification; thus, they are problematic for clinical laboratories to identify. Fifty strains isolated from such wounds were characterized with commercial kits for preformed-enzyme detection, carbohydrate fermentation, and other biochemical tests. PCR fingerprinting was performed on these strains to further characterize subgroups within these species. Bacteroides tectum is a frequent isolate in bite wounds and resembles Prevotella bivia in colony morphology and saccharolytic activity, except that it grows in 20% bile and hydrolyzes esculin. Profile numbers generated by various kits associate B. tectum with P. bivia, Prevotella oralis group, or Prevotella melaninogenica. PCR fingerprinting identified at least four subgroups and confirmed the heterogeneous nature of this species. Prevotella heparinolytica was also frequently isolated from these bite wounds. It produces indole and generates a profile number in preformed-enzyme kits that is usually associated with Bacteroides uniformis. However, it is bile sensitive and quite distinct from the Bacteroides fragilis group of anaerobes. The PCR fingerprint profiles generated by strains of P. heparinolytica were very similar to that of the type strain and to each other. Prevotella zoogleoformans, occasionally isolated from dog and cat bite wounds in humans, resembles P. heparinolytica except for a negative indole test. Clinical laboratories should be aware of the characteristics of these animal species when identifying isolates from animal bite wounds in humans.
Identification and serotyping of Ornithobacterium rhinotracheale
Authors: van Empel P , van den Bosch H , Loeffen P , Storm P
Published: January 2020

In the present study 443 strains of Ornithobacterium rhinotracheale, a causative agent of respiratory disease in fowl, were investigated biochemically and serologically. In both ways O. rhinotracheale could be differentiated from other gram-negative rods and, more particularly, from the Pasteurella-like bacteria potentially pathogenic for fowl. For the biochemical characterization of O. rhinotracheale the API 2ONE identification strip proved to be useful, although O. rhinotracheale is not included in the API system. Serologically, by using monovalent antisera in agar gel precipitation (AGP) tests and enzyme-linked immunosorbent assays (ELISAs), seven serotypes (serotypes A to G) of O. rhinotracheale could be discriminated. The AGP test was chosen as the preferred method to be used for serotyping. Isolates of serotype A were found to be the most prevalent, especially in chickens. Isolates from turkeys were more heterogeneously divided over the serotypes. Some strains showed cross-reactivity between serotypes A, B, and E. Five O. rhinotracheale strains could not be serotyped with the available antisera. Relationships between the geographic origin and the serotypes were found. By the ELISA the presence of antibodies against O. rhinotracheale could be detected in 1-day-old birds as well as in birds with clinical signs, and therefore, it might be useful for diagnostic purposes.
Immunoblot assay for serodiagnosis of Helicobacter pylori infections
Authors: Nilsson I , Ljungh A , Aleljung P , Wadström T
Published: January 2020

An immunoblot assay for the serological diagnosis of Helicobacter pylori infection was evaluated. Serum samples from patients whose gastric biopsy specimens were known to be positive or negative for H. pylori on culture were used to establish interpretive criteria for the immunoblot assay. A panel of sera from patients with diseases other than H. pylori infection and sera from healthy blood donors were included to validate these criteria. All sera were initially assessed in an enzyme immunoassay (Ge-EIA), based on acid glycine-extracted cell surface proteins of H. pylori NCTC 11637. The same antigen extract was used in the immunoblot assay. In addition, the Ge-EIA and the immunoblot assay were compared with a commercially available EIA (Seradyn, Color Vue Pylori). Bands of 110/120 kDa and/or two of five low-molecular-mass proteins (26, 29, 30, 31, and 33 kDa, in any combination) showed a strong correlation with the H. pylori culture-positive patients (97.5%) compared to the correlation obtained with the EIA results (Ge-EIA, 87.5%; Seradyn EIA, 92.5%), and the antibody responses to these proteins were considered specific reactions. In 37 of 40 serum samples from culture-negative patients and also in sera from patients with other disorders, a moderate antibody reactivity to the medium-size proteins (43 to 66 kDa) was observed, and these were considered not valuable for a specific immunoblot assay. Among sera from culture-positive patients, 39 of 40 serum samples were defined to be immunoblot positive, and from among sera from culture-negative patients, 3 of 40 serum samples were defined to be immunoblot positive. The use of sera from patients with negative cultures for H. pylori as negative controls may decrease the sensitivity due to sampling error and false-negative culture results. Immunoblot assay-positive results were detected among 10% of sera from patients with other diseases, whereas they were detected among 42.5% of sera by the Ge-EIA and 47.5% of sera by the Seradyn-EIA. The higher number of EIA-positive sera in this group reflects a possible cross-reactivity (false-positive EIA result). Of the blood donors, representing asymptomatic but possibly colonized subjects, 24% were immunoblot positive. In conclusion, our data indicate that immunoblotting is more sensitive as well as more specific than EIA. Moreover, it permits detection of antibody responses to specific antigens, e.g., the cytotoxin-associated CagA protein, which may have pathological implications.
Molecular typing of Helicobacter pylori isolates from a multicenter U.S. clinical trial by ureC restriction fragment length polymorphism
Authors: Shortridge V D , Stone G G , Flamm R K , Beyer J , Versalovic J , Graham D W , Tanaka S K
Published: January 2020

The molecular typing of 81 pretreatment Helicobacter pylori isolates and the comparison of 18 pretreatment-posttreatment pairs is described by restriction fragment length polymorphism (RFLP) of the ureC gene. The results of our study show the extreme genomic diversity of H. pylori and indicate that infection by H. pylori in the United States does not appear to be limited to a small number of RFLP types.
Evaluation of a new optical immunoassay for diagnosis of neonatal chlamydial conjunctivitis
Authors: Roblin P M , Gelling M , Kutlin A , Tsumura N , Hammerschlag M R
Published: January 2020

The BioStar OIA Chlamydia test (BioStar, Inc., Boulder, Colo.) is a novel immunoassay system that uses changes in reflection of light to directly detect chlamydial antigen in clinical specimens. We compared the optical immunoassay (OIA) with culture for detecting Chlamydia trachomatis in ocular specimens from infants with suspected chlamydial conjunctivitis. We initially performed a retrospective evaluation, testing 152 ocular specimens previously collected for culture with the OIA. The sensitivity and specificity were 94.2 and 97%, respectively. A subsequent prospective study evaluating 37 ocular specimens from infants with suspected C. trachomatis conjunctivitis revealed a sensitivity and specificity of 100 and 92.6%, respectively.
Comparison of QIAamp HCV kit spin columns, silica beads, and phenol-chloroform for recovering human immunodeficiency virus type 1 RNA from plasma
Authors: Shafer R W , Levee D J , Winters M A , Richmond K L , Huang D , Merigan T C
Published: January 2020

Human immunodeficiency virus type 1 (HIV-1) pol mutations are responsible for HIV-1 resistance to current antiretroviral drugs. HIV-1 RNA extraction with QIAamp HCV kit spin columns (Qiagen, Chatsworth, Calif.) followed by reverse transcription-PCR successfully recovered a 1,008-bp pol fragment from the plasma of 31 of 34 HIV-1-infected patients that was suitable for sequencing and recombinant-virus studies. The minimum HIV-1 RNA concentration required for gene recovery was 30 to 40 copies/ml, which was similar to the minimal HIV-1 RNA concentration required when phenol-chloroform or silica beads are used for RNA extraction.
Distribution of colonization factor antigens among enterotoxigenic Escherichia coli strains isolated from patients with diarrhea in Nepal, Indonesia, Peru, and Thailand
Authors: Nirdnoy W , Serichantalergs O , Cravioto A , LeBron C , Wolf M , Hoge C W , Svennerholm A M , Taylor D N , Echeverria P
Published: January 2020

Samples (1,318) of enterotoxigenic Escherichia coli (ETEC) isolated in 1994-1995 from children with diarrhea from Nepal, Indonesia, Peru, and Thailand were examined for colonization factor antigen (CFA) and coli surface (CS) antigens. Fifty-five percent of 361 heat-labile and heat-stable (LT-ST), 14% of 620 LT-only, and 48% of 337 ST-only ETEC had CFA/CS antigens. LT-ST ETEC strains were predominantly in the CFA II group, and ST only strains were in the CFA IV group. Additional studies are needed to identify ETEC strains that do not have CFA/CS antigens.
Doing it right the first time: quality improvement and the contaminant blood culture
Authors: Weinbaum F I , Lavie S , Danek M , Sixsmith D , Heinrich G F , Mills S S
Published: January 2020

The aim of the project was to determine whether the rate of contaminant blood cultures could be reduced by using a team of dedicated phlebotomists. Comparisons were made between adult patients requiring blood cultures for suspected bacteremia on medical and surgical units before and after the introduction and withdrawal of a dedicated blood culture team. The results showed that a significant reduction in the contaminant blood culture rate was achieved by the blood culture team (P < 0.001; chi(2) test). Therefore, in our experience, the rate of contaminant blood cultures can be reduced in a teaching hospital by using a team of dedicated phlebotomists. Calculations made with our data and those published by others suggest that cost savings from reducing false-positive blood cultures are greater than the cost of the blood culture team.
Identification of Burkholderia cepacia isolates from patients with cystic fibrosis and use of a simple new selective medium
Authors: Henry D A , Campbell M E , LiPuma J J , Speert D P
Published: January 2020

We evaluated 819 isolates referred to us as "Burkholderia cepacia" from cystic fibrosis (CF) clinics and research laboratories from five countries; 28 (3.4%) were not B. cepacia. A further 12 (1.5%) organisms appeared to be other Burkholderia species, but identification could not be confirmed by conventional means. The most prevalently misidentified organisms were Stenotrophomonas maltophilia, Alcaligenes xylosoxidans, and Comamonas acidovorans. Many of these organisms grew on oxidation-fermentation polymyxin-bacitracin-lactose (OFPBL) and Pseudomonas cepacia agars, selective media currently used for B. cepacia isolation. We developed a new medium, B. cepacia selective agar (BCSA), which is more enriched for the growth of B. cepacia yet which is more selective against other organisms than currently available selective agars. A total of 190 of 191 (99.5%) isolates of B. cepacia from patients with CF grew on BCSA without vancomycin, whereas 100% grew on OFPBL agar and 179 (94.2%) grew on P. cepacia agar. Of 189 other gram-negative and gram-positive organisms tested, 10 (5.3%) grew on BCSA without vancomycin. The addition of vancomycin to BCSA lowered the false positivity rate to 3.7% without further inhibition of B. cepacia. The false positivity rates for OFPBL and P. cepacia agars were 19.6 and 13.8%, respectively. Isolates of B. cepacia from CF patients grew most quickly on BCSA, with 201 of 205 (98.0%) being readily visible within 24 h, whereas 182 (88.8%) grew on OFPBL agar and 162 (79.0%) grew on P. cepacia agar within 24 h. We propose that the use of BCSA will allow investigators to overcome many of the difficulties associated with the identification of B. cepacia and should be considered for use as a primary isolation agar for specimens from patients with CF.
Diagnosis of invasive candidiasis in neutropenic children with cancer by determination of D-arabinitol/L-arabinitol ratios in urine
Authors: Christensson B , Wiebe T , Pehrson C , Larsson L
Published: January 2020

Determination of D-arabinitol/L-arabinitol ratios (referred to as D/L-arabinitol ratios) in urine as a tool for the diagnosis of invasive candidiasis was investigated in a prospective study comprising 100 children with cancer. The analyses were made by gas chromatography. Positive D/L-arabinitol ratios were found for 10 of 10 children with confirmed invasive candidiasis, 12 of 23 patients undergoing empiric antifungal chemotherapy, and 4 of 67 children not receiving antifungal treatment. D/L-Arabinitol ratios were positive 3 to 31 days (median, 12 days) before the first culture-positive blood sample was drawn or empiric therapy was initiated. The regular monitoring of D/L-arabinitol ratios in urine holds great promise as a sensitive method for diagnosing invasive candidiasis in immunocompromised children with cancer. Moreover, it may be possible to use an early rise in D/L-arabinitol ratios as a basis for the institution of antifungal chemotherapy and as a means of avoiding unnecessary treatment with potentially toxic antifungal agents.
Development of a nested PCR assay for detection of feline infectious peritonitis virus in clinical specimens
Authors: Gamble D A , Lobbiani A , Gramegna M , Moore L E , Colucci G
Published: January 2020

A diagnostic test for feline infectious peritonitis virus (FIPV) infection based on a nested PCR (nPCR) assay was developed and tested with FIPV, feline enteric coronavirus (FECV), canine coronavirus (CCV), and transmissible gastroenteritis virus (TGEV) and clinical fluid samples from cats with effusive feline infectious peritonitis (FIP). The target sequence for the assay is in the S1 region of the peplomer protein E2 gene. A vaccine strain of FIPV and two wild-type FIPV strains tested positive, but FECV, TGEV, and CCV tested negative. Preliminary tests with 12 cats with clinical evidence of effusive FIP and 11 cats with an illness associated with effusions, but attributed to other causes, were performed. Eleven of the 12 cats with effusive FIP tested positive, while 1 was negative. Ten of the 11 cats ill from other causes tested negative, while 1 was positive. On the basis of clinical laboratory and histopathologic criteria, the preliminary sensitivity and specificity of the assay were 91.6 and 94%, respectively.
Aseptic meningitis and encephalitis: the role of PCR in the diagnostic laboratory
Authors: Read S J , Jeffery K J , Bangham C R
Published: January 2020

In this study we have devised a simple and robust PCR strategy to detect a wide range of viruses, bacteria, and parasites, all of which are capable of causing aseptic meningitis and encephalitis. The techniques developed have been used in a routine diagnostic virology laboratory to test prospectively 2,233 cerebrospinal fluid specimens. A virus was detected in 147 specimens of cerebrospinal fluid from 143 patients. Four sets of primers were sufficient to detect the virus in 135 (94%) of the PCR-positive patients. We conclude that with appropriate primers, PCR can be systematically and economically applied to test for a range of organisms in a routine diagnostic laboratory. In our opinion, PCR will soon become the "gold standard" test for viral infections of the central nervous system.
Multiplex PCR detection of vanA, vanB, vanC-1, and vanC-2/3 genes in enterococci
Authors: Patel R , Uhl J R , Kohner P , Hopkins M K , Cockerill F R
Published: January 2020

Vancomycin-resistant enterococci (VRE) are increasingly isolated from clinical specimens. One hundred clinical isolates of enterococci (E. casseliflavus/E. flavescens [n = 10], E. faecalis [n = 34], E. faecium [n = 43], E. avium [n = 1], E. gallinarum [n = 11], and E. raffinosus [n = 1]) were examined for the presence of vanA, vanB, vanC-1, and vanC-2/3 genes by a single multiplex PCR performed directly with colonies from blood agar plates. Six previously characterized VRE strains which carry either vanA, vanB, vanC-1, or vanC-2 genes were used as controls. To discriminate among van genes, the PCR amplicons were digested with MspI and were electrophoresed on agarose gels. Because of significant sequence homology between vanC-2 and vanC-3 genes, this assay is unable to discriminate these genes from each other; therefore, these are referred to as vanC-2/3 genes. PCR products were detected in 63 of the 100 clinical isolates. The restriction fragment length patterns were consistent with vanA for 10 strains, vanB for 30 strains, vanC-1 for 12 strains, vanC-2 for 6 strains, and vanA and vanC-1 for 1 strain. The vancomycin MICs for the isolates with restriction fragment length patterns consistent with vanA and vanB were all > and = 64 micrograms/ml. The vancomycin MICs for the isolates with restriction fragment length patterns consistent with vanC-1 or vanC-2 were 4 to 8 micrograms/ml. The vancomycin MICs for the isolates from which no PCR amplicons were produced were 2 to 4 micrograms/ml. A PCR product was produced in four isolates (vancomycin MICs, 4 to > 256 micrograms/ml) with restriction fragment length patterns differing from those for the control vanA, vanB, vanC-1, and vanC-2 isolates. DNA sequencing of these amplicons revealed that two of the four isolates had nucleic acid sequences which were closely related to the published sequence for the vanB gene and two had nucleic acid sequences which were closely related to the published sequence for the vanC-2 and vanC-3 genes. Multiplex PCR-restriction fragment length polymorphism appears to be a useful and convenient method for rapidly detecting and discriminating genotypes for vancomycin-resistant Enterococcus spp. in the clinical laboratory. In instances in which unusual restriction fragment patterns of PCR amplicons occur, DNA sequencing can be performed to discriminate van genotypes.
Serotypes of Streptococcus pneumoniae causing invasive childhood infections in Bangladesh, 1992 to 1995
Authors: Saha S K , Rikitomi N , Biswas D , Watanabe K , Ruhulamin M , Ahmed K , Hanif M , Matsumoto K , Sack R B , Nagatake T
Published: January 2020

One hundred sixty-five invasive Streptococcus pneumoniae strains were isolated from children under five at Dhaka Shishu (Children's) Hospital during the period 1992 to 1995. Ninety-four strains were from cerebrospinal fluid, and 71 were from blood. More than 91% of the strains were isolated from patients aged 24 months or less. Predominant serotypes were, in descending order 7F, 12F, 14, 15B, 18, 5, and 22A. These comprised 70% of all isolates. The marked differences in serotype distribution in different countries indicate the need for a sentinel surveillance study for the countries of South Asia, particularly Bangladesh, China, India, and Pakistan.
Detection of motility and putative synthesis of flagellar proteins in Salmonella pullorum cultures
Authors: Holt P S , Chaubal L H
Published: January 2020

Salmonella pullorum is a host-adapted pathogen of poultry previously thought to be nonmotile and nonflagellated. We discovered that motility can be induced in this organism under special medium conditions, and this motility was observed in 39 of 44 S. pullorum isolates tested. The migration appeared to occur only on the medium surface and not within the medium itself, indicating that swimming may not be responsible for this event. Agar concentration, carbohydrate concentration and type, and temperature of incubation all affected the motility. Flagellar stains and transmission electron micrographs of the motile S. pullorum culture showed long fibrous appendages resembling flagella extending from the cells, but these appendages were thinner and less numerous than the flagella observed on Salmonella enteritidis. Antisera to G flagellar antigens reacted strongly with the induced-motility S. pullorum culture, indicating that G epitopes were expressed on these cells. These results indicate that, contrary to the paradigm which held that S. pullorum is nonmotile and nonflagellated, motility can be induced in S. pullorum and that the organism appears to have the capacity to produce flagella.
Comparison of Etest and agar dilution method for antimicrobial susceptibility testing of Flavobacterium isolates
Authors: Hsueh P R , Chang J C , Teng L J , Yang P C , Ho S W , Hsieh W C , Luh K T
Published: January 2020

The Etest was evaluated as a possible alternative to the standard agar dilution method for susceptibility testing of nine antimicrobial agents against Flavobacterium species. In studies of 100 clinical isolates, the agreement between the MICs (+/-1 log2 dilution) obtained by the two methods was acceptable for cefotaxime, ceftazidime, amikacin, minocycline, ofloxacin, and ciprofloxacin (> 90%). Conversely, the agreement between the results obtained for piperacillin was limited (84%). The overall agreement was 92.5%.
Kidney and liver transplants from a donor infected with Naegleria fowleri
Authors: Kramer M H , Lerner C J , Visvesvara G S
Published: January 2020

We describe the first reported case of organ transplantation from a boy who had died of undiagnosed Naegleria fowleri infection. While no subsequent amebic infections occurred in the three organ recipients, our report illustrates the need for an improved strategy for evaluating the benefits and risks of transplanting tissues from persons whose illness was likely caused by an infectious agent.
Use of enzyme-linked immunosorbent assay techniques with cross-reacting human sera in diagnosis of murine typhus and spotted fever
Authors: Keysary A , Strenger C
Published: January 2020

Enzyme-linked immunosorbent assay (ELISA) techniques for the determination of immunoglobulin G to rickettsial lipopolysaccharides were developed. These techniques provide a simple and convenient way to serodiagnose Mediterranean spotted fever and murine typhus with a single serum dilution. The results of the ELISAs correlated with the indirect immunofluorescence assay titers of cross-reacting sera.
Microtiter virus isolation and enzyme immunoassays for detection of bovine viral diarrhea virus in cattle serum
Authors: Saliki J T , Fulton R W , Hull S R , Dubovi E J
Published: January 2020

Cattle immunotolerant to and persistently infected (PI) with bovine viral diarrhea (BVD) virus (BVDV) constitute the mechanism by which BVDV persists in cattle herds. Two procedures for using serum to detect PI cattle were developed and evaluated. BVDV was found to remain viable for 7 days in serum samples stored at room temperature. The tests use cell culture virus isolation (VI) in 96-well microtiter plates, followed by immunostaining of cell monolayers with monoclonal antibodies. One technique, the immunoperoxidase monolayer assay (IPMA), forms a red intracellular precipitate while the other, the monolayer enzyme-linked immunosorbent assay (M-ELISA) produces a yellow color in solution. The optimal incubation period for microtiter VI was determined to be 4 days. Optimal IPMA staining was obtained by fixing cell monolayers with 20 to 30% acetone, whereas a simple dry-rehydrate-dry cycle provided optimal M-ELISA staining. The M-ELISA and IPMA had the same sensitivities and specificities, but the M-ELISA was a more rapid procedure and use of a spectrophotometer for reading samples allowed for greater objectivity. When compared to standard VI with routine samples submitted for the diagnosis of BVD, M-ELISA and IPMA had a relative sensitivity of 85% and a relatively specificity of 100%. When only samples from cattle suspected of being PI were considered, these two parameters were 100% for both IPMA and M-ELISA. The two procedures, especially the M-ELISA, are suitable for whole-herd testing to identify PI cattle. The appeal of these tests is derived from the convenience of using serum as a diagnostic sample and the ability to rapidly screen large numbers of samples at low cost.
Comparison of axenic and monoxenic media for isolation of Acanthamoeba
Authors: Penland R L , Wilhelmus K R
Published: January 2020

Acanthamoeba is a genus of ubiquitous, free-living amebae that can be difficult to isolate by standard microbiologic techniques. We retrospectively reviewed the laboratory records of patients with ocular acanthamoebic infection for the period from January 1973 to June 1996 and found that Acanthamoeba isolates were recovered from 73, 71, and 70% of clinical specimens inoculated onto buffered charcoal-yeast extract agar (BCYE), nonnutrient agar with live or dead Escherichia coli, and tryptic soy agar (TSA) with horse or sheep blood, respectively. We then prospectively compared the recovery of a corneal isolate of Acanthamoeba on commercial media from Remel and BBL (TSA with 5% sheep blood, TSA with 5% horse blood, TSA with 5% rabbit blood, V agar, chocolate agar, BCYE, and selective BCYE with polymyxin B, anisomycin, and vancomycin) and on axenic and monoxenic media prepared with live or dead bacteria (Enterobacter aerogenes, E. coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Serratia marcescens, Staphylococcus aureus, and Stenotrophomonas maltophilia). Good recovery of trophozoites was obtained on BCYE, TSA with rabbit blood, TSA with horse blood, and Remel TSA with sheep blood. BBL TSA with horse blood or rabbit blood provided good recovery of cysts. All species of live or dead bacteria yielded good recovery of trophozoites; however, only nonnutrient agar with live P. aeruginosa, live E. aerogenes, or live S. maltophilia gave good recovery of cysts. TSA with either rabbit blood or horse blood, BCYE, and nonnutrient agar prepared with live P. aeruginosa, E. aerogenes, or S. maltophilia offer optimal recovery of Acanthamoeba.
Diagnosis of vascular catheter-related bloodstream infection: a meta-analysis
Authors: Siegman-Igra Y , Anglim A M , Shapiro D E , Adal K A , Strain B A , Farr B M
Published: January 2020

Catheter-related bloodstream infections increased in incidence during the past decade, causing significant morbidity, mortality, and excess hospital costs. Absence of inflammation at the catheter site in most cases makes clinical diagnosis uncertain. The relative accuracy and cost-effectiveness of different microbiologic tests for confirming that bloodstream infection is catheter related have remained unclear. A meta-analysis of published studies was conducted regarding the accuracy of diagnostic test methods using pooled sensitivity and specificity and summary receiver operating characteristic (ROC) curve analysis. The cost for each test was estimated by methods published by the College of American Pathologists. Costs of catheter replacement and antibiotic therapy for false positive results were included in the cost per accurate test result. Twenty-two studies evaluating six test methods met inclusion criteria for the meta-analysis. Accuracy increased in ROC analysis for catheter segment cultures with increasing quantitation (P = 0.03) (i.e., quantitative > semiquantitative > qualitative) largely due to an increase in specificity. The highest Youden index (mean = 0.85) was observed with quantitative catheter segment culture, the only method with pooled sensitivity and specificity above 90%. For blood culture methods, there was no statistically significant trend toward increased accuracy. The unpaired quantitative catheter blood culture offered the lowest cost per accurate test result but was only 78% sensitive. In conclusion, quantitative culture was the most accurate method for catheter segment culture, and unpaired quantitative catheter blood culture was the single most cost-effective test, especially for long-term catheters.
Species identification of enterococci via intergenic ribosomal PCR
Authors: Tyrrell G J , Bethune R N , Willey B , Low D E
Published: January 2020

Accurate species identification of enterococci has become important with the wide prevalence of acquired vancomycin resistance and the presence of less epidemiologically important, inherently vancomycin-resistant enterococci. Using a collection of enterococcal strains, we found that PCR amplification of the intergenic spacer (ITS-PCR) between the 16S and 23S rRNA genes can produce amplicon profiles characteristic of the enterococcus examined. The species examined were group I enterococci (Enterococcus avium, Enterococcus raffinosus, Enterococcus malodoratus, and Enterococcus pseudoavium), group II enterococci (Enterococcus faecalis, Enterococcus faecium, Enterococcus casseliflavus, Enterococcus mundtii, and Enterococcus gallinarum), and group III enterococci (Enterococcus durans and Enterococcus hirae). The enterococcal species in group I, as well as E. faecalis and two strains of E. hirae, were similar and therefore had to be differentiated from each other by Sau3A restriction digests. This produced patterns characteristic of each of these species. The remaining group II and group III enterococcal species produced amplicons characteristic of a particular species except E. gallinarum. The PCR products from E. gallinarum displayed strain-to-strain heterogeneity in the number and size of amplicons. To further test the utility of this technique, 11 phenotypically aberrant strains which had been assigned species identification based on Facklam and Collins-type strain reactions (R.R. Facklam and M.D. Collins, J. Clin. Microbiol. 27:731-734, 1989) were subjected to ITS-PCR. ITS-PCR of the phenotypically aberrant strains identified six strains with reactions consistent with those of type strains. However, five strains were characterized as follows: two strains originally identified as E. mundtii were identified by ITS-PCR as E. casseliflavus, one strain originally identified as E. raffinosus was identified by ITS-PCR as E. durans, one strain originally identified as E. hirae was identified by ITS-PCR as E. faecium [corrected]. We conclude that amplification of the intergenic 23S and 16S rRNA gene regions of enterococci provides a reliable technique for species identification of enterococci.