bluuebunny/crossref_metadata_2025 huggingface dataset:
Clinical comparison of isolator, Septi-Chek, nonvented tryptic soy broth, and direct agar plating combined with thioglycolate broth for diagnosing spontaneous bacterial peritonitis
Authors: Hay J E , Cockerill F R , Kaese D , Vetter E A , Wollan P C , Rakela J , Wilhelm M P
Published: January 2020

Spontaneous bacterial peritonitis is a life-threatening complication of cirrhotic ascites. Optimal patient management depends on the isolation of the causal organism from ascitic fluid. To evaluate culture techniques for the diagnosis of spontaneous bacterial peritonitis, we prospectively compared three blood culture system, the Isolator system, a lysis-centrifugation system, the Septi-Chek system, a biphasic culture system, and a nonvented tryptic soy broth system, all inoculated at the bedside, and our standard method of direct inoculation of specimens after transport to the laboratory onto agar plates and into thioglycolate broth. The results showed that the Septi-Chek and nonvented tryptic soy broth systems each recovered statistically significantly more pathogens than either the Isolator system (P = 0.0084) or the standard method (P = 0.00098). The Isolator system recovered more pathogens than the standard plate method, but this difference was not statistically significant. Both the Isolator system and the standard plate method recovered more contaminating microorganisms than the Septi-Chek or nonvented tryptic soy broth system. The Isolator system required the most processing time compared with the processing times required by any other method.
Comparative evaluation of several techniques for purification of Cryptosporidium parvum oocysts from rat feces
Authors: Suresh P , Rehg J E
Published: January 2020

Three methods of isolating Cryptosporidium parvum oocysts from rat feces were evaluated. Oocysts were initially isolated by sucrose density gradient centrifugation. They were further purified by passage through a glass bead column or a Percoll gradient or by dialysis. Although oocysts recovered from the glass bead column and by dialysis were relatively free of fecal debris, only oocysts recovered from the Percoll gradient were free of bacteria. Recovered oocysts retained their antigenicity and infectivity. The ability to effectively recover oocysts from rat feces suggests that the laboratory rat may be a convenient substitute for ruminants in the propagation and maintenance of C. parvum oocysts for in vitro and in vivo use.
Genetic diversity among Mycobacterium avium complex AccuProbe-positive isolates
Authors: Soini H , Eerola E , Viljanen M K
Published: January 2020

The partial 32-kDa-protein gene sequences of 22 Mycobacterium avium complex (MAC) clinical isolates that were positive by the AccuProbe MAC probe only (not by the M. avium or M. intracellulare probe) were determined. The obtained nucleotide sequences were compared with the published sequences for M. tuberculosis, M. avium, and M. intracellulare by a sequence analysis program. There was a wide range of genetic diversity among the strains studied. Most of them (16 of 22) had sequences similar but not identical to those of M. avium and M. intracellulare. These strains were considered to be true MAC strains. Five strains had sequences in the category of the novel MAIX sequence, which was very different from the sequences of other mycobacteria analyzed thus far. In addition to these strains, one isolate had a sequence that differed greatly from the reference sequences. These results support previous findings showing that the MAC probably contains several additional species. Our results also suggest that the MAC AccuProbe may react with strains that do not belong to the MAC.
Urine sample used for congenital toxoplasmosis diagnosis by PCR
Authors: Fuentes I , Rodriguez M , Domingo C J , del Castillo F , Juncosa T , Alvar J
Published: January 2020

The diagnosis of toxoplasmosis in congenitally infected infants can be difficult; serology is unreliable, and diagnosis must be based on the combination of symptomatology and direct demonstration of the parasite. Four infants suspected of having Toxoplasma gondii infection were studied by serological analysis, tissue culture, and PCR determination. T. gondii was isolated from the urine of one patient. The parasite was detected by PCR in the blood and cerebrospinal fluid of three infants and in the urine in all patients. Because nested PCR proved to be a sensitive, relatively rapid, and specific method and because it can be applied to a variety of different clinical samples, PCR can be a valuable technique for the identification of T. gondii infections in children. The present study indicates that PCR examination of urine, a fluid never before used for diagnosis in this age group, may be valuable in diagnosing cases of congenital toxoplasmosis.
Discordant results from hepatitis C virus genotyping by procedures based on amplification of different genomic regions
Authors: Toniutto P , Pirisi M , Tisminetzky S G , Fabris C , Chinellato E , Gerotto M , Falleti E , Ferroni P , Lombardelli T , Bartoli E , Baralle F
Published: January 2020

We compared the results of genotyping hepatitis C virus (HCV) either by PCR amplification of the core region or by hybridization of PCR-amplified products of the 5' untranslated region (5'UTR assay). Serum samples from 144 Italian anti-HCV-positive patients (106 drug abusers and 38 patients with chronic viral liver disease but no history of drug abuse) were studied. The original core region assay described by Okamoto et al. (H. Y. Okamoto, Y. Sugiyama, S. Okada, K. Kurai, Y. Akahane, Y. Sugai, T. Tanaka, K. Sato, F. Tsuda, Y. Miyakawa, and M. Mayumi, J. Gen. Virol. 73:673-679, 1992) allowed genotyping of 75 of 144 samples. A modified version of Widell et al. (A. Widell, S. Shev, S. Månsson, Y.-Y. Zhang, U. Foberg, G. Norkrans, A. Frydén, O. Weiland, J. Kurkus, and E. Nordenfelt, J. Med. Virol. 44:272-279, 1994) allowed genotyping of 11 of 79 samples (50 of 79 samples remained unclassified by the method of Okamoto et al. In contrast, all 144 samples were genotyped by the 5'UTR assay. Forty-six of 75 (61 percent) of the samples genotyped by the method of Okamoto et al. and 10 of 11 (91 percent) of the samples genotyped by the method of Widell et al. had results consistent with those obtained by the 5'UTR assay. According to the results of direct sequencing, the method of Okamoto et al. erroneously classified seven samples as having mixed infections. In conclusion, HCV genotyping seems more reliable when it is performed by the 5'UTR assay than by either of two core region assays. The major advantage provided by the 5'UTR assay is a much lower proportion of negative or indeterminate results in younger patients with histories of drug abuse or infection by genotypes other than HCV type 1.
Identification of a variant subgroup A strain of respiratory syncytial virus
Authors: Mufson M A , Stanek R J
Published: January 2020

During epidemiologic surveillance of children with respiratory syncytial virus (RSV) disease in Huntington, W.Va., we identified seven strains of a new variant subgroup A RSV (subgroup A-Var) by their reactions in an enzyme immunoassay with two anti-F monoclonal antibodies (MAbs) specific for two epitopes, F1 and F4, generated against the subgroup B RSV. The prototype strain of subgroup A and all other subgroup A field strains from that epidemiologic year failed to react with these two subgroup B MAbs. Additional enzyme immunoassays with 18 subgroup B anti-F MAbs specific for 14 epitopes showed that subgroup A-Var strains also reacted with a MAb specific for the subgroup B F2 epitope. In a radioimmune precipitation assay, the molecular size of the subgroup A-Var F2 subunit of the fusion (F) protein clearly differed from those of both prototype strains of subgroup A and subgroup B RSV. The molecular size of the F2 subunit of subgroup A-Var (24 kDa) was intermediate between the size of the F2 subunit of subgroup A (25 kDa) and that of subgroup B (23 kDa). However, the molecular sizes of the F1 subunits of both subgroup A and subgroup A-Var were identical (54 kDa) and slightly larger than those of the F1 subunits of both subgroups B1 and B2 (53 kDa). These data suggest that subgroup A-Var may represent a distinct RSV A subgroup, analogous to subgroup B1 and B2 RSV, and it is the first-identified naturally occurring subgroup A RSV with an F protein different from that of the prototype A RSV.
Reliability of nucleic acid amplification for detection of Mycobacterium tuberculosis: an international collaborative quality control study among 30 laboratories
Authors: Noordhoek G T , van Embden J D , Kolk A H
Published: January 2020

Nucleic acid amplification to detect Mycobacterium tuberculosis in clinical specimens is increasingly used as a laboratory tool for the diagnosis of tuberculosis. However, the specificity and sensitivity of these tests may be questioned, and no standardized reagents for quality control assessment are available. To estimate the performance of amplification tests for routine diagnosis, we initiated an interlaboratory study involving 30 laboratories in 18 countries. We prepared blinded panels of 20 sputum samples containing no, 100, or 1,000 mycobacterial cells. Each laboratory was asked to detect M. tuberculosis by their routine method of nucleic acid amplification. Only five laboratories correctly identified the presence or absence of mycobacterial DNA in all 20 samples. Seven laboratories detected mycobacterial DNA in all positive samples, and 13 laboratories correctly reported the absence of DNA in the negative samples. Lack of specificity was more of a problem than lack of sensitivity. Reliability was not found to be associated with the use of any particular method. Reliable detection of M. tuberculosis in clinical samples by nucleic acid amplification techniques is possible, but many laboratories do not use adequate quality controls. This study underlines the need for good laboratory practice and reference reagents to monitor the performance of the whole assay, including pretreatment of clinical samples.
Ribosomal DNA typing of Haemophilus ducreyi strains: proposal for a novel typing scheme
Authors: Pillay A , Hoosen A A , Kiepiela P , Sturm A W
Published: January 2020

Local strains of Haemophilus ducreyi were characterized by ribosomal DNA fingerprinting in order to develop a typing scheme for this organism. Chromosomal DNAs from 30 strains were extracted, and restriction fragments were separated by agarose gel electrophoresis after cleavage with BglII, HindIII, HindII, and BstEII. Of the 30 local strains studied, nine BglII, eight HindIII, six HindII, and five BstEII ribotype patterns were observed. In order to develop a typing scheme, we correlated the ribotype patterns obtained with each of the four enzymes for each strain. By this scheme, 13 ribotypes designated b to n were defined among the local strains. This study demonstrates that there is a diversity of ribotypes among H. ducreyi strains in our area, which emphasizes that there are many strains responsible for chancroid. This novel typing scheme provides a useful tool for further epidemiological studies of chancroid.
Development of interpretive criteria and quality control limits for macrolide and clindamycin susceptibility testing of Streptococcus pneumoniae
Authors: Jorgensen J H , Swenson J M , Tenover F C , Barry A , Ferraro M J , Murray P R , Reller L B
Published: January 2020

A six-laboratory collaborative study was conducted to develop MIC and zone diameter quality control limits and interpretive criteria for antimicrobial susceptibility testing of Streptococcus pneumoniae with azithromycin, clarithromycin, dirithromycin, and clindamycin. The MICs of all of the agents plus erythromycin for 302 clinical isolates of pneumococci that had been selected with an emphasis on resistant strains were determined by use of the National Committee for Clinical Laboratory Standards (NCCLS)-recommended broth microdilution procedure. The zone diameters of the isolates were also determined for the same agents except erythromycin by the NCCLS disk diffusion test procedure. Repeated testing of S. pneumoniae ATCC 49619 with different sources and lots of media and disks allowed development of MIC and zone diameter quality control ranges for these agents. Interpretive criteria for the MIC of azithromycin were established and were as follows: susceptible, < or = 0.5 microgram/ml; intermediate, 1 microgram/ml; and resistant, > or = 2 micrograms/ml. The interpretive criteria advocated for the MICs of clarithromycin and clindamycin were as follows: susceptible, < or = 0.25 microgram/ml; intermediate, 0.5 microgram/ml; and resistant, > or = 1 microgram/ml. Comparison of MICs and disk diffusion zone diameters led to the development of interpretive criteria for the zone diameters for azithromycin, clarithromycin, and clindamycin that correlated well with these MIC breakpoints. Testing of this organism collection also led to the reestablishment of the erythromycin MIC breakpoints as being identical to those of clarithromycin, which resulted in equivalent cross-susceptibility and cross-resistance for the three macrolides that are currently marketed in the United States. Thus, the susceptibility of pneumococci to azithromycin and clarithromycin can be predicted accurately by testing only erythromycin in clinical laboratories. This recommendation, as well as the interpretive and quality control criteria that are described, have been accepted by NCCLS and are included in the latest NCCLS susceptibility testing guidelines.
Use of an alkaline phosphatase-conjugated oligonucleotide probe for the gene encoding the bundle-forming pilus of enteropathogenic Escherichia coli
Authors: Nagayama K , Bi Z , Oguchi T , Takarada Y , Shibata S , Honda T
Published: January 2020

An alkaline phosphatase-conjugated 29-base oligonucleotide probe was developed to detect the gene encoding the bundle-forming pilus of enteropathogenic Escherichia coli. The sensitivity and specificity of the probe versus the results of localized adherence in the HEp-2 cell assay and fluorescent actin staining assay positivity were 95.7 and 100%, respectively.
Identification of Yersinia pestis by BBL Crystal Enteric/Nonfermenter Identification System
Authors: Wilmoth B A , Chu M C , Quan T J
Published: January 2020

The BBL Crystal Enteric/Nonfermenter System (Crystal) was used to test 25 archived isolates of Yersinia pestis to obtain a unique biochemical profile code for Y. pestis. The revised Crystal system and the API 20E system were compared by using 12 clinical human isolates of Y. pestis. Crystal correctly identified 11 of the 12 isolates, while API correctly identified 7 of the 12 isolates.
Active surveillance for Vibrio cholerae O1 and vibriophages in sewage water as a potential tool to predict cholera outbreaks
Authors: Madico G , Checkley W , Gilman R H , Bravo N , Cabrera L , Calderon M , Ceballos A
Published: January 2020

The 1991 Peruvian cholera epidemic has thus far been responsible for 600,000 cholera cases in Peru. In an attempt to design a cholera surveillance program in the capital city of Lima, weekly sewage samples were collected between August 1993 and May 1996 and examined for the presence of Vibrio cholerae O1 bacteria and V. cholerae O1 bacteriophages (i.e., vibriophages). During the 144 weeks of surveillance, 6,323 cases of clinically defined cholera were recorded in Lima. We arbitrarily defined an outbreak as five or more reported cases of cholera in a week. The odds of having an outbreak were 7.6 times greater when V. cholerae O1 was present in sewage water during the four previous weeks compared with when it was not (P < 0.001). Furthermore, the odds of having an outbreak increased as the number of V. cholerae O1 isolations during the previous 4 weeks increased (P < 0.001). The odds of having an outbreak were 2.4 times greater when vibriophages were present in sewage water during the four previous weeks compared with when they were not, but this increase was not statistically significant (P = 0.15). The odds of having an outbreak increased as the number of vibriophage isolations during the previous 4 weeks increased (P < 0.05). The signaling of a potential cholera outbreak 1 month in advance may be a valuable tool for implementation of preventive measures. In Peru, active surveillance for V. cholerae O1 and possibly vibriophages in sewage water appears to be a feasible and effective means of predicting and outbreak of cholera.
Multicenter evaluating of a commercially available PCR assay for diagnosing enterovirus infection in a panel of cerebrospinal fluid specimens
Authors: Lina B , Pozzetto B , Andreoletti L , Beguier E , Bourlet T , Dussaix E , Grangeot-Keros L , Gratacap-Cavallier B , Henquell C , Legrand-Quillien M C , Novillo A , Palmer P , Petitjean J , Sandres K , Dubreuil P , Fleury H , Freymuth F , Leparc-Goffart I , Hober D , Izopet J , Kopecka H , Lazizi Y , Lafeuille H , Lebon P , Roseto A , Marchadier E , Masquelier B , Picard B , Puel J , Seigneurin J M , Wattre P , Aymard M
Published: January 2020

Thirteen laboratories participated in blind tests of a panel of 20 coded cerebrospinal fluid specimens (7 uninfected samples, 3 samples infected with 1 50% tissue culture infective dose [TCID50]/0.1 ml [nonenterovirus strains], and 10 samples infected with 10, 1, or 0.1 TCID50/0.1 ml [three different enterovirus serotypes]) on the Amplicor enterovirus PCR assay (Roche Diagnostic Systems). The panel was also evaluated by in-house PCR (two nested-PCR and three one-step PCR assay) or tissue culture (eight laboratories). The viral load was shown to influence greatly the sensitivity of the assay. The average sensitivity of the Amplicor test ranged from 67 to 98% for viral titers of 1 to 10 TCID50/0.1 ml, respectively; titers of 0.1 TCID50/0.1 ml resulted in a sensitivity of only 16%. The overall specificity of the Amplicor test was 98%. The Amplicor assay compared favorably to the five in-house PCR tests (no significant difference in either sensitivity or specificity) and was much more sensitive than tissue culture (P < 0.001), even for high viral loads. It was easy to perform, rapid (about 6 h), well-standardized, and appeared to be suitable for the diagnosis of enterovirus meningitis on a routine basis in laboratories trained in molecular biology techniques.
Novel DNA assay for cytomegalovirus detection: comparison with conventional culture and pp65 antigenemia assay
Authors: Veal N , Payan C , Fray D , Sarol L , Blanchet O , Kouyoumdjian S , Lunel F
Published: January 2020

We compared conventional cytomegalovirus (CMV) isolation, rapid viral culture, a CMV pp65 antigenemia assay, and a novel CMV DNA hybrid capture system (HCS). A total of 309 blood samples from individuals in different risk groups were assessed by at least two of the methods mentioned above. Leukocytes were recovered either after centrifugation in Leucosep tubes containing 1.080 Ficoll for pp65 assay or after simple differential lysis steps for DNA detection. HCS was based on DNA hybridization with a CMV RNA probe and its capture by antibodies to DNA-RNA hybrids. The CMV pp65 lower matrix protein was detected by fluorescence with c10-c11 monoclonal antibody in formalin-fixed leukocytes. Concordant results were observed for 92.9, 78.3, and 82.7% of the patients when comparing (i) viral culture and the pp65 antigenemia assay, (ii) viral culture and HCS, and (iii) the pp65 antigenemia assay and HCS, respectively. Discordant results were observed between a positive HCS result and negative culture and/or pp65 results. These results were associated with relatively low DNA levels (< 20 pg/10(6) cells) and positive viruria. In conclusion, the pp65 antigenemia assay is a rapid and reliable method of detecting CMV and is preferable to culture, but the Murex HCS appears to be more sensitive for CMV detection.
Use of AmpliWax to optimize amplicon sterilization by isopsoralen
Authors: De la Viuda M , Fille M , Ruiz J , Aslanzadeh J
Published: January 2020

The photochemical inactivation of amplicons by isopsoralen (IP-10) has been suggested as a possible means to prevent PCR carryover contamination. To evaluate the technique, serial dilutions of amplicons (10(11) to 10(3)) from the Borrelia burgdorferi OSP A gene were amplified in the presence of 0, 25, 50, and 100 micrograms of IP-10 per ml for 45 cycles. The PCR products were exposed to UV light for 15 min to activate IP-10 and sterilize the amplicons. One microliter of each sterilized sample was reamplified for an additional 45 cycles. The PCR products were then resolved in an agarose gel, blotted onto a nylon membrane, and probed with an alkaline phosphatase-conjugated chemiluminescent probe. Although IP-10 at concentrations of 50 and 100 micrograms/ml effectively sterilized up to 10(11) amplicons, the compound was inhibitory to PCR. IP-10 at a concentration of 25 micrograms/ml had slight inhibitory effect on PCR and did not completely sterilized all of the amplicons. Therefore, in subsequent experiments AmpliWax was substituted for mineral oil, and PCR was performed on 10(9) to 10(3) amplicons as described above. Following the amplification, the PCR tubes were cooled to solidify the AmpliWax and inoculated with various concentrations of IP-10. With this technique, PCR products produced from as many as 10(9) target amplicons were effectively sterilized with 200 micrograms of IP-10 per ml. Similarly, the addition of IP-10 (50 micrograms/ml) before and after PCR was evaluated for the detection of B. burgdorferi in 62 ticks from a region of Southern Connecticut where the organism is highly endemic. PCR performed in the presence of 50 micrograms of IP-10 per ml detected B. burgdorferi-specific DNA in 17 of 62 ticks (27%) following gel electrophoresis and in 34 of 62 ticks (55%) following Southern blot hybridization of the PCR products. In contrast, post-PCR addition of IP-10 detected borrelia-specific DNA in 31 of 62 ticks (50%) following gel electrophoresis and in 46 of 62 ticks (64%) following Southern blot hybridization. We conclude that the replacement of mineral oil with AmpliWax can be useful in eliminating the inhibitory effects of IP-10 and other sterilizing agents for post-PCR sterilization of amplicons.
Identification and subspecific differentiation of Mycobacterium scrofulaceum by automated sequencing of a region of the gene (hsp65) encoding a 65-kilodalton heat shock protein
Authors: Swanson D S , Pan X , Musser J M
Published: January 2020

Mycobacterium scrofulaceum is most commonly recovered from children with cervical lymphadenitis, although it also accounts for approximately 2% of the mycobacterial infections in AIDS patients. Species assignment of M. scrofulaceum isolated by conventional techniques can be difficult and time-consuming. To develop a strategy for rapid species assignment of these organisms, a 360-bp region of the gene (hsp65) encoding a 65-kDa heat shock protein in 37 isolates from diverse sources was sequenced. Eight hsp65 alleles were identified, and these sequences formed phylogenetic clusters and lineages largely distinct from other Mycobacterium species. There was incomplete correlation between serovar designation and hsp65 allele assignment. The hsp65 data correlated strongly with the results of sequence analysis of the gene coding for 16S rRNA. Automated DNA sequencing of a 360-bp region of the hsp65 gene provides a rapid and unambiguous method for species assignment of these acid-fast organisms for diagnostic purposes.
Comparative assessment of Etest for testing susceptibilities of Neisseria gonorrhoeae to penicillin, tetracycline, ceftriaxone, cefotaxime, and ciprofloxacin: investigation using 510(k) review criteria, recommended by the Food and Drug Administration
Authors: Biedenbach D J , Jones R N
Published: January 2020

We evaluated the ability of the Etest (AB Biodisk, Solna, Sweden) method to accurately and reproducibly determine the antimicrobial susceptibility of Neisseria gonorrhoeae. One hundred gonococcal isolates were used to evaluate the diagnostic performance of the Etest compared with the reference agar dilution method for penicillin, tetracycline, ciprofloxacin, and ceftriaxone. Between 92 and 99% of Etest MIC results for all drugs were within +/- 1 log2 dilution of the reference MIC. According to recommended interpretive criteria, ceftriaxone, cefotaxime, and ciprofloxacin had 100% categorical agreement, while penicillin (86%) and tetracycline (85%) categorical agreement percentages were lower because of the large number of strains that were within 0.5 to 1 log2 dilution of the susceptible or resistant breakpoints. Reproducibility data also demonstrated that the Etest was precise (99.1%) when subjected to replicate testing. On the basis of these data, the Etest method provides an effective, simple alternative to the reference agar dilution method for the direct quantification of N. gonorrhoeae susceptibility.
Comprehensive ribotyping scheme for heat-stable serotypes of Campylobacter jejuni
Authors: Fitzgerald C , Owen R J , Stanley J
Published: January 2020

Strains from diverse sources belonging to all 47 heat-stable Penner serotypes of Campylobacter jejuni were examined for polymorphism around the 16S rRNA genes. Penner serotype reference strains and a group of nonserotypeable isolates were included in the study. Complete typeability was obtained; 30 distinct PstI and 42 HaeIII polymorphisms were found. Three bands were detected in almost all strains with these enzymes, confirming that three copies of the 16S rRNA gene are typical for C.jejuni. By combination of the two enzyme polymorphisms, 77 16S ribotypes were defined among the 261 strains analyzed. With two exceptions, no specific association was observed between these ribotypes and heat-stable serotypes. Nine serotypes were homogeneous with respect to the 16S ribotype. Most nonserotypeable strains belonged to ribotypes defined elsewhere in the study. The 16S ribotypes of C.jejuni described here were not found in strains of Campylobacter coli, and vice versa.
Strain-specific reverse transcriptase PCR assay: means to distinguish candidate vaccine from wild-type strains of respiratory syncytial virus
Authors: Zheng H , Peret T C , Randolph V B , Crowley J C , Anderson L J
Published: January 2020

Candidate live-virus vaccines for respiratory syncytial virus are being developed and are beginning to be evaluated in clinical trials. To distinguish candidate vaccine strains from wild-type strains isolated during these trials, we developed PCR assays specific to two sets of candidate vaccine strains. The two sets were a group A strain (3A), its three attenuated, temperature-sensitive variant strains, a group B strain (2B), and its four attenuated, temperature-sensitive variant strains. The PCR assays were evaluated by testing 18 group A wild-type strains, the 3A strains, 9 group B wild-type strains, and the 2B strains. PCR specific to group A wild-type strains amplified only group A wild-type strains, and 3A-specific PCR amplified only 3A strains. PCR specific to group B wild-type strains amplified all group A and group B strains but gave a 688-bp product for group B wild-type strains, a 279-bp product for 2B strains, a 547-bp product for all group A strains, and an additional 688-bp product for some group A strains, including 3A strains. These types of PCR assays can, in conjunction with other methods, be used to efficiently distinguish candidate vaccine strains from other respiratory syncytial virus strains.
Axenic growth and drug sensitivity studies of Balamuthia mandrillaris, an agent of amebic meningoencephalitis in humans and other animals
Authors: Schuster F L , Visvesvara G S
Published: January 2020

A cell-free growth medium for the opportunistic pathogenic ameba Balamuthia mandrillaris is presented. This represents an advance over the use of monkey kidney cells for growth of the amebas and can be helpful in isolation of these amebas from brain tissue from cases in which amebic meningoencephalitis is a diagnostic possibility, as well as for biochemical and molecular biological studies. Three isolates of Balamuthia have been cultured in this medium. The cell-free growth system was also used to screen cultures for sensitivity to a variety of antimicrobial agents. Of the various drugs tested, pentamidine isethionate was most effective against amebas (ca. 90% inhibition after 6 days of exposure), but the drug was amebastatic and not amebacidal in the axenic system at the highest concentration tested (10 micrograms/ml).
Coagglutination test for serotyping Pasteurella haemolytica
Authors: Fodor L , Pénzes Z , Varga J
Published: January 2020

A coagglutination test was described for simple, fast, and reliable detection of Pasteurella haemolytica type-specific antigens in lung lesions even in the absence of viable P. haemolytica. The coagglutinating reagents were prepared by coating protein A-producing Staphylococcus aureus cells with hyperimmune sera raised against P. haemolytica type strains. Bacterial suspensions, saline extracts, and boiled saline extracts of the bacteria were used as antigens. Homologous reactions with all types of antigens were precise. Some cross-reactions were similar to those obtained by the indirect hemagglutination test, and some additional one-way cross-reactions were identified. The coagglutination test was used for serotyping 65 P. haemolytica field strains and for the detection of P. haemolytica type-specific antigens in the lung specimens of 62 calves and 78 sheep. Ninety-four percent of the field strains could be serotyped by the coagglutination test. P. haemolytica type-specific antigens were detected in the lung specimens of 3 calves and 5 sheep that had succumbed to naturally occurring P. haemolytica pneumonia and in the lungs of 20 calves experimentally infected with P. haemolytica A1. The coagglutination test detected type-specific antigens in 36% of the lung specimens of slaughtered field sheep but not in the lungs of slaughtered field cattle with small chronic lung lesions. No reaction occurred in the case of nonpneumonic calves and sheep or when pneumonic lesions were caused by other bacteria. No P. haemolytica strains could be isolated from lung samples that were coagglutination test negative. This test is recommended as an additional method for fast and reliable serotyping of P. haemolytica.
Quantification of low levels of human immunodeficiency virus (HIV) type 1 RNA in P24 antigen-negative, asymptomatic, HIV-positive patients by PCR
Authors: Muñoz-Fernández M A , Navarro J , Montes M G , Cosín J , Zabay J M , Fernández-Cruz E
Published: January 2020

A nested PCR was used to quantify small numbers of human immunodeficiency virus (HIV) type 1 (HIV-1) RNA particles in the serum specimens of 26 p24 antigen-negative, asymptomatic, HIV-positive patients undergoing antiretroviral therapy. Fifteen patients received zidovudine (ZDV) and alpha interferon, and 11 patients received ZDV monotherapy. After PCR, the amounts of RNA were quantified by comparing the endpoint dilutions of serum samples with a standard curve with known amounts of viral particles. Before the beginning of the antiviral therapy, HIV-1 RNA was detected in 92% of the patients. After treatment, a fall in the number of viral particles was detected in patients receiving combination therapy (mean titers +/- standard errors of the means, 3,617 +/- 756 pretherapy versus 1,800 +/- 845 posttherapy; P < 0.05) and in patients receiving monotherapy (3,763 +/- 642 pretherapy versus 1,353 +/- 394 posttherapy; P < 0.05). Our results indicate that PCR with nested primers may be useful for assessing the changes in viremia in HIV-positive patients with low viral load undergoing antiviral therapy.
Validation of catheter semiquantitative culture technique for nonstaphylococcal organisms
Authors: Dooley D P , Garcia A , Kelly J W , Longfield R N , Harrison L
Published: January 2020

The catheter semiquantitative culture roll tip technique has been validated as a discriminator between non-catheter-related bacteremias and catheter-related bacteremias (CRBs) caused by Staphylococcus species. However, this technique has not been specifically validated when used for the evaluation of catheters infected with organisms other than staphylococci. We reviewed catheters that had been submitted for semiquantitative roll tip culture as well as hospital records to determine clinical correlates of infection. Local infection and CRB were defined by standard criteria. Catheter-related sepsis (CRS) was defined as fever, leukocytosis, or hypotension which resolved with catheter removal, without another source of infection. For 195 catheters from 93 patients, gram-negative rods and enterococci were present on 36, fungi were on 25, Corynebacterium species were on 5, Bacillus species were on 3, Staphylococcus species were on 79, and 41 demonstrated no growth. Of 21 episodes of CRB or CRS due to nonstaphylococcal organisms, only 1 (questionable) episode was due to a catheter with < 15 CFU (P < 0.05). Eleven of these 21 episodes of CRB or CRS were due to gram-negative rods and enterococci, of which only the questionable episode was due to a catheter with < 15 CFU. Nine of these 21 episodes of CRB or CRS were due to fungi, none of which were associated with a catheter with < 15 CFU. The data for Staphylococcus species recapitulated published data (none of 21 CRB or CRS episodes were associated with catheters with < 15 CFU) and validated this retrospective technique. The data presented in this study validate the use of the semiquantitative culture technique for the evaluation of catheter-related infections caused by organisms other than staphylococci.
Genome analysis of adenovirus type 3 isolated in Japan
Authors: Shiao S , Aoki K , Isobe K , Tsuzuki W L , Itoh N , Toba K , Kobayashi N , Noguchi Y , Ohno S
Published: January 2020

Adenovirus type 3 (Ad3) isolates, isolated from 45 patients with acute conjunctivitis during the year 1990 in Japan, were studied by DNA restriction enzyme analysis with restriction endonucleases recognizing 6-bp sequences (BamHI, SmaI, HindIII, BglII) and endonucleases recognizing 5- or 4-bp sequences (HinfI and TaqI). All 45 isolates of Ad3 were identified as the genome type Ad3f by six endonucleases. They were further classified into three varieties by HinfI, varieties H1 (87.5%), H2 (8.9%), and H3 (2.2%), and into five varieties by TaqI, varieties T1 (75.6%), T2 (13.3%), T3 (2.2%), T4 (4.5%), and T5 (8.9%). The use of HinfI and TaqI was sufficient to distinguish six subgenome types: types Ad3fH1T1, Ad3fH1T2, Ad3fH1T4, Ad3fH1T5, Ad3fH2T1, and Ad3fH3T3. Among them, Ad3fH1T1 was predominant in areas where the virus is epidemic. The study showed that the same Ad3 genome type, genome type Ad3f, caused acute viral conjunctivitis even in different areas in Japan. The source and the route of infection were suggested to be common in areas where the virus is epidemic.
Escherichia coli O157:H7 in microbial flora of sheep
Authors: Kudva I T , Hatfield P G , Hovde C J
Published: January 2020

We found naturally occurring, potentially virulent Escherichia coli O157:H7 strains in sheep. The incidence of E. coli O157:H7 was transient and ranged from 31% of sheep in June to none in November. The use of a sensitive culture technique and the choice of the proper sampling season were both essential for detecting this bacterium in sheep. DNA hybridizations showed that 80% of the E. coli O157:H7 isolates had at least two of the Shiga-like toxin types I or II or the attaching-effacing lesion genes.
Novel, ligation-dependent PCR assay for detection of hepatitis C in serum
Authors: Hsuih T C , Park Y N , Zaretsky C , Wu F , Tyagi S , Kramer F R , Sperling R , Zhang D Y
Published: January 2020

A simple, sensitive, and specific ligation-dependent PCR (LD-PCR) method for the detection of hepatitis C virus (HCV) RNA in serum is described. The assay uses two DNA capture probes for RNA isolation and two DNA hemiprobes for subsequent PCR. Each capture probe has a 3' sequence complementary to the conserved 5' untranslated region of HCV RNA and a biotin moiety at the 5' end capable of interacting with streptavidin-coated paramagnetic beads. Each hemiprobe contains a sequence complementary to the 5' untranslated region in juxtaposition to one another and a common sequence for PCR primer binding. In guanidinium thiocyanate solutions, the capture probes and the hemiprobes form a hybrid with their target, and the hybrid can be isolated from serum by the binding of the capture probes to the paramagnetic beads in the presence of a magnetic field. The hemiprobes can then be linked to each other by incubation with T4 DNA ligase to form a full probe that serves as a template for a PCR. When serial 10-fold dilutions of synthetic HCV RNA (10(7) to 10 molecules) were tested, there was a good correlation between the amount of PCR product and the initial number of RNA molecules, with a sensitivity of 100 HCV RNA molecules per reaction. Twenty-four specimens that had been tested by either a branched DNA probe (bDNA) assay (13 specimens) or a reverse transcription PCR (RT-PCR) assay (11 specimens) were also analyzed by LD-PCR. The results showed a good correlation among LD-PCR, RT-PCR, and the bDNA assay. However, both LD-PCR and RT-PCR were more sensitive than the bDNA assay when the HCV titer was low.
Novel Borrelia burgdorferi isolates from Ixodes scapularis and Ixodes dentatus ticks feeding on humans
Authors: Anderson J F , Flavell R A , Magnarelli L A , Barthold S W , Kantor F S , Wallich R , Persing D H , Mathiesen D , Fikrig E
Published: January 2020

Seven cultures of Borrelia burgdorferi differing from strains B31 and ZS7 were identified from among 99 isolates from Ixodes scapularis ticks and from white-footed mice (Peromyscus leucopus) and 1 isolate from an Ixodes dentatus tick. Five of the six novel isolates from I. scapularis and the isolate from I. dentatus were from ticks feeding on humans. The six isolates from I. scapularis lacked OspA and OspB, four possessed an OspD band, and two reacted with an anti-OspC monoclonal antibody. Restriction fragment length polymorphisms of HindIII-digested DNAs from six OspA-negative isolates did not hybridize with radiolabeled ospA or LA88 DNA, and only isolate 46047 hybridized with the pG gene. Fragments similar to those recorded for the standard B. burgdorferi sensu stricto strains B31 and ZS7 were obtained with the fla and the HSP70 genes. Pulsed-field gel electrophoresis patterns of DNA digested with MluI included the specific B. burgdorferi sensu stricto band at 135 kbp for the five OspA-negative isolates from I. scapularis ticks. The six novel isolates apparently lack the 55-kbp plasmid encoding OspA. The pG-containing plasmid may be missing from all but isolate 46047. The isolate from the I. dentatus tick was similar to previous isolates from I. dentatus ticks feeding on rabbits. None of the isolates could be recovered from inoculated C3H/HeNCrlBR or white-footed mice. All isolates reacted with sera from humans with early or late Lyme disease. Our studies demonstrate that these borreliae occur in ticks feeding on humans, and therefore, at least some humans in the northeastern United States are likely being exposed to borreliae other than the classic B31-type strains that have thus far been isolated from humans.
Evaluation of enzyme immunoassay for detection of salivary antibody to Helicobacter pylori
Authors: Simor A E , Lin E , Saibil F , Cohen L , Louie M , Pearen S , Donhoffer H A
Published: January 2020

The Helisal test is a quantitative enzyme immunoassay for the measurement of Helicobacter pylori-specific immunoglobulin G antibodies in saliva. This test was evaluated in comparison with culture and histopathologic examination of gastric biopsy specimens obtained from 195 patients who underwent 200 endoscopic procedures for the investigation of gastrointestinal symptoms. Forty-one (21%) patients were found to have peptic ulcer disease, and one other patient had a gastric carcinoma. H. pylori was detected in gastric biopsy specimens obtained from 98 (49%) of the procedures. The sensitivity, specificity, and positive and negative predictive values of the Helisal test were 81, 75, 76, and 80%, respectively. The test was negative for 16 (38%) of the 42 patients with peptic ulcer disease or a gastric malignancy diagnosed at endoscopy. These results suggest that the Helisal assay is only moderately accurate for the detection of H. pylori infection in symptomatic patients.
Determination of stability of Brucella abortus RB51 by use of genomic fingerprint, oxidative metabolism, and colonial morphology and differentiation of strain RB51 from B. abortus isolates from bison and elk
Authors: Jensen A E , Ewalt D R , Cheville N F , Thoen C O , Payeur J B
Published: January 2020

Brucella abortus RB51 and isolates from cattle, bison, and elk were characterized by pulsed-field gel electrophoresis and standard techniques for biotyping Brucella species, which included biochemical, morphological, and antigenic techniques, phage susceptibility, and antibiotic resistance. The objectives were to ascertain the stability of RB51 and to differentiate RB51 from other brucellae. Genomic restriction endonuclease patterns produced by pulsed-field gel electrophoresis demonstrated a unique fingerprint for RB51 relative to other brucellae. Comparisons of the oxidative metabolic profiles of RB51 after time in vivo (14 weeks) and in vitro (75 passages) showed no change in characteristic patterns of oxygen uptake on selected amino acid and carbohydrate substrates. Strain RB51 was biotyped as a typical rough B. abortus biovar 1 (not strain 19) after animal passage or a high number of passages in vitro and remained resistant to rifampin or penicillin and susceptible to tetracycline. No reactions with A or M antiserum or with a monoclonal antibody to the O antigen of Brucella lipopolysaccharides were detected; however, RB51 agglutinated with R antiserum. The results indicate that the genomic fingerprint and rough colonial morphology of RB51 are stable characteristics and can be used to differentiate this vaccine strain from Brucella isolates from cattle, bison, and elk.
Simple differentiation of Vibrio cholerae O139 from V. cholerae O1 and non-O1, non-O139 by modified CAMP test
Authors: Lesmana M , Albert M J , Subekti D , Richie E , Tjaniadi P , Walz S E , Lebron C I
Published: January 2020

Strong positive CAMP reactions were demonstrated by 121 Vibrio cholerae O139 and 504 El Tor isolates, and weak positive CAMP reactions were shown by 235 non-O1, non O139 isolates when these isolates were tested by a modified CAMP technique. Thirty-five classical biotype V. cholerae O1 isolates included in the tests were all CAMP negative.
Detection of extended-spectrum beta-lactamases in clinical isolates of Klebsiella pneumoniae and Escherichia coli
Authors: Jacoby G A , Han P
Published: January 2020

Forty clinical isolates of Escherichia coli and 141 isolates of Klebsiella pneumoniae that either transferred ceftazidime resistance or showed sulbactam enhancement of oxyimino-beta-lactam susceptibility were tested by disk diffusion methodology for susceptibility to aztreonam, cefotaxime, ceftazidime, and cefoxitin. With standard 30 micrograms antibiotic disks, the fraction of these extended-spectrum beta-lactamase (ESBL)-producing isolates testing resistant by National Committee for Clinical Laboratory Standards criteria was lowest (24%) with cefotaxime disks. Forty percent of the E. coli and 29% of the K. pneumoniae isolates appeared susceptible with at least one oxyimino-beta-lactam disk. Ceftazidime and aztreonam disks were equivalent in differentiating ESBL production, and both were superior to cefotaxime disks. Over half the E. Coli and 29% of the K. pneumoniae isolates tested cefoxitin resistant. In 30 isolates, cefoxitin resistance was transmissible and due to a plasmid-mediated AmpC-type beta-lactamase. With a 5-micrograms ceftazidime disk, a breakpoint could be chosen with high sensitivity and specificity for ESBL-producing organisms. Present disk diffusion criteria underestimate the prevalence of ESBL-producing strains.
Selective isolation of vancomycin-resistant enterococci
Authors: van Horn K G , Gedris C A , Rodney K M
Published: January 2020

Broth formulations of two media selective for enterococci, Enterococcel, M-Enterococcosel broths were supplemented with 6 micrograms of vancomycin per ml and evaluated for isolation of vancomycin-resistant enterococci (VRE). Each broth was challenged with various concentrations of Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, and vancomycin-susceptible and vancomycin-resistant enterococci and with 193 perianal specimens obtained from patients at risk in our institution for VRE colonization. Both the Enterococcosel and M-Enterococcus broths with vancomycin detected as few as 1 to 9 CFU of VRE while inhibiting growth of the other organisms tested. Enterococcus faecium organisms (MIC, > 256 micrograms/ml) were recovered from 66 perianal swab cultures in the enterococcosel-vancomycin broth, and VRE were recovered from 62 perianal swab cultures in the M-Enterococcus-vancomycin broth. Enterococcosel-vancomycin broth detected VRE in perianal specimens 48 h earlier than did M-Enterococcus-vancomycin broth. Enterococcosel broth with 6 micrograms of vancomycin per ml can be used for the rapid and selective isolation of VRE from surveillance specimens.
Outbreak of meningitis in weaner pigs caused by unidentified asaccharolytic gram-negative bacterium
Authors: Mohan K , Holmes B , Kock N , Muvavarirwa P
Published: January 2020

Several organisms are known to cause outbreaks of meningitis in pigs, with Haemophilus species being the most frequently implicated. We report such an outbreak in which necropsied pigs manifested an unusual combination of meningitis, tracheitis, and bronchitis. The causative agent appeared to be an asaccharolytic gram-negative nonfermentative bacterium whose classification has yet to be determined. The organism was isolated from the brain and was extremely capnophilic, growing in air only after several serial subcultures.
Detection of Chlamydia trachomatis infections in women by Amplicor PCR: comparison of diagnostic performance with urine and cervical specimens
Authors: Pasternack R , Vuorinen P , Kuukankorpi A , Pitkäjärvi T , Miettinen A
Published: January 2020

We used the Roche Amplicor PCR assay to compare urine and cervical swabs as sample material in the detection of Chlamydia trachomatis causing genital infections. The diagnostic performance of Amplicor PCR was compared with that of cell culture and the Gen-Probe PACE 2 assay with cervical specimens. If discrepant from other results, the specimens negative by PCR were diluted and reanalyzed to reveal PCR inhibitors. Of 666 patients, 39 (5.9%) were confirmed to have chlamydial infection. The respective sensitivity and specificity of Amplicor PCR were as follows: urine specimens, 82.0 and 99.7%; cervical specimens, 82.0 and 99.8%. Those for cell culture with cervical specimens were 84.6 and 100%. For the Gen-Probe PACE 2 assay, the sensitivity and specificity with cervical specimens were 79.5 and 100%, respectively. Without the effect of PCR inhibitors, the sensitivity of PCR with urine would have been 97.4%. Provided that the problems currently caused by inhibitors will be solved, the Amplicor PCR assay with urine specimens offers a tempting alternative for the diagnosis of C. trachomatis infection in women.