bluuebunny/crossref_metadata_2025 huggingface dataset:
Quantitation of human immunodeficiency virus type 1 DNA and RNA by a novel internally controlled PCR assay
Authors: Gupta P , Ding M , Cottrill M , Rinaldo C , Kingsley L , Wolinsky S , Mellors J
Published: January 2020

A novel internally controlled PCR (ICPCR) assay was developed to accurately quantitate human immunodeficiency virus type 1 (HIV-1) DNA and RNA in peripheral blood mononuclear cells and plasma. The ICPCR assay was sensitive and reproducible within a linear range of amplification of 10(0) to 10(3) copies for HIV-1 DNA and 10(1) to 10(4) copies for HIV-1 RNA. The assay detected HIV-1 RNA in plasma and peripheral blood mononuclear cells from all HIV-1 subjects regardless of disease stage. ICPCR was compared with a branched-DNA signal amplification assay for subjects beginning antiretroviral therapy. The reductions in plasma HIV-1 RNA in response to therapy were comparable with the two assays. The ICPCR assay should be useful in monitoring HIV-1 RNA levels both in natural history studies and in clinical trials of antiretroviral agents.
Detection of H-1 parvovirus and Kilham rat virus by PCR
Authors: Besselsen D G , Besch-Williford C L , Pintel D J , Franklin C L , Hook R R , Riley L K
Published: January 2020

H-1 virus and Kilham rat virus (KRV) are autonomous parvoviruses which generally cause subclinical infections in rats and can cause persistent infections in cell cultures. In this study, primer sets specific for either H-1 or KRV were designed on the basis of DNA sequence comparisons of the rodent parvoviruses. The specificities of the H-1 and KRV-specific primer sets were determined by testing viral preparations of seven different parvoviruses and nine other viruses known to infect rodents. The H-1-specific PCR assay amplified the expected 254-bp product only in the presence of H-1 viral DNA and was able to detect as little as 100 fg of H-1 viral DNA. The KRV-specific PCR assay generated the expected 281-bp product only when KRV viral DNA was used as the template and was able to detect as little as 10 pg of KRV viral DNA. Each assay was able to detect its respective virus in tissues from rats experimentally infected with H-1 or KRV. In contrast, no product was amplified by either assay with tissues from mock-infected rats. Our findings indicate that these PCR assays provide rapid, specific, and sensitive methods for the detection of H-1 or KRV infection in rats and cell culture systems.
Detection of porcine reproductive and respiratory syndrome virus in boar semen by PCR
Authors: Christopher-Hennings J , Nelson E A , Nelson J K , Hines R J , Swenson S L , Hill H T , Zimmerman J J , Katz J B , Yaeger M J , Chase C C
Published: January 2020

Porcine reproductive and respiratory syndrome virus (PRRSV) causes a devastating disease in swine. The presence and transmission of PRRSV by boar semen has been demonstrated by using a swine bioassay. In this assay, 4- to 8-week-old pigs were inoculated intraperitoneally with semen from PRRSV-infected boars. Seroconversion of these piglets indicated the presence of PRRSV in semen. Seroconversion in gilts has also been demonstrated following artificial insemination with semen from PRRSV-infected boars. These methods of detecting PRRSV in boar semen are time-consuming, laborious, and expensive. The objective of this study was to develop a reliable and sensitive PCR assay to directly detect PRRSV in boar semen. Primers from open reading frames 1b and 7 of the PRRSV genome were used in nested PCRs. Virus was detected at concentrations as low as 10 infectious virions per ml in PRRSV-spiked semen. Specificity was confirmed by using a nested PCR and a 32P-labeled oligonucleotide probe. The primers did not react with related arteriviruses or other swine viruses. The PCR assay showed good correlation with the swine bioassay, and both methods were superior to virus isolation. To consistently identify PRRSV in boar semen, the cell fraction was separated by centrifugation at 600 x g for 20 min, a lysis buffer without a reducing agent (2-mercaptoethanol) was used, and nondiluted and 1:20-diluted cell fractions were evaluated by PCR. PRRSV was not reliably detected in the seminal plasma fraction of boar semen.
Prevalence of human T-cell lymphotropic virus type 1 (HTLV-1) and HTLV-2 infection among Spanish drug users measured by HTLV-1 assay and HTLV-1 and -2 assay. HTLV-1 and HTLV-2 Spanish Study Group
Authors: Henrard D R , Soriano V , Robertson E , Gutierrez M , Stephens J , Dronda F , Miles F , Pujol E , Buytendorp M , Castro A
Published: January 2020

The prevalence of human T-cell lymphotropic virus type 1 (HTLV-1) and HTLV-2 infection in 1992 and 1993 was determined by testing 2,152 specimens from injection drug users living in 11 geographic areas in Spain. Results obtained by an authentic HTLV-1 and -2 test were compared with those obtained by an HTLV-1 assay. HTLV infection was identified in 7 of 11 regions, with an overall prevalence of 2.5% (range, 0.4 to 11.5%). Fourty-four (81%) of 54 subjects were infected with HTLV-2; the viral strains in the remaining 10 subjects could not be serologically typed. Underestimation of HTLV infection because of the low sensitivities of HTLV-1 enzyme immunoassays for HTLV-2 antibody was relatively low (< 20%). Therefore, previous epidemiologic findings generated with HTLV-1 enzyme immunoassays appear to be reasonably accurate. Our results suggest that the rate of HTLV infection may have been increasing recently among Spanish drug users.
Neisseria meningitidis with decreased susceptibility to penicillin in Saskatchewan, Canada
Authors: Blondeau J M , Ashton F E , Isaacson M , Yaschuck Y , Anderson C , Ducasse G
Published: January 2020

Moderately penicillin-resistant Neisseria meningitidis is rare in North America. We report an outbreak of meningococcal disease in Saskatoon, Saskatchewan, Canada, with serogroup C N. meningitidis. The MICs of penicillin ranged from 0.12 to 0.25 micrograms/ml, and all isolates showing decreased susceptibility had identical genomic fingerprints when they were compared by pulsed-field gel electrophoresis. Our data indicate that N. meningitidis that is moderately resistant to penicillin is prevalent in Saskatchewan, Canada.
Sandwich enzyme-linked immunosorbent assay compared with Pastorex latex agglutination test for diagnosing invasive aspergillosis in immunocompromised patients
Authors: Verweij P E , Stynen D , Rijs A J , de Pauw B E , Hoogkamp-Korstanje J A , Meis J F
Published: January 2020

The performance of a direct sandwich enzyme-linked immunosorbent assay (ELISA) for detecting Aspergillus galactomannan was compared with that of the Pastorex Aspergillus antigen latex agglutination (LA) test by using 532 serum samples from 61 patients at risk for invasive aspergillosis. The ELISA gave positive results earlier in the course of infection than did the LA test. A sensitivity of 70% and a specificity of 86% were obtained for the LA test and corresponding values of 90 and 84% were obtained for the ELISA when a series of serum samples was employed.
Interference by hydroxyethyl starch used for vascular filling in latex agglutination test for cryptococcal antigen
Authors: Millon L , Barale T , Julliot M C , Martinez J , Mantion G
Published: January 2020

The glucuronoxylomannan component of the Cryptococcus neoformans capsular polysaccharide confers serotype specificity, and its detection in cerebrospinal fluid or serum by the latex agglutination test is used for diagnosis. Low-molecular-weight hydroxyethyl starches can be used as an alternative to albumin for vascular filling. This study reports the occurrence of a false-positive result with the Pastorex Cryptococcus test (Sanofi Diagnostics Pasteur, Marnes la Coquette, France) for a patient receiving hydroxyethyl starch characterized by a substitution ratio of 0.6 (Elohes, Biosedra, Sèvres, France).
Isolation of influenza virus in human lung embryonated fibroblast cells (MRC-5) from clinical samples
Authors: de Oña M , Melón S , de la Iglesia P , Hidalgo F , Verdugo A F
Published: January 2020

Ninety-four pharyngeal swab samples corresponding to 94 patients with suspected influenza virus infection were inoculated in Madin-Darby canine kidney (MDCK) cells, the conventional cell system for the isolation of influenza virus, and in fibroblastic human embryo lung (MRC-5) cells, a cell system less commonly used for this purpose but one frequently used in clinical virology laboratories. Both cell preparations were treated with trypsin. Influenza virus was recovered from 15% of the samples inoculated in MDCK cells and from 18% of those inoculated in MRC-5 cells. The use of MRC-5 cells can simplify the search for respiratory viruses and would assist in the rapid detection of influenza virus during new epidemics.
Colorimetric detection of heat-labile toxin-encoding gene of enterotoxigenic Escherichia coli by PCR
Authors: O'Meara D , O'Shaughnessy E , Cryan B , Fanning S
Published: January 2020

In the developing world, enterotoxigenic Escherichia coli (ETEC) strains which produce enterotoxins are a significant cause of morbidity and mortality. Heat-labile (LT) toxin PCR detection methods have been described, but they have limited applications in a routine laboratory setting. A colorimetric DNA method for the rapid amplification and detection of the LT toxin gene in ETEC strains is described. Target amplification together with colorimetric detection would overcome many of the limitations of conventional PCR. This paper describes a colorimetric PCR detection method specific for LT-gene-encoding ETEC strains. DNA was extracted from two representative colonies from each bacterial isolate and amplified by PCR. Digoxigenin was incorporated into the amplification product, permitting a one-step direct detection using anti-digoxigenin alkaline phosphatase-conjugated antibody. This technique was applied to the investigation of 70 E. coli isolates derived from clinical fecal samples obtained from an Irish population. Eleven percent of the samples were LT positive, confirming the applicability of this method. All LT-positive ETEC strains (controls and clinical isolates) were detected, and no false-positive results occurred.
Detection of Chlamydia trachomatis infection in urine samples from men and women by ligase chain reaction
Authors: van Doornum G J , Buimer M , Prins M , Henquet C J , Coutinho R A , Plier P K , Tomazic-Allen S , Hu H , Lee H
Published: January 2020

The suitability of urine specimens from women and men for the detection of Chlamydia trachomatis infection by a ligase chain reaction (LCR)-based assay with plasmid primers was examined with a group of patients attending a sexually transmitted disease clinic in Amsterdam, The Netherlands. Cervical specimens from 15 of 237 (6.3%) women tested positive for C. trachomatis by cell culture. Of the 25 (10.5%) female urine samples that tested positive by the plasmid-LCR assay, 13 were obtained from cervical culture-positive women. Nine of the 12 plasmid-LCR-positive urine samples from cervical culture-negative women were confirmed to be positive by a second LCR assay with primers based on chromosomal DNA. Urethral specimens from 24 of 258 (9.3%) men were positive for C. trachomatis infection by cell culture. Of the 25 (9.7%) urine samples that tested positive by plasmid-LCR, 20 were from culture-positive men. All five of the LCR-positive urine samples from culture-negative men were confirmed to be positive by the LCR with chromosomal DNA primers. Relative to cell culture, testing by plasmid-LCR analysis of male urine samples had a sensitivity of 83.3% and a specificity of 97.9%; after resolution of discordant samples, these values were 86.2 and 100%, respectively. In the study with women, the sensitivities of plasmid-LCR analysis of cervical and urine specimens in comparison with cervical cell culture were 93.3 and 86.7%, respectively. After resolution of discrepant samples, the sensitivities of the plasmid-LCR test for cervical swabs and female urine samples were 96.3 and 92.6%, respectively. These results indicate that the plasmid-LCR-based assay is a very reliable, sensitive, convenient test for the detection of C. trachomatis infection in female and male urine specimens.
Molecular genotyping of methicillin-resistant Staphylococcus aureus via fluorophore-enhanced repetitive-sequence PCR
Authors: Del Vecchio V G , Petroziello J M , Gress M J , McCleskey F K , Melcher G P , Crouch H K , Lupski J R
Published: January 2020

Methicillin resistance in Staphylococcus aureus is a frequent cause of nosocomial and community-acquired infections. Accurate, rapid epidemiologic typing is crucial to the identification of the source and spread of infectious disease and could provide detailed information on the generation of methicillin-resistant S. aureus (MRSA) strains. The high degree of genetic relatedness of MRSA strains has precluded the use of more conventional methods of genetic fingerprinting. A rapid DNA fingerprinting method that exploits PCR amplification from a DNA repeat sequence in MRSA is described. The random chromosomal distribution of this repeat sequence provides an ideal target for detecting DNA fragment patterns specific to individual MRSA strains. Two PCR fingerprinting methods which use an oligonucleotide primer based on a repetitive sequence found in Mycoplasma pneumoniae are presented. The repetitive element sequence-based PCR (rep-PCR) and fluorophore-enhanced rep-PCR (FERP) can identify epidemic strains among background MRSA. The combination of oligonucleotide primers labeled with different fluorescent dyes allowed simultaneous FERP fingerprinting and mecA gene detection. Eight different fingerprint patterns were observed in MRSA strains collected from different sources. These techniques provide a rapid discriminatory means of molecular epidemiologic typing of MRSA involved in nosocomial infections.
Detection of the Staphylococcal mecA gene by chemiluminescent DNA hybridization
Authors: Kolbert C P , Connolly J E , Lee M J , Persing D H
Published: January 2020

A new solution-phase DNA hybridization capture assay for the rapid detection of the mecA gene in clinical isolates of Staphylococcus was compared with multiplex PCR and disk diffusion methods. The assay uses a DNA capture probe immobilized on paramagnetic particles and a second DNA probe labeled with an acridinium ester. Bacteria from 24-h cultures are lysed, and the lysates are hybridized with the DNA probes. After magnetic separation to remove unhybridized labeled probe, the mecA gene is detected by the chemiluminescence of the hybridized probe. Four hundred consecutive staphylococcal isolates were assayed, including 147 S. aureus and 253 coagulase-negative Staphylococcus isolates. Among the S. aureus isolates, 14 of 147 were MecA+ by both the hybridization capture assay and PCR; 133 of 147 were MecA negative by both assays (positive and negative predictive values, 100%). Comparison of disk diffusion results with those obtained by genotypic methods indicated that 14 of 147 S. aureus isolates judged to be resistant were positive by both methods; 119 of 147 were Oxs and negative by both genotypic methods (positive and negative predictive values, 50 and 100%, respectively). The remaining 14 S. aureus isolates were MecA- Oxr; among these, 13 were beta-lactamase hyperproducers. For coagulase-negative staphylococci, 130 of 253 were MecA+ by the hybridization capture assay; 129 of 130 of these isolates were positive by PCR (positive and negative predictive values, 99.2 and 100%, respectively). Comparison with the disk diffusion assay showed that 128 of the coagulase-negative MecA+ isolates were Oxr; 111 of 253 were MecA- and Oxs (positive and negative predictive values, 90.8 and 99.1%, respectively). Thirteen coagulase-negative isolates were MecA-Oxr; among these, three were beta-lactamase hyperproducers. Comparison of the hybridization capture assay results with PCR indicates that the DNA hybridization assay is a sensitive and specific test for the detection of the mecA gene in clinical isolates of Staphylococcus.
Detection of Escherichia coli O157:H7 by multiplex PCR
Authors: Fratamico P M , Sackitey S K , Wiedmann M , Deng M Y
Published: January 2020

In order to develop a PCR assay for Escherichia coli O157:H7, a portion of the 60-MDa plasmid harbored by enterohemorrhagic E. coli (EHEC) was sequenced and PCR primers were designed. A multiplex PCR method was then designed by employing primers specific for the EHEC eaeA gene, conserved sequences of Shiga-like toxins I (SLT-I) and II (SLT-II), and the 60-MDa plasmid. PCR products of 1,087 bp (eaeA), 227 and/or 224 bp (SLT-I and/or SLT-II), and 166 bp (plasmid) were successfully amplified simultaneously in a single reaction. The multiplex PCR method can be used to specifically identify EHEC of serogroup O157.
Molecular typing of Stenotrophomonas (Xanthomonas) maltophilia by DNA macrorestriction analysis and random amplified polymorphic DNA analysis
Authors: Yao J D , Conly J M , Krajden M
Published: January 2020

Stenotrophomonas (Xanthomonas) maltophilia is a multidrug-resistant, nosocomial pathogen for which optimal typing methods in epidemiologic investigations of nosocomial outbreaks have not been defined. We compared DNA macrorestriction analysis by pulsed-field gel electrophoresis (PFGE) with random amplified polymorphic DNA (RAPD) analysis by arbitrarily primed PCR for molecular typing of 109 multidrug-resistant strains of S. maltophilia from multiple outbreaks at our institution over a 10-month period in 1993. PFGE after digestion with restriction endonuclease DraI revealed 62 unique DNA restriction profiles among the 109 strains, with 23, 11, 6, 6, and 3 strains having concordant profiles in each of five types. There were four concordant profiles among 8 strains (2 strains with each profile), while unique profiles were present in each of the remaining 52 strains. Further RAPD analysis with a decanucleotide primer showed the same number of distinct strain types as PFGE but more subtype diversity within each clonal type. We concluded that DNA macrorestriction analysis and RAPD analysis are sufficiently discriminatory and useful for differentiation of S. maltophilia strains in epidemiologic investigations of nosocomial outbreaks. However, RAPD analysis by arbitrarily primed PCR is faster and less laborious method of molecular typing.
Agglutination of Streptococcus suis by sialic acid-binding lectins
Authors: Charland N , Kellens J T , Caya F , Gottschalk M
Published: January 2020

The 35 Streptococcus suis capsular-type reference strains as well as 45 field strains of type 2 were tested with sialic acid-binding lectins from Sambucus nigra (SNA I), Triticum vulgaris, Maackia amurensis, Homarus americanus, and Limax flavus. Only types 1, 1/2, 2, 14, 15, and 16 agglutinated with SNA I and/or the T. vulgaris lectin. All field strains agglutinated only with SNA I. Reaction with SNA I was probably due to the sialic acid moiety since it disappeared after sialidase treatment. These results confirm the presence of sialic acid in S. suis with the possible terminal sequence N-acetylneuraminic acid-alpha(2,6)GalNAc.
Incidence of the highly conserved fib gene and expression of the fibrinogen-binding (Fib) protein among clinical isolates of Staphylococcus aureus
Authors: Bodén Wästfelt M K , Flock J I
Published: January 2020

We have recently described a 19-kDa fibrinogen-binding protein, termed Fib, which is secreted into the extracellular medium by Staphylococcus aureus. In this study, the presence of the Fib protein and the fib gene among clinical isolates of S. aureus and among other staphylococcal species known to interact with fibrinogen was investigated. This task was pursued at the DNA, mRNA, and protein levels. It was found that the fib gene was unique to S. aureus and highly conserved at the nucleotide level. The Fib protein was produced by all S. aureus strains investigated but was not detected in all bovine mastitis strains, because of proteolytic degradation by simultaneously produced staphylococcal proteases. It was concluded that the fib gene was unique to S. aureus and that it could be used in the identification of S. aureus.
Characterization of Hafnia alvei by biochemical tests, random amplified polymorphic DNA PCR, and partial sequencing of 16S rRNA gene
Authors: Ridell J , Siitonen A , Paulin L , Lindroos O , Korkeala H , Albert M J
Published: January 2020

Hafnia alvei strains which possess the attachment-effacement gene (eaeA) may have clinical importance as new diarrhea-causing pathogens and should therefore be differentiated from other H. alvei strains. We characterized diarrheal H. alvei strains, which were positive in the PCR test for the eaeA gene, using biochemical tests not routinely used for identification of members of the family Enterobacteriaceae, and compared them with eaeA-negative strains isolated from different clinical and nonclinical sources to find characteristics useful for identification. Random amplified polymorphic DNA (RAPD)-PCR and partial sequencing of the 16S rRNA gene were utilized to study the genetic diversity of the isolates. The eaeA-positive strains were found to have many characteristic biochemical properties. Negative reactions in the 2-ketogluconate and histidine assimilation tests and a positive reaction in the 3-hydroxybenzoate assimilation test may be useful in routine diagnostics. Nearly identical RAPD-PCR profiles and identical 353-bp fragments of the 16S rRNA genes indicated little genetic diversity among the eaeA-positive strains. The low level of homology (92%) in the partial 16S rRNA genes of eaeA-positive and -negative H. alvei strains raises questions about the taxonomic positioning of eaeA-positive H. alvei.
Development and evaluation of capture immunoglobulin G and M hemadherence assays by using human type O erythrocytes and recombinant parvovirus B19 antigen
Authors: Wang Q Y , Erdman D D
Published: January 2020

The capacity of human parvovirus B19 to agglutinate human type O erythrocytes was used to develop immunoglobulin G and M antibody capture hemadherence assays. When results of these assays were compared with those of corresponding antibody capture enzyme immunoassays using a well-characterized panel of 125 serum specimens, a 96.8% overall agreement was obtained between the two methods.
Identification of Candida species by PCR and restriction fragment length polymorphism analysis of intergenic spacer regions of ribosomal DNA
Authors: Williams D W , Wilson M J , Lewis M A , Potts A J
Published: January 2020

The PCR was used to amplify a targeted region of the ribosomal DNA from 84 Candida isolates. Unique product sizes were obtained for Candida guilliermondii, Candida (Torulopsis) glabrata, and Candida pseudotropicalis. Isolates of Candida albicans, Candida tropicalis, Candida stellatoidea, Candida parapsilosis, and Candida krusei could be identified following restriction digestion of the PCR products.
Relative value of selective group A streptococcal agar incubated under different atmospheres
Authors: Pacifico L , Ranucci A , Ravagnan G , Chiesa C
Published: January 2020

A commercially available selective group A streptococcal agar (ssA) was evaluated for the recovery of group A streptococci (GAS) in comparison with recovery from simultaneous cultures on conventional sheep blood agar (SBA). Both sets of plates were incubated in air, 5% CO2, and anaerobically for 48 h, with a first reading taken at 24 h. A total of 402 (67.0%) GAS were isolated from the 600 specimens that were submitted. Recovery of GAS was significantly greater after 48 h of incubation than after 24 h of incubation for each medium-atmosphere combination. After 48 h of incubation, the sensitivities of GAS detection obtained by each culture technique were as follows: ssA-anaerobic atmosphere, 98.5%; SBA-anaerobic atmosphere, 89.5%; ssA-CO2 atmosphere, 88.0%; SBA-air, 86.5%; SBA-CO2 atmosphere, 82.0%; and ssA-air, 74.6%. There were no cultures positive in air or CO2 which were not positive anaerobically on either medium. The increased sensitivity of detecting positive GAS cultures when incubation was done in an ssA-anaerobic atmosphere for 48 h uncovered patients truly infected with the organisms.
Prior enrichment of human immunodeficiency virus DNA with probe DNA particles for efficient PCR diagnosis
Authors: Tano H , Fan K , Kitajima M A , Kasai K , Tamatsukuri S , Furuichi Y , Hayashi T , Kondo M , Imai M
Published: January 2020

We report here a novel method by which one can recover human immunodeficiency virus (HIV) DNA in a small volume (approximately 50 microliters) of solution from 8 ml of crude cell lysate which contains as few as several HIV DNA copies. The method uses the specific binding of HIV DNA to HIV probe DNA particles. The HIV DNA thus concentrated on the particles can be subjected to the PCR assay. The method also enables the examination of 100 individual blood specimens in a combined form.
D-mannitol in cerebrospinal fluid of patients with AIDS and cryptococcal meningitis
Authors: Megson G M , Stevens D A , Hamilton J R , Denning D W
Published: January 2020

Cryptococcal meningitis (CM) is associated with raised intracranial pressure which is linked with serious neurological sequelae. Cryptococcus neoformans produces D-mannitol in vitro and in experimental meningitis in rabbits. Mannitol present in the cerebrospinal fluid (CSF) of CM patients could exacerbate raised intracranial pressure and contribute to neurological damage. To link CSF mannitol to cryptococcal infection, levels of mannitol in the CSF of AIDS patients with CM were measured by gas-liquid chromatography. Mannitol was detected in 19 of 21 samples (range, 1.5 to 26.2 mg/liter), but there was no quantitative correlation between the mannitol concentration and the cryptococcal antigen titer.
Microbiological findings about pulmonary cryptosporidiosis in two AIDS patients
Authors: Dupont C , Bougnoux M E , Turner L , Rouveix E , Dorra M
Published: January 2020

There is no known treatment for pulmonary cryptosporidiosis, a rare complication of intestinal cryptosporidiosis in AIDS patients. We report two cases of cryptosporidiosis which were unusual because (i) extracellular invasive forms of the parasite were found in the bronchoalveolar lavage and (ii) the outcome was favorable in one patient after treatment with azithromycin.
Rapid identification of Campylobacter species by restriction fragment length polymorphism analysis of a PCR-amplified fragment of the gene coding for 16S rRNA
Authors: Cardarelli-Leite P , Blom K , Patton C M , Nicholson M A , Steigerwalt A G , Hunter S B , Brenner D J , Barrett T J , Swaminathan B
Published: January 2020

Restriction fragment length polymorphism analysis of a PCR-amplified DNA fragment of the gene coding for 16S rRNA was performed on 148 previously characterized strains of Campylobacter, Helicobacter, Arcobacter, and Wolinella succinogenes and 13 Campylobacter-like isolates. These strains included clinical, animal, and environmental isolates. PCR amplification generated a 283-bp fragment from all species. The amplicon from each strain was digested with six restriction endonucleases (AccI, AvaI, DdeI, HaeIII, HpaII, XhoI). DdeI was useful for the initial grouping of the strains. Additional discrimination within the different DdeI groups was obtained with AccI, HaeIII, HpaII, and XhoI digestions. The PCR-restriction fragment length polymorphism analysis allowed for the discrimination of members of the genus Campylobacter from members of closely related genera and discrimination between Campylobacter species. The proposed method is simple and rapid and can be useful for the routine identification of Campylobacter-like organisms in clinical or epidemiologic studies.
Growth inhibition of Mycobacterium tuberculosis by polyoxyethylene stearate present in the BACTEC pyrazinamide susceptibility test
Authors: Miller M A , Thibert L , Desjardins F , Siddiqi S H , Dascal A
Published: January 2020

We have previously found that approximately 3.5% of 428 clinical isolates of Mycobacterium tuberculosis yield uninterpretable results in the BACTEC pyrazinamide (PZA) susceptibility test system, because of inadequate growth. We tested the hypothesis that polyoxyethylene stearate (POES), the ingredient of the reconstituting fluid for the test, was the cause of this growth inhibition. A total of 15 isolates known for their previously uninterpretable results and 100 randomly chosen clinical isolates were tested in parallel both with and without POES. Repeat testing of the isolates with previously uninterpretable results yielded results in the presence of POES in only seven (47%). In the absence of POES, all gave interpretable results but one such result showed false resistance. For the other 100 clinical isolates, interpretable results were obtained with and without POES, but growth was enhanced in the absence of POES, especially in the PZA-susceptible strains. This was evidenced by a decreased time to attain a growth index of 200 in the control vial (4.9 days without POES versus 5.8 days with POES; P < 0.001) and a higher mean growth index ratio on the day of interpretation of the test (7.4% without POES versus 2.2% with POES; P < 0.001). However, the enhanced growth without POES led to 20 susceptible strains being misinterpreted as either resistant or borderline. We suggest that isolates of M. tuberculosis which yield uninterpretable results in the BACTEC PZA test system should be retested both with and without POES. If interpretable results indicating PZA resistance are obtained only in the absence of POES, the result should be confirmed by a pyrazinamidase assay or by the conventional proportion method. Routine omission of POES from the BACTEC test for all clinical strains is discouraged because of the unacceptably high false-resistance rates.
Molecular technique for rapid identification of mycobacteria
Authors: Avaniss-Aghajani E , Jones K , Holtzman A , Aronson T , Glover N , Boian M , Froman S , Brunk C F
Published: January 2020

Identification of mycobacteria through conventional microbiological methods is cumbersome and time-consuming. Recently we have developed a novel bacterial identification method to accurately and rapidly identify different mycobacteria directly from water and clinical isolates. The method utilizes the PCR to amplify a portion of the small subunit rRNA from mycobacteria. The 5' PCR primer has a fluorescent label to allow detection of the amplified product. The PCR product is digested with restriction endonucleases, and an automated DNA sequencer is employed to determine the size of the labeled restriction fragments. Since the PCR product is labeled only at the 5' end, the analysis identifies only the restriction fragment proximal to the 5' end. Each mycobacterial species has a unique 5' restriction fragment length for each specific endonuclease. However, frequently the 5' restriction fragments from different species have similar or identical lengths for a given endonuclease. A set of judiciously chosen restriction enzymes produces a unique set of fragments for each species, providing us with an identification signature. Using this method, we produced a library of 5' restriction fragment sizes corresponding to different clinically important mycobacteria. We have characterized mycobacterial isolates which had been previously identified by biochemical test and/or nucleic acid probes. An analysis of these data demonstrates that this protocol is effective in identifying 13 different mycobacterial species accurately. This protocol has the potential of rapidly (less than 36 h) identifying mycobacterial species directly from clinical specimens. In addition, this protocol is accurate, sensitive, and capable of identifying multiple organisms in a single sample.
Isolation of Arthrobacter spp. from clinical specimens and description of Arthrobacter cumminsii sp. nov. and Arthrobacter woluwensis sp. nov
Authors: Funke G , Hutson R A , Bernard K A , Pfyffer G E , Wauters G , Collins M D
Published: January 2020

Arthrobacter spp. are very widely distributed in the environment (e.g., soil) but have not been described as causing disease in humans. Over a 6-year period, two reference laboratories isolated or received 11 strains which were eventually identified as belonging to the genus Arthrobacter. These strains had been initially identified as Centers for Disease Control and Prevention coryneform group B-1 and B-3 bacteria (whitishgrayish colonies of 2 mm or greater in diameter after 24 h of incubation, respiratory metabolism, absent or weak acid production from sugars, and hydrolysis of gelatin). However, chemotaxonomic investigations revealed lysine as the diamino acid of the cell wall and the presence of branched cellular fatty acids (with anteiso-pentadecanoic acid predominating) which was compatible with an assignment of the 11 isolates to the genus Arthrobacter only. Peptidoglycan and 16S rRNA gene sequence analyses demonstrated that three of the strains studied were representatives of a new Arthrobacter species for which the name Arthrobacter cumminsii sp. nov. is proposed and that one other strain represented a second new Arthrobacter species for which the name Arthrobacter woluwensis sp. nov. is proposed. This report is the first on the isolation of Arthrobacter spp. from clinical specimens.
Effect of hydrocortisone succinate on growth of Chlamydia pneumoniae in vitro
Authors: Tsumura N , Emre U , Roblin P , Hammerschlag M R
Published: January 2020

We examined the effect of hydrocortisone succinate on the growth of three isolates of Chlamydia pneumoniae in vitro. There was a significant increase in the number of inclusions seen in two of the C. pneumoniae strains in the presence of hydrocortisone. There was no significant increase in the number of inclusions with various concentrations of hydrocortisone over time. The addition of hydrocortisone did not affect the in vitro activities of azithromycin, erythromycin, and doxycycline against C. pneumoniae.
Detection of Chlamydia trachomatis and Neisseria gonorrhoeae by ligase chain reaction-based assays with clinical specimens from various sites: implications for diagnostic testing and screening
Authors: Buimer M , van Doornum G J , Ching S , Peerbooms P G , Plier P K , Ram D , Lee H H
Published: January 2020

Ligase chain reaction (LCR)-based tests for the diagnosis of Chlamydia trachomatis and Neisseria gonorrhoeae infections in men and women attending a sexually transmitted disease clinic were evaluated. LCR testing of urethral swab and urine specimens from men and cervical swab and urine specimens from women was compared with culture of male urethral swabs and female cervical and urethral swabs, respectively. An expanded "gold standard" was defined as a positive culture or at least one specimen confirmed to be positive by LCR testing. The prevalence of C. trachomatis infection as detected by cell culture was 7.0% among 614 men and 5.0% among 602 women. By LCR, these values increased to 11.4 and 9.9% with urethral swabs and urine, respectively, for men and 9.6 and 9.1% with cervical swabs and urine, respectively, for women. Relative to the expanded gold standard, the sensitivity of cell culture with male urethral swabs or female cervical swabs was 57.3 and 45.5%, respectively, compared with corresponding values of 93.3 and 87.9% for LCR. The sensitivity of LCR with urine specimens was 77.3 and 78.8% for men and women, respectively. The prevalence of N. gonorrhoeae infection as detected by culture was 5.9% among 220 men and 2.9% among 383 women. The corresponding values were 8.2 and 5.5%, respectively, by LCR testing of swabs. Prevalence values by LCR testing of urine were 7.3% for men and 2.9% for women. The sensitivity of culture was 72.2% for men and 50.0% for women. The sensitivities of LCR were 100% with male urethral swabs, 95.4% with female cervical swabs, 88.9% with male urine, and 50.0% with female urine. These results indicate that the LCR-based assays represent a major improvement in C. trachomatis and N. gonorrhoeae diagnostics. The sensitivity of testing of urethral or cervical swabs by LCR was markedly greater than that by culture. The sensitivity of testing female or male urine specimens was equal to or greater than that of culturing cervical or urethral specimens. LCR testing of urine specimens may prove useful for screening for C. trachomatis.
Diagnostic antigenemia tests for penicilliosis marneffei
Authors: Kaufman L , Standard P G , Jalbert M , Kantipong P , Limpakarnjanarat K , Mastro T D
Published: January 2020

Disseminated penicilliosis marneffei is an emerging opportunistic mycosis seen in severely immunocompromised human immunodeficiency virus (HIV)-infected patients and is caused by the dimorphic fungus Penicillium marneffei. Early diagnosis and treatment improve clinical outcome. Proper diagnosis is complicated by nonspecific signs and symptoms and by difficulties in histologic recognition and species identification of the pathogen. Since no established immunodiagnostic methods for penicilliosis marneffei are available, we attempted to develop separate immunodiffusion tests to detect P. marneffei antigens and antibodies in patient serum specimens and a latex agglutination test for antigenemia. Antigens consisted of 2-week-old fission arthroconidial filtrates produced in Pine's broth at 37 degrees C. Rabbit antisera were prepared against the 10 x -concentrated filtrate antigens. Studies were carried out with 17 serum specimens from HIV-seropositive adult Thai patients with penicilliosis marneffei and 15 control serum specimens from Thai persons free of HIV and P. marneffei infection. The immunodiffusion tests detected P.marneffei antigenemia in 10 (58.8%) of 17 patients, whereas the latex agglutination test detected antigenemia in 13 (76.5%) of the 17 patients. Antibody was demonstrated in only 2 of the 17 patient sera. All of the tests appeared to be highly specific, since none were positive with sera from 15 Thai control patients, six serum samples containing cryptococcal antigen, or six urine specimens positive for Histoplasma polysaccharide antigens.
Study of Abbott Toxo IMx system for detection of immunoglobulin G and immunoglobulin M toxoplasma antibodies: value of confirmatory testing for diagnosis of acute toxoplasmosis
Authors: Liesenfeld O , Press C , Flanders R , Ramirez R , Remington J S
Published: January 2020

We compared the Abbott Toxo immunoglobulin G (IgG) and IgM IMx assays with the Sabin-Feldman dye test and an IgM enzyme-linked immunosorbent assay (ELISA) in 398 serum samples previously tested in our laboratory (retrospective group) and 1,000 consecutive serum samples, tested as they were received in our laboratory in 1995 (prospective group). In the retrospective group, the IgG IMx had a sensitivity of 100%, specificity of 99.0%, positive predictive value of 99.0%, negative predictive value of 100%, and overall agreement of 99.5%. The percentages for the IgM IMx were 97.8, 99.0, 98.9, 98, and 98.4%, respectively. In the prospective group, the IgG IMx had a sensitivity of 97.8%, specificity of 98.7%, positive predictive value of 97.8%, negative predictive value of 98.7%, and overall agreement of 98.4%. The percentages for the IgM IMx were 88.3, 95.9, 74.7, 98.3, and 95%. A serological profile (IgA and IgE antibodies and the differential agglutination [AC/HS] test) was performed in an attempt to resolve discrepancies. Of 21 serum samples that were positive in the IgM IMx and negative in the IgM ELISA, 19 had serological profiles which were most compatible with an infection acquired in the distant past. Of 8 serum samples which were positive in the IgM ELISA and negative in the IgM IMx, 5 had serological profiles which were most compatible with an infection acquired in the distant past. Of the 55 serum samples that were positive in both IgM tests, 21 were from patients who had serological profiles which were most compatible with an infection acquired in the distal past. In conclusion, our data highlight the importance of confirmatory testing for the diagnosis of recently acquired infection with Toxoplasma gondii. When compared with the dye test and IgM ELISA, the Toxo IgG and IgM IMx assays, respectively, revealed high overall agreement in the retrospective and prospective study.
Two different 16S rRNA genes in a mycobacterial strain
Authors: Ninet B , Monod M , Emler S , Pawlowski J , Metral C , Rohner P , Auckenthaler R , Hirschel B
Published: January 2020

Sequencing of the gene coding for 16S rRNA (16S rDNA) is a well-established method used to identify bacteria, particularly mycobacteria. Unique sequences allow identification of a particular genus and species. If more than one 16S rDNA is present on one mycobacterial genome, their sequences are assumed to be strictly or almost identical. We have isolated a slowly growing Mycobacterium strain, "X", identified by conventional biochemical tests as Mycobacterium terrae. Identification by amplification and direct sequencing of 16S rDNA yielded ambiguous results in two variable regions, suggesting the presence of different copies of the sequenced gene. Total DNA was digested by restriction enzymes and hybridized after Southern blotting to a probe representing about two-thirds of the 16S rDNA. Two copies of 16S rDNA were identified and cloned. By sequencing, the clones were of two different types, A and B, differing in 18 positions. Oligonucleotides specific to each copy of the 16S rDNA were used to distinguish the positions of the two genes observed in the Southern blot. We conclude that Mycobacterium strain "X" has two different copies of 16S rDNA. Variations in the sequence between two copies of 16S rDNA gene have been described in archaeobacteria, but not in mycobacteria. When placed in a phylogenetic tree together with other slowly growing mycobacteria gene A shows a common root with M. terrae, whereas gene B is placed separately.
Assessment of routine use of an anaerobic bottle in a three-component, high-volume blood culture system
Authors: Hellinger W C , Cawley J J , Alvarez S , Hogan S F , Harmesen W S , Ilstrup D M , Cockerill F R
Published: January 2020

The relative value of routine anaerobic blood culture for recovery of organisms and identification of episodes of bloodstream infection was assessed in a three-component, high-volume blood culture system which employs aerobic and anaerobic bottles of BacT/Alert (Organon-Teknika, Durham, N.C.) and aerobic cultures of Isolator (Wampole Laboratories, Cranbury, N.J.). The results of 5,595 blood culture sets from patients with suspected bloodstream infection were analyzed. Compared with either the aerobic BacT/Alert bottle or aerobic culture of Isolator, the BacT/Alert anaerobic bottle recovered significantly fewer isolates (242 versus 294, P < 0.05; 242 versus 298, P < 0.05) but did not detect significantly fewer episodes of bloodstream infection (141 versus 157, P > 0.05; 141 versus 147, P > 0.05). The BacT/Alert anaerobic bottle recovered significantly more isolates of obligately anaerobic bacteria (16 versus 4, P < 0.05; 16 versus 0, P < 0.05) and detected significantly more episodes of bloodstream infection caused by obligately anaerobic bacteria (10 versus 3, P < 0.05; 10 versus 0, P < 0.05) than either the aerobic bottle of BacT/Alert or the aerobic culture of Isolator. The combination of the BacT/Alert anaerobic bottle and the aerobic culture of Isolator recovered as may isolates (374 versus 377) and detected as many episodes of bloodstream infection (194 versus 191) as the combination of the aerobic bottle of BacT/Alert and the aerobic culture of Isolator, and both of these combinations identified at least 8% more isolates and detected at least 3% more bloodstream infections than the combination of the BacT/Alert aerobic and anaerobic bottles. Further analysis of the data revealed that the utility of the BacT/Alert anaerobic bottle, especially when combined with the aerobic culture of Isolator, resulted from not only enhanced recovery of obligately anaerobic bacteria but also effective recovery of facultatively anaerobic bacteria. These results demonstrate the utility of the anaerobic BacT/Alert bottle for detecting bloodstream infection caused by either facultatively anaerobic bacteria or obligately anaerobic bacteria and support the routine inclusion of anaerobic blood culture in the three-component blood culture system used in our hospital.
Multicentric epidemiological study of Aspergillus fumigatus isolates by multilocus enzyme electrophoresis
Authors: Rodriguez E , De Meeüs T , Mallie M , Renaud F , Symoens F , Mondon P , Piens M A , Lebeau B , Viviani M A , Grillot R , Nolard N , Chapuis F , Tortorano A M , Bastide J M
Published: January 2020

The genotypes of 63 isolates of Aspergillus fumigatus obtained from three hospitals in different geographical areas and of eight culture collection strains were determined by multilocus enzyme electrophoresis. Twelve of the 17 enzymatic loci studied were polymorphic, giving rise to 48 different electrophoretic types. The existence of fixed multilocus genotypes, significant heterozygote deficits and excesses at the different loci, and linkage disequilibria within subpopulations strongly suggests a clonal reproduction mode for A. fumigatus. Numerical analysis of the comparison and disposition of the different electrophoretic types demonstrates a significant genetic differentiation between the three sampling sites. However, no correlation could be found between geographical distances and genetic differentiation. On account of the multiple discriminatory markers, multilocus enzyme electrophoresis typing seems to be a very powerful tool for epidemiological and reproductive mode studies of A. fumigatus.
Clinical isolates of Histoplasma capsulatum from Indianapolis, Indiana, have a recombining population structure
Authors: Carter D A , Burt A , Taylor J W , Koenig G L , White T J
Published: January 2020

A sample of 30 clinical isolates of Histoplasma capsulatum was analyzed to determine (i) whether genetic exchange is important in the life cycle of this fungus and (ii) whether distinct subpopulations which correlate with disease severity or host immune status exist. Eleven biallelic molecular markers were developed, with the frequency of the least common allele at each molecular locus ranging from 10 to 50%. Every isolate had a different, unique multilocus genotype. Data analysis indicated that frequent recombination occurs within the Indianapolis, Ind., population. There were no associations between isolates from the immunocompromised population or from those with different clinical manifestations of histoplasmosis.
Genotypic characterization of sequential Aspergillus fumigatus isolates from patients with cystic fibrosis
Authors: Verweij P E , Meis J F , Sarfati J , Hoogkamp-Korstanje J A , Latgé J P , Melchers W J
Published: January 2020

Twenty-three sequential Aspergillus fumigatus sputum isolates, which had been collected over a period of 2 years, from two patients with cystic fibrosis were genotyped by random amplified polymorphic DNA PCR and restriction fragment length polymorphism analysis. In patient B, one genotype was predominantly present in the sputum samples, while in the other patient up to nine different genotypes were identified. This study suggests that different patterns of colonization with A.fumigatus exist in patients with cystic fibrosis.
Assessment of enzyme immunoassay versus radioimmunoassay for detection of Legionella pneumophila serogroup 1 antigen in frozen urine specimens
Authors: Chang F Y , Stout J E , Yu V L
Published: January 2020

An enzyme immunoassay (EIA) was compared with a radioimmunoassay (RIA) for detection of Legionella pneumophila serogroup 1 antigen in 37 frozen urine samples. Among 18 samples positive by the RIA at the time of collection, 50% (9 of 18) of stored specimens retained positivity when retested by the RIA whereas 78% (14 of 18) of stored specimens retained positivity when retested by the EIA (P < 0.02). A false-negative result occurred after storage for 5 months or less if the original RIA result showed weak positivity.